WO2024256715A1 - Reagent for in situ hybridization quality control - Google Patents

Reagent for in situ hybridization quality control Download PDF

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WO2024256715A1
WO2024256715A1 PCT/EP2024/066706 EP2024066706W WO2024256715A1 WO 2024256715 A1 WO2024256715 A1 WO 2024256715A1 EP 2024066706 W EP2024066706 W EP 2024066706W WO 2024256715 A1 WO2024256715 A1 WO 2024256715A1
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sequence
signal
monitoring
probe
analyte
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Seiichiro Watanabe
Lawrence Greenfield
Christian Korfhage
Cheng Frank Zhong
Frank Reinecke
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Resolve Biosciences GmbH
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Resolve Biosciences GmbH
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6841—In situ hybridisation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/682—Signal amplification

Definitions

  • RNAscope® reference ⁇ guide: ⁇ Guidelines ⁇ and ⁇ Protocols ⁇ for ⁇ Obtaining ⁇ Optimized ⁇ RNA ⁇ in ⁇ situ ⁇ Hybridization ⁇ Results ⁇ with ⁇ Any ⁇ Tissue ⁇ Type ⁇ from ⁇ Any ⁇ Species. ⁇ ACD.
  • the ⁇ disclosure ⁇ pertains ⁇ to ⁇ monitoring ⁇ probes comprising ⁇ at ⁇ least ⁇ one ⁇ identifier ⁇ element (t) ⁇ comprising ⁇ a ⁇ nucleotide ⁇ sequence ⁇ which ⁇ is ⁇ essentially ⁇ complementary ⁇ to ⁇ at ⁇ least ⁇ a ⁇ section ⁇ of ⁇ the ⁇ unique ⁇ set ⁇ identifier ⁇ sequence ⁇ of ⁇ an ⁇ identifier ⁇ element ⁇ (T) ⁇ of ⁇ a ⁇ corresponding ⁇ decoding ⁇ element.
  • ⁇ In ⁇ one ⁇ embodiment ⁇ the ⁇ nucleotide ⁇ acid ⁇ sequence of ⁇ the ⁇ monitoring ⁇ probe is ⁇ attached ⁇ to ⁇ a ⁇ particle ⁇ selected ⁇ from ⁇ a ⁇ non-magnetic ⁇ bead ⁇ particle, ⁇ magnetic ⁇ bead ⁇ particle, ⁇ a ⁇ streptavidin ⁇ coated ⁇ particle, ⁇ a ⁇ biotin ⁇ coated ⁇ particle, ⁇ and/or ⁇ any ⁇ combination ⁇ thereof.
  • In ⁇ yet ⁇ a ⁇ further ⁇ embodiment ⁇ the ⁇ monitoring ⁇ probe is ⁇ immobilized ⁇ on ⁇ a ⁇ slide ⁇ surface through ⁇ a ⁇ poly-lysine ⁇ coating ⁇ of ⁇ the ⁇ slide ⁇ surface.
  • the ⁇ monitoring ⁇ probe is ⁇ immobilized ⁇ to ⁇ the ⁇ poly-lysine ⁇ coated ⁇ slide ⁇ surface ⁇ using ⁇ homobifunctional ⁇ or ⁇ heterobifunctional ⁇ cross-linking ⁇ reagents ⁇ via ⁇ a ⁇ reactive ⁇ group.
  • In ⁇ yet ⁇ a ⁇ further ⁇ embodiment ⁇ the ⁇ monitoring ⁇ probe or ⁇ the ⁇ slide ⁇ surface comprise ⁇ a ⁇ reactive ⁇ group ⁇ consisting ⁇ of ⁇ an ⁇ alkyne ⁇ or ⁇ azide, ⁇ and ⁇ the ⁇ other ⁇ contact ⁇ surface ⁇ comprises ⁇ the ⁇ partner ⁇ azide or ⁇ alkyne, ⁇ such ⁇ that ⁇ the ⁇ immobilization ⁇ can ⁇ occur ⁇ using ⁇ click ⁇ chemistry.
  • In ⁇ yet ⁇ a ⁇ further ⁇ embodiment ⁇ the ⁇ size ⁇ of ⁇ the ⁇ monitoring ⁇ probe is ⁇ 250 ⁇ nm ⁇ or ⁇ less; ⁇ 225 ⁇ nm ⁇ or ⁇ less; ⁇ 210 ⁇ nm ⁇ or ⁇ less; ⁇ but ⁇ at ⁇ least ⁇ 90 ⁇ nm, ⁇ at ⁇ least ⁇ 130 ⁇ nm ⁇ at ⁇ least ⁇ 180 ⁇ nm, ⁇ in ⁇ one ⁇ embodiment ⁇ the ⁇ size ⁇ of ⁇ the ⁇ monitoring ⁇ probe is ⁇ about ⁇ 200 ⁇ nm.
  • the method ⁇ to ⁇ monitor ⁇ an ⁇ in ⁇ situ hybridization ⁇ multiplex ⁇ reaction ⁇ comprises ⁇ at ⁇ least ⁇ one ⁇ monitoring ⁇ probe as ⁇ described ⁇ herein, ⁇ wherein ⁇ the ⁇ in ⁇ situ hybridization ⁇ multiplex ⁇ reaction ⁇ is ⁇ characterized ⁇ by ⁇ comprising: ⁇ a.) a ⁇ binding ⁇ element which ⁇ is ⁇ essentially ⁇ complementary ⁇ to ⁇ at ⁇ least ⁇ a ⁇ section ⁇ of ⁇ an ⁇ analyte ⁇ nucleotide ⁇ sequence ⁇ to ⁇ be ⁇ detected ⁇ and ⁇ an ⁇ identifier ⁇ element ⁇ (T) ⁇ comprising ⁇ a ⁇ nucleotide ⁇ sequence ⁇ which ⁇ is ⁇ unique ⁇ to ⁇ the ⁇ analyte ⁇ nucleotide ⁇ sequence; ⁇ and/or b.) a ⁇ decoding ⁇ element comprising ⁇ an ⁇ identifier ⁇ element ⁇ (t) ⁇ comprising ⁇ a ⁇ nucleotide ⁇ sequence ⁇ which ⁇ is ⁇ essentially ⁇ comp
  • the in-situ hybridization ⁇ multiplex ⁇ reaction ⁇ is ⁇ described ⁇ in ⁇ more ⁇ details ⁇ in ⁇ this ⁇ description, ⁇ however, ⁇ it ⁇ should ⁇ be ⁇ mentioned ⁇ in ⁇ a ⁇ first ⁇ overview, ⁇ that ⁇ the ⁇ monitoring ⁇ probe ⁇ of ⁇ the ⁇ present ⁇ RES-PA18-PCT ⁇ invention ⁇ is ⁇ designed ⁇ to ⁇ allow ⁇ the ⁇ monitoring ⁇ of ⁇ the ⁇ correct ⁇ in ⁇ situ hybridization ⁇ multiplex ⁇ reaction ⁇ by ⁇ being ⁇ part ⁇ of ⁇ the ⁇ reaction, ⁇ thereby ⁇ being ⁇ exposed ⁇ to ⁇ the ⁇ same ⁇ reactants ⁇ and ⁇ reactions ⁇ steps ⁇ as ⁇ used ⁇ in ⁇ the ⁇ in ⁇ situ hybridization ⁇ multiplex ⁇ reaction.
  • In ⁇ one ⁇ embodiment ⁇ the ⁇ method/ ⁇ monitoring ⁇ probe comprises ⁇ a ⁇ set ⁇ of ⁇ monitoring ⁇ probes, ⁇ wherein ⁇ for ⁇ each ⁇ of ⁇ the ⁇ different ⁇ identifier ⁇ elements ⁇ (t) ⁇ of ⁇ the ⁇ decoding ⁇ elements the set
  • the ⁇ disclosure ⁇ encompasses ⁇ a method, ⁇ wherein ⁇ the monitoring ⁇ probe as ⁇ disclosed ⁇ herein, ⁇ can ⁇ be ⁇ detected ⁇ by ⁇ a ⁇ signal ⁇ element ⁇ with ⁇ a ⁇ fluorophore ⁇ being ⁇ the ⁇ same ⁇ or ⁇ being ⁇ different ⁇ from ⁇ the ⁇ fluorophore ⁇ used ⁇ in ⁇ the ⁇ detection ⁇ of ⁇ the ⁇ target ⁇ nucleic ⁇ acid ⁇ sequence ⁇ that ⁇ permits ⁇ normalizing ⁇ the ⁇ signal ⁇ of ⁇ the ⁇ immobilized ⁇ monitoring ⁇ probe.
  • the ⁇ delta ⁇ G ⁇ value ⁇ of ⁇ the ⁇ monitoring ⁇ probes is ⁇ adjusted ⁇ by ⁇ the ⁇ sequence ⁇ length ⁇ or ⁇ sequence ⁇ composition.
  • ⁇ In ⁇ yet ⁇ a ⁇ further ⁇ embodiment ⁇ the ⁇ delta ⁇ G ⁇ value ⁇ of ⁇ the ⁇ monitoring ⁇ probes is ⁇ adjusted ⁇ by ⁇ using ⁇ modified ⁇ bases ⁇ which ⁇ have ⁇ higher ⁇ stability ⁇ than ⁇ standard ⁇ bases.
  • temperature ⁇ fluctuation ⁇ can ⁇ be ⁇ read-out ⁇ by ⁇ specific ⁇ change ⁇ in ⁇ the ⁇ fluorescence ⁇ and/or ⁇ color ⁇ facilitated ⁇ by ⁇ the ⁇ direct ⁇ and/or ⁇ indirect ⁇ interaction ⁇ of ⁇ the ⁇ monitoring ⁇ probe ⁇ with ⁇ the signal ⁇ oligonucleotide.
  • the ⁇ kit ⁇ according ⁇ is ⁇ adapted for ⁇ monitoring ⁇ a ⁇ tissue ⁇ sample ⁇ preparation.
  • the control ⁇ slide ⁇ can ⁇ be ⁇ used for ⁇ assessing ⁇ in-situ ⁇ hybridization ⁇ instrument installations.
  • the ⁇ present ⁇ disclosure pertains ⁇ to a ⁇ reagent ⁇ (monitoring ⁇ probe ⁇ or ⁇ monitoring ⁇ probe ⁇ set) ⁇ for ⁇ methods ⁇ and ⁇ kits ⁇ for ⁇ detection ⁇ of ⁇ a ⁇ polymorphic analyte ⁇ in ⁇ a ⁇ sample ⁇ by ⁇ specific ⁇ signal-encoding ⁇ of ⁇ said ⁇ analyte by ⁇ in-situ ⁇ hybridization.
  • ⁇ the ⁇ detected ⁇ signal ⁇ may ⁇ label ⁇ a ⁇ gene ⁇ family, ⁇ a ⁇ single ⁇ gene, ⁇ a ⁇ specific ⁇ genetic ⁇ locus, ⁇ a ⁇ benign ⁇ or ⁇ malign ⁇ genetic ⁇ variation, ⁇ genetic ⁇ specificity ⁇ or ⁇ biomarker ⁇ in ⁇ a ⁇ cluster ⁇ of ⁇ patients, ⁇ a ⁇ virus-genome, ⁇ a ⁇ certain ⁇ subset ⁇ of ⁇ nucleic ⁇ acid ⁇ variation ⁇ (e.g. ⁇ a ⁇ group ⁇ of ⁇ SNPs, ⁇ a ⁇ mutation, ⁇ a ⁇ virus-variant, ⁇ a ⁇ gene-variant, ⁇ a ⁇ transcript, ⁇ isoforms, ⁇ splice-variants, ⁇ etc.).
  • the ⁇ technology ⁇ allows ⁇ the ⁇ high-throughput ⁇ detection ⁇ of ⁇ nucleic ⁇ acids ⁇ with ⁇ variable ⁇ sequences ⁇ of ⁇ at ⁇ least ⁇ 60%, ⁇ at ⁇ least ⁇ 65%, ⁇ at ⁇ least ⁇
  • this ⁇ disclosure ⁇ provides in ⁇ vitromethods for ⁇ diagnosis ⁇ of ⁇ a ⁇ disease ⁇ in ⁇ plants ⁇ selected ⁇ from ⁇ the ⁇ group ⁇ comprising: ⁇ diseases ⁇ caused ⁇ by ⁇ biotic ⁇ stress, ⁇ preferably ⁇ by ⁇ infectious ⁇ and/or ⁇ parasitic ⁇ origin, ⁇ or ⁇ diseases ⁇ caused ⁇ by ⁇ abiotic ⁇ stress, ⁇ preferably ⁇ caused ⁇ by ⁇ nutritional ⁇ deficiencies ⁇ and/or ⁇ unfavourable environment, ⁇ said ⁇ method ⁇ comprising ⁇ the ⁇ use ⁇ of ⁇ the ⁇ multiplex ⁇ method ⁇ according ⁇ to ⁇ the ⁇ present ⁇ disclosure.
  • ⁇ this ⁇ disclosure ⁇ relates to ⁇ optical ⁇ multiplexing ⁇ systems ⁇ suitable ⁇ for ⁇ the ⁇ method ⁇ according ⁇ to ⁇ the present ⁇ disclosure, ⁇ comprising ⁇ at ⁇ least: - at ⁇ least ⁇ one ⁇ reaction ⁇ vessel ⁇ for ⁇ containing ⁇ the ⁇ kits ⁇ or ⁇ part ⁇ of ⁇ the ⁇ kits ⁇ according ⁇ to ⁇ the ⁇ present ⁇ disclosure; - a ⁇ detection ⁇ unit ⁇ comprising ⁇ a ⁇ microscope, ⁇ in ⁇ particular ⁇ a ⁇ fluorescence ⁇ microscope - a ⁇ camera - a ⁇ liquid ⁇ handling ⁇ device.
  • ⁇ the ⁇ unique ⁇ tags are ⁇ design ⁇ as ⁇ follow: - No ⁇ cross-hybridization ⁇ between ⁇ all ⁇ oligonucleotides ⁇ of ⁇ the ⁇ process ⁇ (probes, ⁇ decoders, ⁇ readout), ⁇ so ⁇ that ⁇ all ⁇ tag ⁇ sequences ⁇ are ⁇ usable ⁇ together ⁇ (compatible) - No ⁇ cross-hybridization ⁇ between ⁇ connector ⁇ elements ⁇ (bridges) ⁇ of ⁇ different ⁇ unique ⁇ tags - Stability ⁇ of ⁇ hybridization ⁇ of ⁇ the ⁇ unique ⁇ tags ⁇ should ⁇ be ⁇ in ⁇ a ⁇ narrow ⁇ range: ⁇ as ⁇ stable ⁇ as ⁇ possible ⁇ (fast ⁇ hybridization, ⁇ i.e., short ⁇ cycle ⁇ times) ⁇ but ⁇ significantly ⁇ different ⁇ (in ⁇ this ⁇ case ⁇ less ⁇ stable) ⁇ than ⁇ the ⁇ primary ⁇ probe ⁇ (for ⁇ differential ⁇ denaturation, ⁇ without ⁇ removing ⁇ primary ⁇ probes).
  • the ⁇ present ⁇ description ⁇ pertains ⁇ in ⁇ particular ⁇ to ⁇ the ⁇ usage ⁇ of ⁇ a ⁇ set ⁇ of ⁇ labeled ⁇ and ⁇ unlabeled ⁇ nucleic ⁇ acid ⁇ sequences ⁇ for ⁇ specific ⁇ quantitative ⁇ and/or ⁇ spatial ⁇ detection ⁇ of ⁇ different ⁇ analytes ⁇ in ⁇ parallel ⁇ via ⁇ specific ⁇ hybridization.
  • the ⁇ so ⁇ called ⁇ “multi-decoders” ⁇ allows ⁇ the ⁇ recruiting ⁇ of ⁇ more ⁇ than ⁇ just ⁇ one ⁇ signal ⁇ oligonucleotide ⁇ and ⁇ therefore ⁇ can ⁇ generate ⁇ new ⁇ signal ⁇ types ⁇ by ⁇ utilizing ⁇ the ⁇ combination ⁇ of ⁇ two ⁇ or ⁇ more ⁇ different ⁇ signal-oligonucleotides ⁇ without ⁇ decreasing ⁇ the ⁇ brightness ⁇ of ⁇ the ⁇ signals.
  • due ⁇ to ⁇ the ⁇ use ⁇ of ⁇ a ⁇ first ⁇ set ⁇ of ⁇ analyte-specific ⁇ probes ⁇ according ⁇ to ⁇ step ⁇ A1 i.e. ⁇ the ⁇ transcript ⁇ plexity ⁇ of ⁇ A1 ⁇ which ⁇ is ⁇ at ⁇ least ⁇ 10 ⁇ times ⁇ higher in ⁇ numbers than ⁇ the ⁇ number ⁇ of ⁇ probes
  • Fig. ⁇ 2 Embodiment ⁇ where ⁇ the ⁇ analyte ⁇ is ⁇ a ⁇ protein, and ⁇ the ⁇ probe ⁇ set ⁇ comprises ⁇ proteins ⁇ (here: ⁇ antibodies) ⁇ specifically ⁇ binding ⁇ to ⁇ the ⁇ analyte. ⁇ The ⁇ probes ⁇ comprise ⁇ a ⁇ unique ⁇ identifier ⁇ sequence ⁇ allowing ⁇ hybridization ⁇ of ⁇ decoding ⁇ oligonucleotides.
  • Fig. ⁇ 3 Flowchart ⁇ of ⁇ the ⁇ method ⁇ according ⁇ to ⁇ the ⁇ disclosure.
  • Fig. ⁇ 4 Alternative ⁇ options ⁇ for ⁇ the ⁇ application ⁇ of ⁇ decoding ⁇ and ⁇ signal ⁇ oligonucleotides.
  • Fig. ⁇ 5 Example ⁇ for ⁇ signal ⁇ encoding ⁇ of ⁇ three ⁇ different ⁇ nucleic ⁇ acid ⁇ sequences ⁇ by ⁇ two ⁇ different ⁇ signal ⁇ types ⁇ and ⁇ three ⁇ detection ⁇ rounds; ⁇ in ⁇ this ⁇ example, ⁇ the ⁇ encoding ⁇ scheme ⁇ includes ⁇ error ⁇ detection.
  • Fig. ⁇ 6 Number ⁇ of ⁇ generated ⁇ code ⁇ words ⁇ (logarithmic ⁇ scale) ⁇ against ⁇ number ⁇ of ⁇ detection ⁇ cycles.
  • Fig. ⁇ 7 Calculated ⁇ total ⁇ efficiency ⁇ of ⁇ a ⁇ 5-round ⁇ encoding ⁇ scheme ⁇ based ⁇ on ⁇ single ⁇ step ⁇ efficiencies.
  • Fig. ⁇ 8 Comparison ⁇ of ⁇ relative ⁇ transcript ⁇ abundances ⁇ between ⁇ different ⁇ experiments.
  • Fig. ⁇ 9 Correlation ⁇ of ⁇ relative ⁇ transcript ⁇ abundances ⁇ between ⁇ different ⁇ experiments.
  • Fig. ⁇ 10 Comparison ⁇ of ⁇ intercellular ⁇ distribution ⁇ of ⁇ signals.
  • Fig. ⁇ 11 Comparison ⁇ of ⁇ intracellular ⁇ distribution ⁇ of ⁇ signals.
  • Fig. ⁇ 12 Distribution ⁇ pattern ⁇ of ⁇ different ⁇ cell ⁇ cycle ⁇ dependent ⁇ transcripts.
  • the ⁇ number ⁇ of ⁇ codewords ⁇ for ⁇ merFISH ⁇ does ⁇ not ⁇ exponentially ⁇ increase ⁇ with ⁇ the ⁇ number ⁇ of ⁇ detection ⁇ cycles ⁇ but ⁇ gets ⁇ less ⁇ effective ⁇ with ⁇ each ⁇ added ⁇ round. ⁇ In ⁇ contrast, ⁇ the ⁇ number ⁇ of ⁇ codewords ⁇ for ⁇ intronSeqFISH, ⁇ the ⁇ method ⁇ of ⁇ the ⁇ present ⁇ disclosure ⁇ without ⁇ using ⁇ multi-decoders, ⁇ the method ⁇ with ⁇ multi-decoders ⁇ increases ⁇ exponentially. ⁇ The ⁇ slope ⁇ of ⁇ the ⁇ curve ⁇ for ⁇ the ⁇ method ⁇ using ⁇ multi-decoders ⁇ is ⁇ much ⁇ higher ⁇ than ⁇ that ⁇ of ⁇ the ⁇ prior ⁇ invention, ⁇ leading ⁇ to ⁇ more ⁇ than ⁇ 20 ⁇ 000 ⁇ 000 ⁇ times ⁇ more ⁇ code ⁇ words ⁇ usable ⁇ after ⁇ 20 ⁇ rounds ⁇ of ⁇ detection.
  • Fig. ⁇ 19 ⁇ Visual ⁇ representation ⁇ of ⁇ mRNA ⁇ and ⁇ control ⁇ particle.
  • Fig ⁇ 20 ⁇ Particle ⁇ attachment ⁇ to ⁇ microscope ⁇ slights.
  • Fig. ⁇ 21 ⁇ Assay ⁇ for ⁇ parameter ⁇ optimization.
  • Fig. ⁇ 22 ⁇ Example ⁇ for ⁇ the ⁇ use ⁇ of ⁇ ZEN’s ⁇ DFIs.
  • Fig. ⁇ 23 ⁇ Results ⁇ of ⁇ MC ⁇ Run ⁇ of ⁇ 1-plex ⁇ particle
  • Fig. ⁇ 24 ⁇ Results ⁇ of ⁇ MC ⁇ Run ⁇ of ⁇ 1-plex ⁇ particle, ⁇ 6-plex ⁇ particle, ⁇ 12-plex ⁇ particle
  • Fig. ⁇ 25 ⁇ MC ⁇ Run ⁇ of ⁇ 19-plex ⁇ particles
  • Fig.26 ⁇ Embodiments ⁇ of ⁇ the ⁇ monitoring ⁇ probe ⁇ according ⁇ to ⁇ the ⁇ present ⁇ disclosure. ⁇ A) ⁇ Monitoring ⁇ probes ⁇ (1) ⁇ attached ⁇ to ⁇ a ⁇ particle ⁇ (3) ⁇ like ⁇ a ⁇ magnetic ⁇ bead, ⁇ which ⁇ is ⁇ immobilized ⁇ to ⁇ a ⁇ slide ⁇ surface ⁇ (2). ⁇ On ⁇ the ⁇ right ⁇ a ⁇ tissue ⁇ sample ⁇ (4) ⁇ is ⁇ place ⁇ on ⁇ top ⁇ of ⁇ the ⁇ particles. ⁇ B) ⁇ shows ⁇ a ⁇ specific ⁇ embodiment ⁇ of ⁇ the ⁇ particle, ⁇ wherein ⁇ the ⁇ monitoring ⁇ pro
  • D) ⁇ shows ⁇ a ⁇ specific ⁇ embodiment ⁇ of ⁇ the ⁇ particle, ⁇ wherein ⁇ the ⁇ monitoring ⁇ probe ⁇ comprises ⁇ a ⁇ target ⁇ nucleic ⁇ acid ⁇ sequence ⁇ (1c) ⁇ allowing ⁇ the ⁇ binding/hybridization ⁇ of ⁇ a ⁇ binding ⁇ element ⁇ (7).
  • ⁇ the ⁇ present ⁇ disclosure ⁇ pertains ⁇ to ⁇ oligo ⁇ attached ⁇ beads ⁇ (monitoring ⁇ probes) ⁇ and ⁇ uses ⁇ thereof ⁇ to ⁇ create ⁇ ground ⁇ truth.
  • Altered ⁇ delta ⁇ G ⁇ values ⁇ can ⁇ be ⁇ accomplished ⁇ by ⁇ selecting ⁇ the ⁇ base ⁇ composition ⁇ of ⁇ the ⁇ sequence, ⁇ altering ⁇
  • ⁇ customer ⁇ samples ⁇ can ⁇ be ⁇ applied ⁇ directly ⁇ to ⁇ slides ⁇ containing ⁇ attached ⁇ control ⁇ beads.
  • ⁇ In ⁇ these ⁇ cases, ⁇ the ⁇ control ⁇ bead ⁇ will: 1. Serve ⁇ as ⁇ an ⁇ internal ⁇ control ⁇ to ⁇ monitor ⁇ the ⁇ entire ⁇ workflow ⁇ used ⁇ by ⁇ the ⁇ customer ⁇ in ⁇ running ⁇ their ⁇ sample. 2. Monitor ⁇ the ⁇ efficiency ⁇ of ⁇ the ⁇ tissue ⁇ preparation ⁇ process ⁇ in ⁇ permeabilizing ⁇ the ⁇ tissue ⁇ sufficiently ⁇ to ⁇ allow ⁇ diffusion ⁇ of ⁇ the ⁇ assay ⁇ oligonucleotides ⁇ into ⁇ and ⁇ through ⁇ the ⁇ tissues.
  • the ⁇ present ⁇ disclosure ⁇ also ⁇ describes ⁇ the ⁇ usage ⁇ of ⁇ at ⁇ least ⁇ one set ⁇ of ⁇ nucleic ⁇ acid ⁇ sequences ⁇ for ⁇ specific ⁇ quantitative ⁇ and/or ⁇ spatial ⁇ detection ⁇ of ⁇ different ⁇ analytes ⁇ in ⁇ parallel ⁇ via ⁇ specific ⁇ hybridization.
  • the ⁇ technology allows ⁇ the ⁇ discrimination ⁇ of ⁇ more ⁇ different ⁇ analytes ⁇ than ⁇ different ⁇ detection ⁇ signals ⁇ are ⁇ available.
  • the ⁇ discrimination ⁇ may ⁇ be ⁇ realized ⁇ via ⁇ sequential ⁇ signal-coding ⁇ of ⁇ the ⁇ analytes ⁇ achieved ⁇ by ⁇ several ⁇ cycles ⁇ of ⁇ specific ⁇ hybridization, ⁇ detection ⁇ of ⁇ signals ⁇ and ⁇ selective ⁇ elution ⁇ of ⁇ the ⁇ hybridized ⁇ nucleic ⁇ acid ⁇ sequences.
  • an ⁇ “slide” ⁇ is ⁇ any ⁇ surface ⁇ which ⁇ can ⁇ be ⁇ used ⁇ for ⁇ in-situ ⁇ hybridization. ⁇ In ⁇ some ⁇ embodiments ⁇ the ⁇ slide ⁇ is ⁇ a ⁇ microscope ⁇ slide, ⁇ in ⁇ some ⁇ embodiments ⁇ made ⁇ out ⁇ of ⁇ glass.
  • thee ⁇ cell ⁇ is ⁇ a ⁇ prokaryotic ⁇ cells ⁇ or ⁇ a ⁇ eukaryotic ⁇ cell, ⁇ in ⁇ particular ⁇ a ⁇ mammalian ⁇ cell, ⁇ in ⁇ particular ⁇ a ⁇ human ⁇ cell.
  • the ⁇ biological ⁇ tissue, ⁇ biological ⁇ cells, ⁇ extracts ⁇ and/or ⁇ part ⁇ of ⁇ cells ⁇ are ⁇ fixed.
  • In ⁇ particular, ⁇ the ⁇ analytes ⁇ are ⁇ fixed ⁇ in ⁇ a ⁇ permeabilized ⁇ sample, ⁇ such ⁇ as ⁇ a ⁇ cell-containing ⁇ sample.
  • an ⁇ "oligonucleotide ⁇ as ⁇ used ⁇ herein, ⁇ refers ⁇ to ⁇ s ⁇ short ⁇ nucleic ⁇ acid ⁇ molecule, ⁇ such ⁇ as ⁇ DNA, ⁇ PNA, ⁇ LNA ⁇ or ⁇ RNA.
  • the ⁇ length ⁇ of ⁇ the ⁇ oligonucleotides ⁇ is ⁇ within ⁇ the ⁇ range ⁇ 4-200 ⁇ nucleotides ⁇ (nt), ⁇ preferably ⁇ 6-80 ⁇ nt, ⁇ more ⁇ preferably ⁇ 8-60 ⁇ nt, ⁇ more ⁇ preferably ⁇ 10-50 ⁇ nt, ⁇ more ⁇ preferably ⁇ 12 ⁇ to ⁇ 35 ⁇ depending ⁇ on ⁇ the ⁇ number ⁇ of ⁇ consecutive ⁇ sequence ⁇ elements.
  • the ⁇ oligonucleotides ⁇ may ⁇ be ⁇ linear ⁇ or ⁇ may ⁇ comprise ⁇ hairpin ⁇ or ⁇ loop ⁇ structures.
  • the ⁇ oligonucleotides ⁇ may ⁇ comprise ⁇
  • the ⁇ "analyte-specific ⁇ probe" ⁇ consists ⁇ of ⁇ at ⁇ least ⁇ one, ⁇ preferably ⁇ at ⁇ least ⁇ two ⁇ elements, ⁇ namely ⁇ the ⁇ so-called ⁇ binding ⁇ element ⁇ (S) ⁇ which ⁇ specifically ⁇ interacts ⁇ with ⁇ one ⁇ of ⁇ the ⁇ analytes, ⁇ and ⁇ a ⁇ so-called ⁇ identifier ⁇ element ⁇ (T) ⁇ comprising ⁇ the ⁇ 'unique ⁇ identifier ⁇ sequence'.
  • the ⁇ binding ⁇ element ⁇ (S) ⁇ may ⁇ be ⁇ a ⁇ nucleic ⁇ acid ⁇ such ⁇ as ⁇ a ⁇ hybridization ⁇ sequence ⁇ or ⁇ an ⁇ aptamer, ⁇ or ⁇ a ⁇ peptidic ⁇ structure ⁇ such ⁇ as ⁇ an ⁇ antibody.
  • the ⁇ “monitoring ⁇ probe” ⁇ consists ⁇ of ⁇ at ⁇ least ⁇ one ⁇ element, ⁇ namely ⁇ the ⁇ so-called ⁇ identifier ⁇ element ⁇ (T) ⁇ comprising ⁇ the ⁇ 'unique ⁇ identifier ⁇ sequence'.
  • the ⁇ monitoring ⁇ probe ⁇ is ⁇ attached ⁇ to ⁇ a ⁇ surface, ⁇ selected ⁇ from ⁇ a ⁇ particle ⁇ (e.g., ⁇ a ⁇ magnetic ⁇ or ⁇ nonmagnetic ⁇ bead), ⁇ a ⁇ functional ⁇ group ⁇ (e.g., streptavidin, ⁇ biotin, ⁇ a ⁇ hybridization ⁇ sequence ⁇ or ⁇ an ⁇ aptamer, ⁇ or ⁇ a ⁇ peptidic ⁇ structure ⁇ such ⁇ as ⁇ an ⁇ antibody) ⁇ and ⁇ a ⁇ surface ⁇ (e.g., ⁇ a ⁇ slide), ⁇ as ⁇ well ⁇ as ⁇ any ⁇ combination ⁇ thereof.
  • the ⁇ monitoring ⁇ probe ⁇ may ⁇ also ⁇ comprise ⁇ a ⁇ so-called ⁇ binding
  • the ⁇ "unique ⁇ identifier ⁇ sequence" ⁇ as ⁇ comprised ⁇ by ⁇ the ⁇ analyte-specific ⁇ probe ⁇ and ⁇ the ⁇ monitoring ⁇ probe ⁇ is ⁇ unique ⁇ in ⁇ its ⁇ sequence ⁇ compared ⁇ to ⁇ other ⁇ unique ⁇ identifiers.
  • "Unique" ⁇ in ⁇ this ⁇ context ⁇ means ⁇ that ⁇ it ⁇ specifically ⁇ identifies ⁇ only ⁇ one ⁇ analyte, ⁇ such ⁇ as ⁇ Cyclin ⁇ A, ⁇ Cyclin ⁇ D, ⁇ Cyclin ⁇ E ⁇ etc., ⁇ or, ⁇ alternatively, ⁇ it ⁇ specifically ⁇ identifies ⁇ only ⁇ a ⁇ group ⁇ of ⁇ analytes, ⁇ independently ⁇ whether ⁇ the ⁇ group ⁇ of ⁇ analytes ⁇ comprises ⁇ a ⁇ gene ⁇ family ⁇ or ⁇ not. ⁇ Therefore, ⁇ the ⁇ analyte ⁇ or ⁇ a ⁇ group ⁇ of ⁇ analytes ⁇ to ⁇ be ⁇ encoded ⁇ by ⁇ this ⁇ unique ⁇ identifier ⁇ can ⁇ be ⁇ distinguished ⁇ from ⁇
  • a ⁇ "decoding ⁇ oligonucleotide” ⁇ or ⁇ an ⁇ “adapter” ⁇ or ⁇ a ⁇ /adapter ⁇ segment” ⁇ consists ⁇ of ⁇ at ⁇ least ⁇ one, ⁇ preferably ⁇ at ⁇ least ⁇ two ⁇ sequence ⁇ elements. ⁇ One ⁇ sequence ⁇ element ⁇ that ⁇ can ⁇ specifically ⁇ bind ⁇ to ⁇ a ⁇ unique ⁇ identifier ⁇ sequence, ⁇ referred ⁇ to ⁇ as an ⁇ “identifier ⁇ connector ⁇ element ⁇ “(t) ⁇ or "first ⁇ connector ⁇ element” ⁇ (t), ⁇ and ⁇ a ⁇ second ⁇ sequence ⁇ element ⁇ specifically ⁇ binding ⁇ to ⁇ a ⁇ signal ⁇ oligonucleotide, ⁇ referred ⁇ to ⁇ as ⁇ "translator ⁇ element” ⁇ (c). ⁇ The ⁇ length ⁇ of ⁇ the ⁇ sequence ⁇ elements ⁇ is ⁇ within ⁇ the ⁇ range ⁇ 8- 60 ⁇ nt, ⁇ preferably ⁇ 12-40 ⁇ nt, ⁇ more ⁇ preferably ⁇ 14-20 ⁇ nt, ⁇ depending ⁇ on ⁇ the
  • ⁇ the ⁇ decoding ⁇ oligonucleotide ⁇ in ⁇ the ⁇ kits ⁇ and/or ⁇ methods ⁇ of ⁇ the ⁇ present ⁇ disclosure ⁇ may ⁇ be ⁇ a ⁇ “multi-decoder”.
  • ⁇ the ⁇ decoding ⁇ oligonucleotide ⁇ is ⁇ a ⁇ multi-decoder ⁇ comprising ⁇ - an ⁇ identifier ⁇ connector ⁇ element ⁇ (t) ⁇ comprising ⁇ a ⁇ nucleotide ⁇ sequence ⁇ which ⁇ is ⁇ essentially ⁇ complementary ⁇ to ⁇ at ⁇ least ⁇ a ⁇ section ⁇ of ⁇ the ⁇ unique ⁇ identifier ⁇ sequence ⁇ of ⁇ the ⁇ identifier ⁇ element ⁇ (T) ⁇ of ⁇ the ⁇ corresponding ⁇ analyte-specific ⁇ probe ⁇ set, ⁇ and - at ⁇ least ⁇ one, ⁇ preferably ⁇ at ⁇ least ⁇ two translator ⁇ elements ⁇ (c) ⁇ comprising ⁇ each ⁇ a ⁇ nucleotide ⁇ sequence ⁇ allowing ⁇ a ⁇ specific ⁇ hybridization ⁇ of ⁇ a ⁇ different ⁇ signal ⁇ oligonucleotide.
  • ⁇ the ⁇ first ⁇ translator ⁇ element ⁇ binds ⁇ a ⁇ different ⁇ signal ⁇ oligonucleotide ⁇ as ⁇ the ⁇ second ⁇ translator ⁇ element.
  • ⁇ the ⁇ signal ⁇ oligonucleotides ⁇ differ ⁇ in ⁇ the ⁇ signal ⁇ element ⁇ comprised ⁇ in ⁇ the ⁇ signal ⁇ oligonucleotide, ⁇ e.g. ⁇ in ⁇ the ⁇ kind ⁇ of ⁇ the ⁇ fluorophore.
  • In ⁇ a ⁇ monitoring ⁇ probe ⁇ set ⁇ the ⁇ monitoring ⁇ probes ⁇ are ⁇ identical ⁇ in ⁇ the ⁇ binding ⁇ element ⁇ (S) ⁇ but ⁇ may ⁇ comprise ⁇ a ⁇ different ⁇ identifier ⁇ element ⁇ (T) ⁇ for ⁇ specifically ⁇ interacting ⁇ with ⁇ the ⁇ surface ⁇ on ⁇ which ⁇ they ⁇ are ⁇ immobilized, ⁇ but ⁇ for ⁇ specifically ⁇ interacting ⁇ with ⁇ different ⁇ decoding ⁇ elements.
  • “Selective ⁇ denaturation” may ⁇ be ⁇ the ⁇ process ⁇ of ⁇ eliminating ⁇ bound ⁇ decoding ⁇ oligonucleotides ⁇ and ⁇ signal ⁇ oligonucleotides ⁇ with ⁇ highest ⁇ efficiency ⁇ while ⁇ at ⁇ the ⁇ same ⁇ time ⁇ the ⁇ target ⁇ specific ⁇ probes ⁇ have ⁇ to ⁇ stay ⁇ hybridized ⁇ with ⁇ the ⁇ highest ⁇ efficiency.
  • the ⁇ total ⁇ efficiency ⁇ of ⁇ these ⁇ two ⁇ combined ⁇ events ⁇ may ⁇ to ⁇ be ⁇ at ⁇ least ⁇ 0.22 ⁇ for ⁇ two ⁇ detection ⁇ cycles, ⁇ 0.37 ⁇ for ⁇ three ⁇ detection ⁇ cycles, ⁇ 0.47 ⁇ for ⁇ four ⁇ detection ⁇ cycles, ⁇ 0.55 ⁇ for ⁇ five ⁇ detection ⁇ cycles, ⁇ 0.61 ⁇ for ⁇ six ⁇ detection ⁇ cycles, ⁇ 0.65 ⁇ for ⁇ seven ⁇ detection ⁇ cycles, ⁇ 0.69 ⁇ for ⁇ eight ⁇ detection ⁇ cycles, ⁇ 0.72 ⁇ for ⁇ nine ⁇ det
  • a ⁇ single ⁇ set refers ⁇ to ⁇ a ⁇ plurality ⁇ of ⁇ oligonucleotides.
  • An ⁇ "analyte ⁇ specific ⁇ probe ⁇ set" refers ⁇ to ⁇ a ⁇ plurality ⁇ of ⁇ moieties ⁇ or ⁇ sub-jects, ⁇ e.g., analyte-specific ⁇ probes ⁇ that ⁇ are ⁇ different ⁇ from ⁇ each ⁇ other ⁇ and ⁇ bind ⁇ to ⁇ independent ⁇ regions ⁇ of ⁇ the ⁇ analyte.
  • a "decoding ⁇ oligonucleotide ⁇ set” refers ⁇ to ⁇ a ⁇ plurality ⁇ of ⁇ decoding ⁇ oligonucleotides ⁇ specific ⁇ for ⁇ a ⁇ certain ⁇ unique ⁇ identifier ⁇ needed ⁇ to ⁇ realize ⁇ the ⁇ encoding ⁇ independent ⁇ of ⁇ the ⁇ length ⁇ of ⁇ the ⁇ code ⁇ word. ⁇ Each ⁇ and ⁇ all ⁇ of ⁇ the ⁇ decoding ⁇ oligonucleotides ⁇ included ⁇ in a ⁇ "decoding ⁇ oligonucleotide ⁇ set” ⁇ bind ⁇ to ⁇ the ⁇ same ⁇ unique ⁇ identifier ⁇ element ⁇ (T) ⁇ of ⁇ the ⁇ analyte-specific ⁇ probe.
  • the ⁇ codewords ⁇ for ⁇ each ⁇ analyte may ⁇ be ⁇ assigned ⁇ sequentially, ⁇ or ⁇ may ⁇ be ⁇ assigned ⁇ at ⁇ random. ⁇ For ⁇ instance, ⁇ a ⁇ first ⁇ analytemay ⁇ be ⁇ assigned ⁇ to ⁇ 101, ⁇ while ⁇ a ⁇ second ⁇ nucleic ⁇ acid ⁇ target ⁇ may ⁇ be ⁇ assigned ⁇ to ⁇ 110. ⁇
  • ⁇ the ⁇ codewords ⁇ may ⁇ be ⁇ assigned ⁇ using ⁇ an ⁇ error- detection ⁇ system ⁇ or ⁇ an ⁇ error- correcting ⁇ system, ⁇ such ⁇ as a ⁇ Hamming ⁇ system, ⁇ a ⁇ Golay ⁇ code, ⁇ or ⁇ an ⁇ extended ⁇ Hamming ⁇ system ⁇ (or ⁇ a ⁇ SECDED ⁇ system, ⁇ i.e., ⁇ single ⁇ error ⁇ correction, ⁇ double ⁇ error ⁇ detection).
  • an ⁇ error- detection ⁇ system ⁇ or ⁇ an ⁇ error- correcting ⁇ system such ⁇ as a ⁇ Hamming ⁇ system, ⁇ a ⁇ Golay ⁇ code, ⁇ or ⁇
  • Essentially ⁇ complementary ⁇ means, ⁇ when ⁇ referring ⁇ to ⁇ two ⁇ nucleotide ⁇ sequences, ⁇ that ⁇ both ⁇ sequences ⁇ can ⁇ specifically ⁇ hybridize ⁇ to ⁇ each ⁇ other ⁇ under ⁇ stringent ⁇ conditions, ⁇ thereby ⁇ forming ⁇ a ⁇ hybrid ⁇ nucleic ⁇ acid ⁇ molecule ⁇ with ⁇ a ⁇ sense ⁇ and ⁇ an ⁇ antisense ⁇ strand ⁇ connected ⁇ to ⁇ each ⁇ other ⁇ via ⁇ hydrogen ⁇ bonds ⁇ (Watson-and-Crick ⁇ base ⁇ pairs).
  • ⁇ essentially ⁇ complementary ⁇ includes ⁇ not ⁇ only ⁇ perfect ⁇ base-pairing ⁇ along ⁇ the ⁇ entire ⁇ strands, ⁇ i.e., perfect ⁇ complementary ⁇ sequences ⁇ but ⁇ also ⁇ imperfect ⁇ complementary ⁇ sequences ⁇ which, ⁇ however, ⁇ still ⁇ have ⁇ the ⁇ capability ⁇ to ⁇ hybridize ⁇ to ⁇ each ⁇ other ⁇ under ⁇ stringent ⁇ conditions.
  • the ⁇ decoding ⁇ oligonucleotides ⁇ of ⁇ a ⁇ particular ⁇ set ⁇ differ ⁇ from ⁇ the ⁇ decoding ⁇ oligonucleotides ⁇ of ⁇ another ⁇ set. ⁇ This ⁇ means, ⁇ the ⁇ decoding ⁇ oligonucleotides ⁇ of ⁇ set ⁇ 1 ⁇ bind ⁇ to ⁇ the ⁇ analyte-specific ⁇ probes ⁇ of ⁇ above ⁇ set ⁇ 1 ⁇ of ⁇ analyte-specific ⁇ probes, ⁇ the ⁇ decoding ⁇ oligonucleotides ⁇ of ⁇ set ⁇ 2 ⁇ binds ⁇ to ⁇ the ⁇ analyte-specific ⁇ probes ⁇ of ⁇ above ⁇ set ⁇ 2 ⁇ of ⁇ analyte-specific ⁇ probes, ⁇ the ⁇ decoding ⁇ oligonucleotides ⁇ of ⁇ set ⁇ 3 ⁇ bind ⁇ to ⁇ the ⁇ analyte-specific ⁇ probes ⁇ of ⁇ above ⁇ set ⁇ 3 ⁇ of ⁇ analyte-specific ⁇ probes, ⁇ etc. ⁇ It ⁇ is ⁇ also ⁇ understood ⁇ that
  • the ⁇ decoding ⁇ oligonucleotides ⁇ of ⁇ a ⁇ particular ⁇ set ⁇ differ ⁇ from ⁇ the ⁇ decoding ⁇ oligonucleotides ⁇ of ⁇ another ⁇ set.
  • the ⁇ term ⁇ "quencher” ⁇ or ⁇ "quencher ⁇ dye” ⁇ or ⁇ "quencher ⁇ molecule refers ⁇ to ⁇ a ⁇ dye ⁇ or ⁇ an ⁇ equivalent ⁇ molecule, ⁇ such ⁇ as ⁇ nucleoside ⁇ guanosine ⁇ (G) ⁇ or ⁇ 2'-deoxyguanosine ⁇ (dG), ⁇ which ⁇ is ⁇ capable ⁇ of ⁇ reducing ⁇ the ⁇ fluorescence ⁇ of ⁇ a ⁇ fluorescent ⁇ reporter ⁇ dye ⁇ or ⁇ donor ⁇ dye.
  • the ⁇ quencher ⁇ is ⁇ a ⁇ fluorescent ⁇ dye ⁇ its ⁇ fluorescence ⁇ wavelength ⁇ is ⁇ typically ⁇ substantially ⁇ different ⁇ from ⁇ that ⁇ of ⁇ the ⁇ reporter ⁇ dye ⁇ and ⁇ the ⁇ quencher ⁇ fluorescence ⁇ is ⁇ usually ⁇ not ⁇ monitored ⁇ during ⁇ an ⁇ assay. ⁇
  • the ⁇ method ⁇ is ⁇ particularly ⁇ qualified ⁇ to ⁇ monitor, ⁇ encode, ⁇ identify, ⁇ detect, ⁇ count, or ⁇ quantify ⁇ the ⁇ in ⁇ situ ⁇ analysis ⁇ of ⁇ analytes ⁇ or ⁇ single ⁇ analyte ⁇ molecules ⁇ in ⁇ a ⁇ biological ⁇ sample, ⁇ i.e., such ⁇ as ⁇ a ⁇ sample ⁇ which ⁇ contains ⁇ nucleic ⁇ acids ⁇ or ⁇ proteins ⁇ as ⁇ said ⁇ analytes.
  • the ⁇ biological ⁇ sample may ⁇ be ⁇ in ⁇ a ⁇ form ⁇ as ⁇ it ⁇ is ⁇ in ⁇ its ⁇ natural ⁇ environment ⁇ (i.e., liquid, ⁇ semi-liquid, ⁇ solid ⁇ etc.), ⁇ or ⁇ processed, ⁇ e.g., as ⁇ a ⁇ dried ⁇ film ⁇ on ⁇ the ⁇ surface ⁇ of ⁇ a ⁇ device ⁇ which ⁇ may ⁇ be ⁇ re-liquefied ⁇ before ⁇ the ⁇ method ⁇ is ⁇ carried ⁇ out. ⁇ In ⁇ another ⁇ embodiment ⁇ of ⁇ the ⁇
  • ⁇ in ⁇ some ⁇ embodiments, ⁇ the ⁇ cell ⁇ and/or ⁇ the ⁇ tissue ⁇ is ⁇ fixed ⁇ prior ⁇ to ⁇ introducing ⁇ the ⁇ probes, ⁇ e.g., ⁇ to ⁇ preserve ⁇ the ⁇ positions ⁇ of ⁇ the ⁇ analytes ⁇ like ⁇ nucleic ⁇ acids ⁇ within ⁇ the ⁇ cell.
  • ⁇ a ⁇ cell ⁇ may ⁇ be ⁇ fixed ⁇ using ⁇ chemicals ⁇ such ⁇ as ⁇ formaldehyde, ⁇ paraformaldehyde, ⁇ glutaraldehyde, ⁇ ethanol, ⁇ methanol, ⁇ acetone, ⁇ acetic ⁇ acid, ⁇ or ⁇ the ⁇ like.
  • This ⁇ measure ⁇ has ⁇ the ⁇ advantage ⁇ that ⁇ the ⁇ analytes ⁇ to ⁇ be ⁇ encoded, ⁇ e.g., the ⁇ nuclei ⁇ acids ⁇ or ⁇ proteins, ⁇ are ⁇ immobilized ⁇ and ⁇ cannot ⁇ escape. ⁇ In ⁇ doing ⁇ so, ⁇ the ⁇ analytes ⁇ then ⁇ prepared ⁇ for ⁇ a ⁇ better ⁇ detection ⁇ or ⁇ encoding ⁇ by ⁇ the ⁇ method ⁇ according ⁇ to ⁇ the ⁇ disclosure. ⁇ In ⁇ yet ⁇ a ⁇ further ⁇ embodiment ⁇ within ⁇ the ⁇ set ⁇ of ⁇ analyte-specific ⁇ probes ⁇ the ⁇ individual ⁇ analyte- specific ⁇ probes ⁇ comprise ⁇ binding ⁇ elements ⁇ (S1, ⁇ S2, ⁇ S3, ⁇ S4, ⁇ S5) ⁇ which ⁇ specifically ⁇ interact ⁇ with ⁇ different ⁇ sub-structures ⁇ of ⁇ one ⁇ of ⁇ the ⁇ analytes ⁇ to ⁇ be ⁇ encoded.
  • the ⁇ present ⁇ disclosure ⁇ pertains ⁇ to ⁇ kit ⁇ for ⁇ multiplex ⁇ analyte ⁇ encoding, ⁇ comprising: (A1) at ⁇ least ⁇ a ⁇ first ⁇ set of ⁇ analyte-specific ⁇ probes ⁇ for ⁇ encoding ⁇ different ⁇ analytes, ⁇ each ⁇ set ⁇ of ⁇ analyte-specific ⁇ probes ⁇ interacting ⁇ with ⁇ a ⁇ different ⁇ analyte, ⁇ wherein ⁇ if ⁇ the ⁇ analyte ⁇ is ⁇ a ⁇ nucleic ⁇ acid ⁇ each ⁇ set ⁇ of ⁇ analyte-specific ⁇ probes ⁇ comprises ⁇ analyte-specific ⁇ probes ⁇ which ⁇ specifically ⁇ interact ⁇ with ⁇ different ⁇ sub-structures ⁇ of ⁇ the ⁇ same ⁇ analyte, ⁇ each ⁇ analyte- specific ⁇ probe ⁇ comprising ⁇ (aa) ⁇ a ⁇ binding ⁇ element ⁇ (S) ⁇ that ⁇ specifically ⁇ interacts ⁇ with ⁇ one ⁇ of ⁇ the ⁇ different ⁇
  • RES-PA18-PCT ⁇ A ⁇ multiplex ⁇ method ⁇ or ⁇ assay ⁇ allow ⁇ the ⁇ simultaneously ⁇ measurement ⁇ of ⁇ multiple ⁇ analytes ⁇ according ⁇ to ⁇ the ⁇ present ⁇ disclosure it ⁇ may ⁇ be ⁇ used ⁇ to ⁇ determine ⁇ the ⁇ presence ⁇ or ⁇ absence ⁇ of ⁇ a ⁇ plurality ⁇ of ⁇ predetermined ⁇ (known) ⁇ analytes ⁇ like ⁇ nucleic ⁇ acid ⁇ target ⁇ sequences ⁇ in ⁇ a ⁇ sample. ⁇ An analyte ⁇ may ⁇ be “predetermined” ⁇ in ⁇ that ⁇ its ⁇ sequence ⁇ is known ⁇ to ⁇ design ⁇ a ⁇ probe ⁇ that ⁇ binds ⁇ to ⁇ the that ⁇ target.
  • the ⁇ unique ⁇ tag ⁇ can ⁇ be ⁇ identified ⁇ by ⁇ various ⁇ techniques, ⁇ including ⁇ hybridization, ⁇ e.g., with ⁇ labelled probes, ⁇ directly ⁇ or ⁇ indirectly ⁇ or ⁇ by ⁇ sequencing ⁇ (by ⁇ synthesis, ⁇ ligation).
  • the ⁇ kit ⁇ does ⁇ not ⁇ comprise ⁇ sets ⁇ of ⁇ analyte-specific ⁇ probes ⁇ as ⁇ defined ⁇ under ⁇ item ⁇ A1) and ⁇ A2).
  • ⁇ if ⁇ the ⁇ analyte ⁇ in ⁇ the ⁇ kits ⁇ or ⁇ methods ⁇ according ⁇ to ⁇ the ⁇ present disclosure is ⁇ a ⁇ nucleic ⁇ acid
  • ⁇ the ⁇ present ⁇ disclosure is ⁇ generally ⁇ directed ⁇ to ⁇ a ⁇ methods ⁇ including ⁇ acts ⁇ of ⁇ exposing ⁇ a ⁇ sample ⁇ to ⁇ a ⁇ plurality ⁇ of ⁇ analyte-specific ⁇ probes; ⁇ for ⁇ each ⁇ of ⁇ the ⁇ analyte-specific ⁇ probes, ⁇ determining ⁇ binding ⁇ of ⁇ the ⁇ analyte-specific ⁇ probes ⁇ within ⁇ the ⁇ sample; ⁇ creating ⁇ codewords ⁇ based ⁇ on ⁇ the binding ⁇ of ⁇ the ⁇ analyte-specific ⁇ probes, ⁇ the ⁇ decoding ⁇ oligonucleotides ⁇ and ⁇ the ⁇ signal ⁇ oligonucleotides; ⁇ and ⁇ for ⁇ at ⁇ least ⁇ some ⁇ of ⁇ the ⁇ codewords, ⁇ matching ⁇ the ⁇ codeword ⁇ to ⁇ a ⁇ valid ⁇ codeword.
  • To ⁇ create ⁇ such ⁇ a ⁇ zero ⁇ (0) ⁇ in ⁇ a ⁇ codeword ⁇ for ⁇ an ⁇ individual ⁇ analyte ⁇ the ⁇ kit ⁇ may ⁇ comprise: ⁇ (D) at ⁇ least ⁇ a ⁇ set ⁇ of ⁇ non-signal ⁇ decoding ⁇ oligonucleotides ⁇ for ⁇ binding ⁇ to ⁇ a ⁇ particular ⁇ identifier ⁇ element ⁇ (T) ⁇ of ⁇ analyte-specific ⁇ probes, ⁇ wherein ⁇ the ⁇ decoding ⁇ oligonucleotides ⁇ in ⁇ the ⁇ same ⁇ set ⁇ of ⁇ non-signal ⁇ decoding ⁇ oligonucleotides ⁇ interacting ⁇ with ⁇ the ⁇ same ⁇ different ⁇ identifier ⁇ element ⁇ (T), wherein ⁇ each ⁇ non-signal ⁇ decoding ⁇ oligonucleotide ⁇ comprises ⁇ an ⁇ identifier ⁇ connector ⁇ e
  • ⁇ the ⁇ different ⁇ sets ⁇ of ⁇ decoding ⁇ oligonucleotides ⁇ may ⁇ be ⁇ comprised ⁇ in ⁇ a ⁇ pre-mixture ⁇ of ⁇ different ⁇ sets ⁇ of ⁇ decoding ⁇ oligonucleotides ⁇ or ⁇ exist ⁇ separately.
  • ⁇ the ⁇ different ⁇ sets ⁇ of ⁇ analyte-specific ⁇ probes ⁇ may ⁇ be ⁇ comprised ⁇ in ⁇ a ⁇ pre-mixture ⁇ of ⁇ different ⁇ sets ⁇ of ⁇ analyte-specific ⁇ probes ⁇ or ⁇ exist ⁇ separately.
  • As ⁇ mentioned ⁇ above ⁇ the ⁇ analyte ⁇ to ⁇ be ⁇ encoded ⁇ may ⁇ be a ⁇ nucleic ⁇ acid, ⁇ preferably ⁇ DNA, ⁇ PNA ⁇ or ⁇ RNA, ⁇ in ⁇ particular mRNA, ⁇ a peptide, ⁇ polypeptide, ⁇ a ⁇ protein, ⁇ and/or ⁇ mixures thereof.
  • the binding ⁇ element ⁇ (S) ⁇ may ⁇ comprise ⁇ moieties ⁇ which ⁇ are ⁇ affinity ⁇ moieties ⁇ from ⁇ affinity ⁇ substances ⁇ or ⁇ affinity ⁇ substances ⁇ in ⁇ their ⁇ entirety ⁇ selected ⁇ from ⁇ the ⁇ group ⁇ consisting ⁇ of ⁇ antibodies, ⁇ antibody ⁇ fragments, ⁇ anticalin ⁇ proteins, ⁇ receptor ⁇ ligands, enzyme ⁇ substrates, ⁇ lectins, ⁇ cytokines, ⁇ lymphokines, ⁇ interleukins, ⁇ angiogenic ⁇ or ⁇ virulence ⁇ factors, ⁇ allergens, ⁇ peptidic ⁇ allergens, ⁇ recombinant ⁇ allergens, ⁇ allergen-idiotypical ⁇ antibodies, ⁇ autoimmune-provoking ⁇ structures, ⁇ tissue-rejection-inducing ⁇ structures, ⁇ immunoglobulin ⁇ constant ⁇ regions ⁇ and ⁇ combinations ⁇ thereof.
  • ⁇ the ⁇ binding ⁇ element ⁇ (S) ⁇ may ⁇ comprise ⁇ or ⁇ is ⁇ an ⁇ antibody ⁇ or ⁇ an ⁇ antibody ⁇ fragment ⁇ selected ⁇ from ⁇ the ⁇ group ⁇ consisting ⁇ of ⁇ Fab, ⁇ scFv; ⁇ single ⁇ domain, ⁇ or ⁇ a ⁇ fragment ⁇ thereof, ⁇ bis ⁇ scFv, ⁇ F(ab)2, ⁇ F(ab)3, ⁇ minibody, ⁇ diabody, ⁇ triabody, ⁇ tetrabody ⁇ and ⁇ tandab.
  • ⁇ the ⁇ method ⁇ according ⁇ to ⁇ the ⁇ present ⁇ disclosure ⁇ comprises ⁇ selectively ⁇ removing ⁇ the ⁇ decoding ⁇ oligonucleotides ⁇ and ⁇ signal ⁇ oligonucleotides ⁇ from ⁇ the ⁇ sample, ⁇ thereby ⁇ essentially ⁇ maintaining ⁇ the ⁇ specific ⁇ binding ⁇ of ⁇ the ⁇ analyte-specific ⁇ probes ⁇ to ⁇ the ⁇ analyte ⁇ to ⁇ be ⁇ encoded.
  • all ⁇ steps ⁇ are ⁇ performed ⁇ sequentially.
  • some ⁇ steps ⁇ may ⁇ be ⁇ performed ⁇ simultaneously, ⁇ in ⁇ particular ⁇ the ⁇ contacting ⁇ steps ⁇ A) ⁇ to ⁇ C), ⁇ in ⁇ particular ⁇ B) ⁇ and ⁇ C).
  • said ⁇ encoding ⁇ scheme ⁇ may ⁇ be predetermined ⁇ and ⁇ allocated ⁇ to ⁇ the ⁇ analyte ⁇ to ⁇ be ⁇ encoded.
  • ⁇ the ⁇ code ⁇ words ⁇ obtained ⁇ for ⁇ the ⁇ individual ⁇ analytes ⁇ in ⁇ the ⁇ performed ⁇ cycles ⁇ comprise ⁇ the ⁇ detected ⁇ signals ⁇ and ⁇ additionally ⁇ at ⁇ least ⁇ one ⁇ element ⁇ corresponding ⁇ to ⁇ no ⁇ detected ⁇ signal like ⁇ 0,1 ⁇ or ⁇ 0,1,2 ⁇ etc. ⁇ (see ⁇ also ⁇ Fig. ⁇ 13 ⁇ and ⁇ Fig. ⁇ 14).
  • In ⁇ this ⁇ cycle ⁇ the ⁇ position ⁇ has ⁇ the ⁇ value ⁇ zero ⁇ (0).
  • ⁇ at ⁇ least ⁇ for ⁇ one ⁇ individual ⁇ analyte ⁇ a ⁇ position ⁇ of ⁇ the ⁇ code ⁇ word ⁇ is ⁇ zero ⁇ (0).
  • the ⁇ ⁇ code ⁇ word ⁇ zero ⁇ (0) ⁇ is ⁇ generated ⁇ ⁇ by ⁇ using ⁇ no ⁇ decoding ⁇ oligonucleotides ⁇ ⁇ having ⁇ an ⁇ identifier ⁇ connector ⁇ element ⁇ (t) ⁇ comprising ⁇ a ⁇ nucleotide ⁇ sequence ⁇ which ⁇ is ⁇ essentially ⁇ complementary ⁇ to ⁇ at ⁇ least ⁇ a ⁇ section ⁇ of ⁇ the ⁇ unique ⁇ identifier ⁇ sequence ⁇ of ⁇ the ⁇ identifier ⁇ element ⁇ (T) ⁇ of ⁇ a ⁇ corresponding ⁇ analyte-specific ⁇ probe ⁇ for ⁇ an ⁇ individual ⁇ analyte.
  • oligonucleotides having ⁇ an ⁇ identifier ⁇ connector ⁇ element ⁇ (t) ⁇ comprising ⁇ a ⁇ nucleotide ⁇ sequence ⁇ which ⁇ is ⁇ essentially ⁇ complementary ⁇ to ⁇ at ⁇ least ⁇ a ⁇ section ⁇ of ⁇ the ⁇ unique ⁇ identifier ⁇ sequence ⁇ of ⁇ the ⁇ identifier ⁇ element ⁇ (T) ⁇ of ⁇ a ⁇ corresponding ⁇ analyte-specific ⁇ probe ⁇ for ⁇ an ⁇ individual ⁇ analyte ⁇ are ⁇ used.
  • the ⁇ sample ⁇ is ⁇ contacted ⁇ with ⁇ at ⁇ least ⁇ one, ⁇ preferably ⁇ at ⁇ least ⁇ two different ⁇ sets ⁇ of ⁇ signal ⁇ oligonucleotides, ⁇ wherein ⁇ the ⁇ signal ⁇ oligonucleotides ⁇ in ⁇ each ⁇ set ⁇ comprise ⁇ a ⁇ different ⁇ signal ⁇ element ⁇ and ⁇ comprise ⁇ a ⁇ different ⁇ connector ⁇ element ⁇ (C).
  • the ⁇ different ⁇ sets ⁇ of ⁇ non-signal ⁇ oligonucleotides may ⁇ be ⁇ comprised ⁇ in ⁇ a ⁇ pre- mixture ⁇ of ⁇ different ⁇ sets ⁇ of ⁇ non-signal ⁇ oligonucleotides ⁇ or ⁇ exist ⁇ separately.
  • the ⁇ decoding ⁇ oligonucleotides in ⁇ a ⁇ particular ⁇ set ⁇ of ⁇ decoding ⁇ oligonucleotides ⁇ interacts ⁇ with ⁇ identical ⁇ identifier ⁇ elements ⁇ (T) ⁇ which ⁇ are ⁇ unique ⁇ to ⁇ a ⁇ particular ⁇ analyte.
  • ⁇ the ⁇ different ⁇ sets ⁇ of ⁇ decoding ⁇ oligonucleotides ⁇ may ⁇ be ⁇ comprised ⁇ in ⁇ a ⁇ pre- mixture ⁇ of ⁇ different ⁇ sets ⁇ of ⁇ decoding ⁇ oligonucleotides ⁇ or ⁇ exist ⁇ separately as ⁇ well ⁇ as ⁇ the ⁇ different ⁇ sets ⁇ of ⁇ analyte-specific ⁇ probes ⁇ may ⁇ be ⁇ comprised ⁇ in ⁇ a ⁇ pre-mixture ⁇ of ⁇ different ⁇ sets ⁇ of ⁇ analyte- specific ⁇ probes ⁇ or ⁇ exist ⁇ separately as ⁇ well the ⁇ different ⁇ sets ⁇ of ⁇ signal ⁇ oligonucleotides ⁇ may ⁇ be ⁇ comprised ⁇ in ⁇ a ⁇ pre-mixture ⁇ of ⁇ different ⁇ sets ⁇ of ⁇ signal ⁇ oligonucleotides ⁇ or ⁇ exist ⁇ separately.
  • ⁇ the ⁇ analyte ⁇ specific ⁇ probes ⁇ may ⁇ be incubated ⁇ with ⁇ the ⁇ sample, ⁇ thereby ⁇ allowing ⁇ a ⁇ specific ⁇ binding ⁇ of ⁇ the ⁇ analyte ⁇ specific ⁇ probes ⁇ to ⁇ the ⁇ analytes ⁇ to ⁇ be ⁇ encoded, ⁇ further ⁇ the ⁇ decoding ⁇ oligonucleotides ⁇ may ⁇ be incubated ⁇ with ⁇ the ⁇ sample, ⁇ thereby ⁇ allowing ⁇ a ⁇ specific ⁇ hybridization ⁇ of ⁇ the ⁇ decoding ⁇ oligonucleotides ⁇ to ⁇ identifier ⁇ elements ⁇ (T) ⁇ of ⁇ the ⁇ respective ⁇ analyte-specific ⁇ probes, ⁇ further the ⁇ signal oligonucleotides ⁇ may ⁇ be incubated ⁇ with ⁇ the ⁇ sample, ⁇ thereby ⁇ allowing ⁇ a ⁇ specific ⁇ RES-PA18-PCT ⁇ hybridization ⁇ of ⁇ the ⁇ signal ⁇ oli
  • ⁇ the ⁇ analyte ⁇ to ⁇ be ⁇ encoded ⁇ may ⁇ be a ⁇ nucleic ⁇ acid, ⁇ preferably ⁇ DNA, ⁇ PNA, ⁇ RNA, ⁇ in ⁇ particular ⁇ mRNA, ⁇ a ⁇ peptide, ⁇ polypeptide, ⁇ a ⁇ protein or ⁇ combinations ⁇ thereof. Therefore, ⁇ the ⁇ binding ⁇ element ⁇ (S) ⁇ may ⁇ comprise ⁇ an ⁇ amino ⁇ acid ⁇ sequence ⁇ allowing ⁇ a ⁇ specific ⁇ binding ⁇ to ⁇ the ⁇ analyte ⁇ to ⁇ be ⁇ encoded.
  • the ⁇ binding ⁇ element ⁇ (S) ⁇ is ⁇ an ⁇ antibody ⁇ or ⁇ an ⁇ antibody ⁇ fragment ⁇ selected ⁇ from ⁇ the ⁇ group ⁇ consisting ⁇ of ⁇ Fab, ⁇ scFv; ⁇ single ⁇ domain, ⁇ or ⁇ a ⁇ fragment ⁇ thereof, ⁇ bis ⁇ scFv, ⁇ Fab ⁇ 2, ⁇ Fab ⁇ 3, ⁇ minibody, ⁇ diabody, ⁇ triabody, ⁇ tetrabody ⁇ and ⁇ tandab.
  • ⁇ the ⁇ signal ⁇ caused ⁇ by ⁇ the ⁇ signal ⁇ element therefore ⁇ in ⁇ particular ⁇ the ⁇ binding ⁇ of ⁇ the ⁇ signal ⁇ oligonucleotides ⁇ to ⁇ the ⁇ decoding ⁇ oligonucleotides, ⁇ interacting ⁇ with ⁇ the ⁇ corresponding ⁇ analyte ⁇ probes, ⁇ bound ⁇ to ⁇ the ⁇ respective ⁇ analyte ⁇ is ⁇ determined ⁇ by: (a) Imaging ⁇ at ⁇ least ⁇ a ⁇ portion ⁇ of ⁇ the ⁇ sample; ⁇ and/or (b) Using ⁇ an ⁇ optical ⁇ imaging ⁇ technique; ⁇ and/or (c) Using ⁇ a ⁇ fluorescence ⁇ imaging ⁇ technique; ⁇ and/or (d) Multi-color ⁇ fluorescence ⁇ imaging ⁇ technique; ⁇ and/or ⁇ (e) Super-resolution ⁇ fluorescence ⁇ imaging ⁇ technique.
  • the ⁇ kits ⁇ and ⁇ method ⁇ according to ⁇ the ⁇ present ⁇ disclosure ⁇ may ⁇ be ⁇ used ⁇ ideally ⁇ for ⁇ in ⁇ vitromethods ⁇ for ⁇ diagnosis ⁇ of ⁇ a ⁇ disease ⁇ selected ⁇ from ⁇ the ⁇ group ⁇ comprising ⁇ cancer, ⁇ neuronal ⁇ diseases, ⁇ cardiovascular ⁇ diseases, ⁇ inflammatory ⁇ diseases, ⁇ autoimmune ⁇ diseases, ⁇ diseases ⁇ due ⁇ to ⁇ a ⁇ viral ⁇ or ⁇ bacterial ⁇ infection, ⁇ skin ⁇ diseases, ⁇ skeletal ⁇ muscle ⁇ diseases, ⁇ dental ⁇ diseases, and ⁇ prenatal ⁇ diseases.
  • ⁇ the ⁇ kits ⁇ and ⁇ method ⁇ according to ⁇ the ⁇ present ⁇ disclosure ⁇ may ⁇ be ⁇ used ⁇ also ⁇ ideally ⁇ for ⁇ in ⁇ vitromethods ⁇ for ⁇ screening, ⁇ identifying ⁇ and/or ⁇ testing ⁇ a ⁇ substance ⁇ and/or ⁇ drug ⁇ comprising: (a) contacting ⁇ a ⁇ test ⁇ sample ⁇ comprising ⁇ a ⁇ sample ⁇ with ⁇ a ⁇ substance ⁇ and/or ⁇ drug ⁇ (b) detecting ⁇ different ⁇ analytes ⁇ in ⁇ a ⁇ sample ⁇ by ⁇ sequential ⁇ signal-encoding ⁇ of ⁇ said ⁇ analytes ⁇ with ⁇ a ⁇ method ⁇ according ⁇ to ⁇ the ⁇ present ⁇ disclosure.
  • optical ⁇ multiplexing ⁇ system ⁇ suitable ⁇ for ⁇ the ⁇ method ⁇ according ⁇ to ⁇ the ⁇ present ⁇ disclosure comprising ⁇ at ⁇ least: - a reaction ⁇ vessel ⁇ for ⁇ containing ⁇ the ⁇ kits ⁇ or ⁇ part ⁇ of ⁇ the ⁇ kits ⁇ according ⁇ to ⁇ the ⁇ present ⁇ disclosure; - a ⁇ detection ⁇ unit ⁇ comprising ⁇ a ⁇ microscope, ⁇ in ⁇ particular ⁇ a ⁇ fluorescence ⁇ microscope; - a ⁇ camera; - a ⁇ liquid ⁇ handling ⁇ device.
  • optical ⁇ multiplexing ⁇ system ⁇ may ⁇ comprise ⁇ further ⁇ a ⁇ heat ⁇ and ⁇ cooling ⁇ device and/or ⁇ a ⁇ robotic ⁇ system.
  • ⁇ the ⁇ method ⁇ according ⁇ to ⁇ the ⁇ present ⁇ disclosure ⁇ encodes a ⁇ nucleic ⁇ acid ⁇ analyte, ⁇ such ⁇ as ⁇ an ⁇ mRNA, ⁇ e.g., such ⁇ an ⁇ mRNA ⁇ coding ⁇ for ⁇ a ⁇ particular ⁇ protein.
  • the ⁇ disclosure is ⁇ now ⁇ further ⁇ explained ⁇ by ⁇ means ⁇ of ⁇ embodiments ⁇ resulting ⁇ in ⁇ additional ⁇ features, ⁇ characteristics ⁇ and ⁇ advantages ⁇ of ⁇ the ⁇ disclosure.
  • the ⁇ technology ⁇ allows ⁇ distinguishing ⁇ a ⁇ higher ⁇ number ⁇ of ⁇ analytes ⁇ than ⁇ different ⁇ signals ⁇ are ⁇ available.
  • the ⁇ process ⁇ preferably ⁇ includes ⁇ at ⁇ least ⁇ one, ⁇ preferably ⁇ at ⁇ least ⁇ two consecutive ⁇ rounds ⁇ of ⁇ specific ⁇ binding, ⁇ signal ⁇ detection ⁇ and ⁇ selective ⁇ denaturation ⁇ (if ⁇ a ⁇ next ⁇ round ⁇ is ⁇ required), ⁇ eventually ⁇ producing ⁇ a ⁇ signal ⁇ code.
  • the ⁇ decoding ⁇ oligonucleotide ⁇ transcribes ⁇ the ⁇ information
  • ⁇ the ⁇ present ⁇ disclosure ⁇ pertains ⁇ to ⁇ a ⁇ method ⁇ of ⁇ assigning ⁇ an ⁇ analyte ⁇ to ⁇ a ⁇ position ⁇ in ⁇ an ⁇ image, ⁇ comprising ⁇ assigning ⁇ a ⁇ fluorescence ⁇ pattern ⁇ to ⁇ the ⁇ analyte, ⁇ observing ⁇ the ⁇ fluorescence ⁇ pattern ⁇ at ⁇ the ⁇ position ⁇ in ⁇ the ⁇ image, ⁇ and ⁇ assigning ⁇ the ⁇ analyte ⁇ to ⁇ the ⁇ position, ⁇ in ⁇ particular ⁇ wherein ⁇ observing ⁇ the ⁇ fluorescence ⁇ pattern ⁇ comprises ⁇ repeating ⁇ steps ⁇ of ⁇ labeling ⁇ the ⁇ position ⁇ using ⁇ a ⁇ fluorophore ⁇ tagged ⁇ oligo ⁇ drawn ⁇ from ⁇ a ⁇ re-accessible pool, ⁇ performing ⁇ a ⁇ single ⁇ excitation ⁇ at ⁇ the ⁇ position ⁇ in ⁇ the ⁇ image, ⁇ and ⁇ contacting ⁇ the ⁇ analyte ⁇ to ⁇ a ⁇ denaturant, ⁇ in ⁇ particular ⁇ wherein ⁇ observing ⁇ the ⁇ the ⁇
  • ⁇ the ⁇ present ⁇ disclosure ⁇ pertains ⁇ to ⁇ a ⁇ method ⁇ of ⁇ assigning ⁇ coded ⁇ fluorescence ⁇ patterns ⁇ to ⁇ a ⁇ plurality ⁇ of ⁇ target ⁇ analytes ⁇ in ⁇ a ⁇ cell, ⁇ comprising: ⁇ subjecting ⁇ the ⁇ cell ⁇ to ⁇ a ⁇ plurality ⁇ of ⁇ detection ⁇ rounds, ⁇ each ⁇ detection ⁇ round ⁇ comprising: ⁇ contacting ⁇ the ⁇ cell ⁇ to ⁇ representatives ⁇ of ⁇ the ⁇ same ⁇ at ⁇ least ⁇ one, ⁇ preferably ⁇ at ⁇ least ⁇ two populations ⁇ of ⁇ tagged ⁇ fluorescence ⁇ moieties, ⁇ and ⁇ removing ⁇ the ⁇ fluorescent ⁇ moieties ⁇ after ⁇ a ⁇ single ⁇ excitation ⁇ event, ⁇ in ⁇ particular - wherein ⁇ the ⁇ number ⁇ of ⁇ patterns ⁇ detectable ⁇ increases ⁇ exponentially ⁇ with ⁇ the ⁇ number ⁇ of ⁇ detection ⁇ rounds, ⁇ - wherein ⁇ the ⁇ fluorescence ⁇ moieties
  • ⁇ the ⁇ present ⁇ disclosure ⁇ pertains ⁇ to ⁇ a ⁇ method ⁇ of ⁇ assigning ⁇ coded ⁇ fluorescence ⁇ patterns ⁇ to ⁇ a ⁇ plurality ⁇ of ⁇ target ⁇ analytes ⁇ in ⁇ a ⁇ cell, ⁇ comprising: ⁇ contacting ⁇ a ⁇ target ⁇ to ⁇ a ⁇ bipartite ⁇ labeling ⁇ probe, ⁇ the ⁇ bipartite ⁇ labeling ⁇ probe ⁇ comprising ⁇ a ⁇ target- specific ⁇ moiety ⁇ and ⁇ a ⁇ fluorophore-specifying ⁇ moiety; ⁇ contacting ⁇ the ⁇ bipartite ⁇ labeling ⁇ probe ⁇ to ⁇ a ⁇ first ⁇ aliquot ⁇ of ⁇ a ⁇ fluorophore ⁇ reservoir ⁇ comprising ⁇ no ⁇ more ⁇ than ⁇ two ⁇ populations ⁇ of ⁇ fluorophores; ⁇ replacing
  • ⁇ replacing ⁇ the ⁇ fluorophore ⁇ specifying ⁇ moiety ⁇ in ⁇ the ⁇ bipartite ⁇ probe ⁇ comprises ⁇ denaturing ⁇ a ⁇ binding ⁇ between ⁇ a ⁇ target-specific ⁇ moiety ⁇ and ⁇ a ⁇ fluorophore-specifying ⁇ moiety ⁇ after ⁇ subjecting ⁇ the ⁇ bipartite ⁇ labeling ⁇ probe ⁇ bound ⁇ to ⁇ a ⁇ RES-PA18-PCT ⁇ fluorophore ⁇ of ⁇ the ⁇ fluorophore ⁇ to ⁇ excitation ⁇ energy.
  • ⁇ replacing ⁇ the ⁇ fluorophore ⁇ specifying ⁇ moiety ⁇ in ⁇ the ⁇ bipartite ⁇ probe ⁇ comprises ⁇ drawing ⁇ from ⁇ one ⁇ of ⁇ no ⁇ more ⁇ than ⁇ two ⁇ fluorophore ⁇ specifying ⁇ moiety ⁇ reservoirs.
  • Step ⁇ 1 ⁇ Applying ⁇ the ⁇ at ⁇ least ⁇ 20 ⁇ analyte- or ⁇ target-specific ⁇ probe ⁇ sets.
  • ⁇ a ⁇ probe ⁇ set ⁇ of ⁇ 5 ⁇ different ⁇ probes ⁇ is ⁇ shown, ⁇ each ⁇ comprising ⁇ a ⁇ sequence ⁇ element ⁇ complementary ⁇ to ⁇ an ⁇ individual ⁇ subsequence ⁇ of ⁇ the ⁇ target ⁇ nucleic ⁇ acid ⁇ sequence ⁇ (S1 ⁇ to ⁇ S5).
  • S1 ⁇ to ⁇ S5 ⁇ In ⁇ this ⁇ example, ⁇ the ⁇ regions ⁇ do ⁇ not ⁇ overlap.
  • Step ⁇ 5 ⁇ Hybridization ⁇ of ⁇ decoding ⁇ oligonucleotides.
  • the ⁇ decoding ⁇ oligonucleotides ⁇ are ⁇ hybridized ⁇ with ⁇ the ⁇ unique ⁇ identifier ⁇ sequences ⁇ of ⁇ the ⁇ probes ⁇ (T) ⁇ via ⁇ their ⁇ complementary ⁇ first ⁇ sequence ⁇ elements ⁇ (t). ⁇ After ⁇ incubation, ⁇ the ⁇ decoding ⁇ oligonucleotides ⁇ provide ⁇ the ⁇ translator ⁇ sequence ⁇ element ⁇ (c) ⁇ for ⁇ a ⁇ subsequent ⁇ hybridization ⁇ step.
  • Step ⁇ 6 Eliminating ⁇ the ⁇ excess ⁇ of ⁇ decoding ⁇ oligonucleotides. ⁇ After ⁇ hybridization, ⁇ the ⁇ unbound ⁇ decoding ⁇ oligonucleotides ⁇ are ⁇ eliminated, ⁇ e.g. ⁇ by ⁇ washing ⁇ steps.
  • Step ⁇ 7 ⁇ Applying ⁇ the ⁇ signal ⁇ oligonucleotide. ⁇ The ⁇ signal ⁇ oligonucleotides ⁇ are ⁇ applied. ⁇ The ⁇ signal ⁇ oligonucleotides ⁇ comprise ⁇ at ⁇ least ⁇ one ⁇ second ⁇ connector ⁇ element ⁇ (C) ⁇ that ⁇ is ⁇ essentially ⁇ complementary ⁇ to ⁇ the ⁇ translator ⁇ sequence ⁇ element ⁇ (c) ⁇ and ⁇ at ⁇ least ⁇ one ⁇ signal ⁇ element ⁇ that ⁇ provides ⁇ a ⁇ detectable ⁇ signal ⁇ (F).
  • Step ⁇ 8 ⁇ Hybridization ⁇ of ⁇ the ⁇ signal ⁇ oligonucleotides.
  • the ⁇ signal ⁇ oligonucleotides ⁇ are ⁇ hybridized ⁇ via ⁇ the ⁇ complementary ⁇ sequence ⁇ connector ⁇ element ⁇ (C) ⁇ to ⁇ the ⁇ translator ⁇ element ⁇ (c) ⁇ of ⁇ decoding ⁇ oligonucleotide.
  • Step ⁇ 9 ⁇ Eliminating ⁇ the ⁇ excess ⁇ of ⁇ signal ⁇ oligonucleotides. ⁇ After ⁇ hybridization, ⁇ the ⁇ unbound ⁇ signal ⁇ oligonucleotides ⁇ are ⁇ eliminated, ⁇ e.g. ⁇ by ⁇ washing ⁇ steps.
  • Step ⁇ 12 ⁇ Eliminating ⁇ the ⁇ denatured ⁇ decoding ⁇ oligonucleotides.
  • Step ⁇ 1 Target ⁇ nucleic ⁇ acids: ⁇ In ⁇ this ⁇ example ⁇ three ⁇ different ⁇ target ⁇ nucleic ⁇ acids ⁇ (A), ⁇ (B) ⁇ and ⁇ (C) ⁇ have ⁇ to ⁇ be ⁇ detected ⁇ and ⁇ differentiated ⁇ by ⁇ using ⁇ only ⁇ two ⁇ different ⁇ types ⁇ of ⁇ signal ⁇ oligonucleotides.
  • the ⁇ three ⁇ different ⁇ nucleic ⁇ acid ⁇ sequences ⁇ are ⁇ encoded ⁇ by ⁇ three ⁇ rounds ⁇ of ⁇ detection ⁇ with ⁇ three ⁇ different ⁇ signal ⁇ types ⁇ (1), ⁇ (2) ⁇ and ⁇ (1/2) ⁇ and ⁇ a ⁇ resulting ⁇ hamming ⁇ distance ⁇ of ⁇ 3 ⁇ to ⁇ allow ⁇ for ⁇ error ⁇ de
  • Step ⁇ 3 Hybridization ⁇ of ⁇ the ⁇ decoding ⁇ oligonucleotides ⁇ and ⁇ multi-decoders: ⁇ For ⁇ each ⁇ unique ⁇ identifier ⁇ present, ⁇ a ⁇ certain ⁇ decoding ⁇ oligonucleotide ⁇ or ⁇ multi-decoder ⁇ is ⁇ applied ⁇ specifically ⁇ hybridizing ⁇ to ⁇ the ⁇ corresponding ⁇ unique ⁇ identifier ⁇ sequence ⁇ by ⁇ its ⁇ first ⁇ RES-PA18-PCT ⁇ sequence ⁇ element ⁇ (here ⁇ (t1) ⁇ to ⁇ (T1), ⁇ (t2) ⁇ to ⁇ (T2) ⁇ and ⁇ (t3) ⁇ to ⁇ (T3)).
  • Each ⁇ of ⁇ the ⁇ decoding ⁇ oligonucleotides ⁇ or ⁇ multi-decoders ⁇ provides ⁇ a ⁇ translator ⁇ or ⁇ two ⁇ translator ⁇ elements ⁇ that ⁇ define ⁇ the ⁇ signals ⁇ that ⁇ will ⁇ be ⁇ generated ⁇ after ⁇ hybridization
  • Step ⁇ 4 ⁇ Hybridization ⁇ of ⁇ signal ⁇ oligonucleotides: ⁇ For ⁇ each ⁇ type ⁇ of ⁇ translator ⁇ element, ⁇ a ⁇ signal ⁇ oligonucleotide ⁇ with ⁇ a ⁇ certain ⁇ signal, ⁇ differentiable ⁇ from ⁇ signals ⁇ of ⁇ other ⁇ signal ⁇ oligonucleotides, ⁇ is ⁇ applied. ⁇ This ⁇ signal ⁇ oligonucleotide ⁇ can ⁇ specifically ⁇ hybridize ⁇ to ⁇ the ⁇ corresponding ⁇ translator ⁇ element. ⁇ The ⁇ illustration ⁇ in ⁇ Fig. ⁇ 16 ⁇ summarizes ⁇ steps ⁇ 7 ⁇ to ⁇ 9 ⁇ of ⁇ Fig. ⁇ 3 Step ⁇ 5: ⁇ Signal ⁇ detection ⁇ for ⁇ the ⁇ encoding ⁇ scheme: ⁇ The ⁇ different ⁇ signals ⁇ are ⁇ detected. ⁇ Note ⁇ that ⁇ in ⁇ this ⁇ example ⁇ the nucleic ⁇ acids ⁇ (A), ⁇ (B) ⁇ and ⁇ (C) ⁇ can ⁇ already ⁇ be ⁇ distinguished ⁇ after ⁇ the ⁇
  • Step ⁇ 6 ⁇ Selective ⁇ denaturation: ⁇ The ⁇ decoding ⁇ (and ⁇ signal) ⁇ oligonucleotides ⁇ and/or ⁇ multi- decoders ⁇ of ⁇ all ⁇ nucleic ⁇ acid ⁇ sequences ⁇ to ⁇ be ⁇ detected ⁇ are ⁇ selectively ⁇ denatured ⁇ and ⁇ eliminated ⁇ as ⁇ described ⁇ in ⁇ steps ⁇ 11 ⁇ and ⁇ 12 ⁇ of ⁇ Fig.3. Afterwards ⁇ the ⁇ unique ⁇ identifier ⁇ sequences ⁇ of ⁇ the ⁇ different ⁇ probe ⁇ sets ⁇ can ⁇ be ⁇ used ⁇ for ⁇ the ⁇ next ⁇ round ⁇ of ⁇ hybridization ⁇ and ⁇ detection.
  • This ⁇ way ⁇ three ⁇ different ⁇ nucleic ⁇ acids ⁇ can ⁇ be ⁇ distinguished in ⁇ three ⁇ detection ⁇ rounds ⁇ with ⁇ two ⁇ different ⁇ signals, ⁇ allowing ⁇ an ⁇ error ⁇ detection ⁇ and ⁇ correction.
  • Step ⁇ 2 ⁇ Hybridization ⁇ of ⁇ the ⁇ probe ⁇ sets. ⁇ For ⁇ each ⁇ target ⁇ nucleic ⁇ acid, ⁇ an ⁇ own ⁇ probe ⁇ set ⁇ is ⁇ applied, ⁇ specifically ⁇ hybridizing ⁇ to ⁇ the ⁇ corresponding ⁇ nucleic ⁇ acid ⁇ sequence ⁇ of ⁇ interest. ⁇ Each ⁇ probe ⁇ set ⁇ provides ⁇ a ⁇ unique ⁇ identifier ⁇ sequence ⁇ (T1), ⁇ (T2) ⁇ or ⁇ (T3). ⁇ This ⁇ way ⁇ each ⁇ different ⁇ target ⁇ nucleic ⁇ acid ⁇ is ⁇ uniquely ⁇ labeled. ⁇ In ⁇ this ⁇ example ⁇ sequence ⁇ (T) ⁇ is ⁇ labeled ⁇ with ⁇ (T1), ⁇ sequence ⁇ (B) ⁇ with ⁇ (T2) ⁇ and ⁇ sequence ⁇ (C) ⁇ with ⁇ (T3). ⁇ The ⁇ illustration ⁇ summarizes steps ⁇ 1 ⁇ to ⁇ 3 ⁇ of ⁇ Figure ⁇ 3.
  • Step ⁇ 3 ⁇ Hybridization ⁇ of ⁇ the ⁇ decoding ⁇ oligonucleotides.
  • each ⁇ unique ⁇ identifier ⁇ present ⁇ a ⁇ certain ⁇ decoding ⁇ oligonucleotide ⁇ is ⁇ applied ⁇ specifically ⁇ hybridizing ⁇ to ⁇ the ⁇ corresponding ⁇ unique ⁇ identifier ⁇ sequence ⁇ by ⁇ its ⁇ first ⁇ sequence ⁇ element ⁇ (here ⁇ (t1) ⁇ to ⁇ (T1), ⁇ (t2) ⁇ to ⁇ (T2) ⁇ and ⁇ (t3) ⁇ to ⁇ (T3)).
  • Each ⁇ of ⁇ the ⁇ decoding ⁇ oligonucleotides provides ⁇ a ⁇ translator ⁇ element ⁇ that ⁇ defines ⁇ the ⁇ signal ⁇ that ⁇ will ⁇ be ⁇ generated ⁇ after ⁇ hybridization ⁇ of ⁇ signal ⁇ oligonucleotides.
  • Step ⁇ 4 ⁇ Hybridization ⁇ of ⁇ signal ⁇ oligonucleotides. ⁇ For ⁇ each ⁇ type ⁇ of ⁇ translator ⁇ element, ⁇ a ⁇ signal ⁇ oligonucleotide ⁇ with ⁇ a ⁇ certain ⁇ signal ⁇ (2), ⁇ differentiable ⁇ from ⁇ signals ⁇ of ⁇ other ⁇ signal ⁇ oligonucleotides, ⁇ is ⁇ applied. ⁇ This ⁇ signal ⁇ oligonucleotide ⁇ can ⁇ specifically ⁇ hybridize ⁇ to ⁇ the ⁇ corresponding ⁇ translator ⁇ element. ⁇ The ⁇ illustration ⁇ summarizes ⁇ steps ⁇ 7 ⁇ to ⁇ 9 ⁇ of ⁇ Figure ⁇ 3.
  • Step ⁇ 6 ⁇ Selective ⁇ denaturation.
  • the ⁇ method ⁇ according ⁇ to ⁇ the ⁇ disclosure combines ⁇ the ⁇ advantages ⁇ of ⁇ seqFISH (mainly ⁇ complete ⁇ freedom ⁇ concerning ⁇ the ⁇ encoding ⁇ scheme) ⁇ with ⁇ all ⁇ advantages ⁇ of ⁇ methods ⁇ using ⁇ only ⁇ one ⁇ specific ⁇ hybridization ⁇ event ⁇ while ⁇ eliminating ⁇ the ⁇ major ⁇ problems ⁇ of ⁇ such ⁇ methods.
  • the ⁇ efficiency ⁇ E ⁇ of ⁇ the ⁇ whole ⁇ encoding ⁇ process ⁇ can ⁇ be ⁇ described ⁇ by ⁇ the ⁇ following ⁇ equation: RES-PA18-PCT ⁇ Based ⁇ on ⁇ this ⁇ equation ⁇ the ⁇ efficiency ⁇ of ⁇ each ⁇ single ⁇ step ⁇ can ⁇ be ⁇ estimated ⁇ for ⁇ a ⁇ given ⁇ total ⁇ efficiency ⁇ of ⁇ the ⁇ method. ⁇ The ⁇ calculation ⁇ is ⁇ hereby ⁇ based ⁇ on ⁇ the ⁇ assumption, ⁇ that ⁇ each ⁇ process ⁇ has ⁇ the ⁇ same ⁇ efficiency. ⁇ The ⁇ total ⁇ efficiency ⁇ describes ⁇ the ⁇ portion ⁇ of ⁇ successfully ⁇ decodable ⁇ signals ⁇ of ⁇ the ⁇ total ⁇ signals ⁇ present.

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Abstract

A monitoring probe comprising a nucleotide sequence, which corresponds to a translator (c) allowing the hybridisation of a signal oligonucleotide; an identifier element allowing the hybridisation of a decoding clement, wherein the Identifier comprises a sequence which is essentially complementary to a section of the unique set identifier of an identifier element (T) of a corresponding decoder; a target nucleic add sequence allowing the hybridisation of a binding element. Both probe and target may be immobilised. The target may be obtained from a tissue sample. The probe may be used to monitor ambient temperature ranges. The probe supervises the quality (QC) of manufactured reagents and kits in the field of in-situ hybridisation during all stages of manufacturing. Kits and control slides comprising the monitoring probes.

Description

RES-PA18-PCT^ REAGENT^FOR^IN^SITU^HYBRIDIZATION^QUALITY^CONTROL FIELD^OF^THE^DISCLOSURE In^ order^ to^ supervise^ the^ quality^ of^manufactured^ reagents^ and^ kits,^ especially^ in^ the^ field^ of^ laboratory^(especially^in^diagnostic^labs)^and/or^medical^applications,^an^effective^quality^control^ (QC)^ during^ all^ stages^ of^ manufacturing^ is^ essential^ and^ a^ requirement^ for^ pharmaceutical^ approval.^A^good^QC^ensures^ that^ the^produced^materials^will^meet^ the^ intended^performance^ when^used^by^a^customer.^ Especially^in^the^technical^field^of^in^situ^hybridization,^surrogate^QC^methods^(pH,^ionic^strength,^ absorbance,^ orthogonal^ analytical^ assays)^ are^ used^ to^ measure^ individual^ kit^ components.^ However, these^methods^do^not^measure^the^overall^performance^of^the^entire^assay^or^workflow.^ Failure^ of^ assay^ performance^ due^ to^ interactions^ between^ the^ individual^ kit^ components,^ interaction^between^the^kits^with^the^instrument,^user^error,^laboratory^environmental^conditions^ outside^the^desired^assay^range^or^use^of^unacceptable^additional^components^supplied^by^the^ user^are^missed^when^controls^are^used^which^do^not^monitor^ the^entire^workflow.^Especially^ unforeseen^misuse^and/or^unforeseen^interactions^are^difficult^to^monitor^with^QC-test methods which^monitor^only^very^limited^portions^of^the^assay.^ On^ the^ other^ hand,^more^ holistic^ quality^ controls for^ in^ situ^ hybridization^ validation,^ require^ carrying^out^the^entire^workflow^to^assess^the^final^performance^data^of^a^sample.^These^controls^ typically^run^in^parallel^to^the^customer’s^test^with^standardized^tissue^and/or^cell^line^samples.^ Although^these^types^of^controls^can^monitor^interactions^among^the^individual^kit^components,^ environment,^and^instrument,^they^give^little^information^as^to^the^source^of^performance^failure.^ Variability,^for^example^inherent^in^tissue^and^cell^ lines, make^it^difficult^to^rule^out^the^sample^ itself^as^a^source^of^failed^performance.^In^addition,^failure^of^the^entire^workflow^performance^ gives^little^insight^as^to^which^part^of^the^workflow^caused^the^failure.^Because^of^the^external^ nature^of^these^types^of^QC,^they^need^to^be^run^as^a^separate^sample^and^cannot^be^used^as^an^ internal^control. There^is^the^need^for^a^nonbiological,^easy^to^manufacture,^set^of^reagents^that^can^monitor^the^ entire^in^situ^hybridization^workflow^and^can^be^designed^to^identify different^critical^steps^of^the^ process.^ RES-PA18-PCT^ The^present^disclosure^provides^reagents^and^methods^which^overcome^these^problems. BACKGROUND In^the^prior^art^some^approaches^for^QC-assays^have^been^established. Wilcox,^JN^(1993).^Fundamental^Principles^of^In^Situ^Hybridization.^J^of^Histochem.^Cytochem.^41^ (12):^1725^– 1733^suggest^the^use^of^control^tissue^as^an^exogenous^control.^As^a^negative^control,^ the^article^recommends^the^use^of^an^RNA^target^that^is^not expressed^in^the^tissue^of^interest.^ Alternatively,^the^use^of^probes^to^the^antisense^sequence^can^serve^as^a^negative^control.^As^a^ positive^ control,^ the^ article^ recommends^ the^ use^ of^ an^ RNA^ target^ of^ known^ distribution^ and^ expression^level.^However,^this^method^has^the^disadvantage^that^the^use^of^a^gene^expressed^in^ some^tissue^precludes^ the^ability^ to^measure^expression^of^ that^gene,^ is^qualitative^due^ to^not^ knowing^the^exact^level^of^expression^and^cannot^be^randomly^distributed^across^the^sample. Rosenstraus,^M;^Wang,^Z;^Chang,^S-Y;^DeBonville,^D;^and^Spadaro,^JP^(1998).^An^Internal^Control^ for^Routine^Diagnostic^PCR:^Design,^Properties,^and^Effect^on^Clinical^Performance.^J.^Clin.^Micro.^ 36^(1):^191^– 197,^describe^ the^use^of^ internal^control^ for^PCR^diagnostic^ tests^consisting^of^a^ synthetic^exogenous^RNA^spiked^into^the^clinical^sample^at^the^initiation^of^sample^preparation,^ prior^ to^ amplification.^ However,^ this^ method^ has^ the^ disadvantage^ that^ the^ internal^ control^ described^measures^ all^ steps^ required^ for^purification^of^nucleic^ acid^ and^amplification^of^ the^ purified^ target.^ Spiking^ in^ of^ an^ exogenous^ RNA^ control^ does^ not^ specifically^ measure^ hybridization^efficiency.^ Buckwalter,^SP;^Sloan,^LM;^Cunningham,^SA;^Espy,^MJ;^Uhl,^JR;^Jones,^MF;^Vetter,^EA;^Mandrekar,^J;^ Cockerill^III,^FR;^Pritt,^BS;^Patel,^R;^Wengenack,^NL.^(2014).^Inhibition^Controls^for^Qualitative^Real- Time^PCR^Assays:^Are^They^Necessary^for^All^Specimen^Matrices?^J.^Clin.^Micro.^52^(6):^2139^– 2143^describe^the^diversity^of^internal^controls^used^in^amplification^reactions,^such^as^plasmids,^ whole^viruses,^genomic^DNA,^and^whole^organisms.^It^describes^the^frequency^of^clinical^samples^ demonstrating^inhibition.^However,^the^article^only^describes^the^use^of^controls^for^monitoring^ inhibition^of^amplification. Zambenedetti,^MR;^Pavoni,^DP;^Dallabona,^AC;^Dominguez,^AC;^Poersch,^CdO;^Pragoso,^SP;^ and^ Krieger,^MA^ (2017).^ Internal^Control^ for^Real-Time^Polymerase^Chain^Reaction^Based^on^MS2^ Bacteriophage^ for^ RNA^Viruses^Diagnostics.^Mem^ Inst^ Oswaldo^ Cruz,^ Rio^ de^ Janeiro.^ 112^ (5):^ RES-PA18-PCT^ 339-347^describe^that^an^internal^control^is^necessary^to^differentiate^the^true^negative^results^ from^a^failure^in^the^workflow.^The^article^also^assesses^the^importance^of^using^an^internal^control^ to^assess^ the^sample^preparation^steps^prior^ to^amplification.^However,^ the^described^ internal^ control^measures^failure^of^amplification^without^differentiating^which^steps^within^the^process^ result^in^failed^performance. Xie,^ F;^ Timme,^ KA;^ and^ Wood,^ JR.^ (2018).^ Using^ Single^ Molecule^ mRNA^ Fluorescent^ in^ Situ^ Hybridization^(RNA-FISH)^ to^Quantify^mRNAs^ in^ Individual^Murine^Oocytes^ and^Embryos.^ Sci.^ Rep.^8^(1):^7930^– 7941 describe the^use^of^naturally^expressed^genes^as^a^positive^control^and^ specially^ designed^ non^ hybridizing^ probes^ as^ a^ negative^ control.^ Alternative^ approaches^ for^ designing^ negative^ controls^ include^ running^ samples^ omitting^ some^ of^ the^ probes.^ These^ approaches^require^the^use^of^a^second^sample. However,^the^use^of^an^internally^expressed^gene^ does^not^permit^absolution^quantification^of^the^degree^of^inhibition^since^the^exact^number^of^ target^genes^are^unknown.^Omission^of^a^probe^set^on^a^second^sample^requires the^processing^of^ a^second^sample. Tang,^ Z;^ Gu,^ J;^ Tang,^ G;^ and^ Medeiros,^ LJ.^ (2018).^ Quality^ Assurance/Quality^ Control^ of^ Fluorescence^ in^ Situ^ Hybridization^ Tests^ in^ Hematologic^ Malignancies.^ OBM^ Genetics.^ 2^ (4):^ doi:10.21926/obm.genet.1804038 describes the^ use^ of^ quality^ assurance/quality^ control^ (QA/QC)^measurements^ applied^ for^ FISH^ tests^ by^ following^ the^ American^ College^ of^ Medical^ Genetics^ and^ Genomics^ (ACMGG)^ technical^ standards^ and^ guideline.^ The^ ACMGG^ guidelines^ recommend^the^use^of^both^an^external^positive^and^external^negative^control^for^each^batch^of^ clinical^samples.^However,^external^controls^require^separate^samples^to^be^run,^which^reduces^ sample^throughput^and^increases^the^cost^of^the^test.^In^addition,^the^use^of^an^external^control^ does^not^serve^as^controls^for^the^assay^using^the^actual^sample. Aralar,^A;^Yuan,^Y;^Chen,^K;^Geng,^Y;^Velez^DO;^Sinha,^M;^Lawrence,^SM;^and^Fraley,^SI.^ (2020)^ Improving^ Quantitative^ Power^ in^ Digital^ PCR^ through^ Digital^ High-Resolution^Melting.^ J.^ Clin.^ Micro.^58^(6):^e00325-20 describe^the^use^of^an^exogenous^internal^control.^It^defines^an^internal^ control^ (IC)^as^a^nontarget^DNA^sequence^present^in^each^reaction^mixture^and^ is^coamplified^ simultaneously^with^the^target^sequence.^Furthermore,^it^describes^that^without^an^IC,^a^negative^ reaction^(no^signal)^can^mean^that^there^was^no^target^sequence^present^in^the^reaction^mixture^ or^ that^ the^ reaction^ was^ inhibited,^ resulting^ in^ a^ false-negative^ call. However,^ as^ with^ other^ amplification-based^internal^controls,^the^approach^measures^all^steps^required^for^amplification.^ RES-PA18-PCT^ It^lacks^the^ability^to^identify^which^steps^during^sample^preparation^and^amplification^resulted^ in^the^failure/inhibition. Young,^AP;^Jackson,^DJ;^and^Wyeth,^RC.^(2022)^A^Technical^Review^and^Guide^to^RNA^Fluorescence^ in^ situ^ Hybridization.^ Peer^ J.^ Mar^ 19;8:e8806;^ (Section^ on^ Controls^ for^ an^ ISH^ experiment);^ describes^the^use^of^a^positive^control^against^an^internally^expressed^gene. As^a^negative^control,^ the^publication^suggests^the^use^of^RNase^treatment^of^a^negative^control^sample.^The^publication^ suggests^an^alternative^negative^control^would^be^to^include^a^probe^to^the^antisense^sequence. However,^using^probes^against^genes^expressed^ in^some^tissues^precludes^ the^ability^to^detect^ expression^ of^ that^ gene^ in^ any^ tissue.^ In^ addition,^ it^ is^ a^ relative^ internal^ control^ in^ that^ it^ is^ unknown^the^exact^level^of^expression^of^the^targeted^positive^control^gene.^This^form^of^positive^ control^is^dependent^on^where^within^the^tissue^the^gene^is^expressed^and^cannot^be^distributed^ randomly^across^the^slide.^A^separate^treatment^of^a^sample^is^used^to^include^a^negative^control^ in^a^run. Li,^ J.^ (2005).^ Internal^ Control^ for^ In^ Situ^ Hybridization.^ United^ States^ Patent^ Application^ 2005/0272032^A1^Publication^Date:^Dec.^8,^2005 describe^ a^method^for^monitoring^the^quality^of^ in^ situ^ hybridization^ analysis^ of^ a^ nuclear^ DNA^ target^ in^ a^ tissue^ or^ cell^ sample^ using^ a^ mitochondrial^DNA^probe^as^an^internal^control.^The^invention^also^provides^a^reagent^for^in^situ^ hybridization^detection^of^a^nuclear^DNA^target^and^mitochondrial^DNA^target^in^a^tissue^or^cell^ sample. However,^ the^ approach^uses^nontargeted^DNA^ sequences^ inherent^ in^ the^ sample.^The^ absolute^amount^of^internal^control^target^is^unknown,^therefore^efficiency^cannot^be^determined. FISH^Reference^ Standard.^ Horizon.^ Catalogue^No.^ HD717Certificate^ of^ Analysis describes^ as^ a^ quality^control,^Horizon’s^Fish^Standard^uses^negative^and^positive^cell^ line^cores^and^assesses^ genomic^aberrations^specific^to^the^FISH^assay. However,^the^use^of^cell^lines^to^qualify^this^quality^ control^is^dependent^on^biological^variability.^In^addition,^failure^to^pass^the^qualification^gives^no^ indication^as^to^what^step^of^the^entire^workflow^failed. Commercially^available^PeptiQuant™^Plus.^ instrument^performance^kits.^The^product^literature^ describes^ the^ use^ of^ a^ panel^ of^ peptides^ spiked^ into^ samples^ as^ quality^ controls^ for^ mass^ spectrometry.^The^approach^enables^multiplexing,^spiking^a^known^quantity^into^the^sample^and^ covers^a^broad^dynamic^range^of^masses. However,^the^approach^is^limited^to^mass^spectrometry. RES-PA18-PCT^ Commercially^ available^ RNAscope®^ reference^ guide:^ Guidelines^ and^ Protocols^ for^ Obtaining^ Optimized^RNA^in^situ^Hybridization^Results^with^Any^Tissue^Type^from^Any^Species.^ACD. This^ recommends^ the^ use^ of^ two^ levels^ of^ quality^ control:^ A^ technical^ workflow^ check^ and^ a^ sample/RNA^ quality^ check.^ The^ technical^ quality^ control^ check^ ensures^ that^ the^ assay^ and^ reagents^ are^ working^ correctly.^ Recommends^ using^ a^ control^ sample^ tested^ on^ two^ separate^ slides:^ one^ slide^ with^ a^ housekeeping^ gene^ positive^ control^ probe^ and^ another^ slide^ with^ a^ nonspecific^bacterial^gene^negative^control.^They^recommend^checking^tissue^sample^quality^with^ positive^and^negative^controls^before^running^the^experiment.^Their^recommended^positive^and^ negative^controls^are^genes^inherently^expressed^in^the^sample. However,^it^requires^the^use^of^ additional^positive^and^negative^samples.^Decreases^throughput.^Increases^overall^cost^of^running^ a^ sample^ with^ control.^ When^ using^ additional^ control^ samples,^ it^ does^ not^ serve^ as^ controls^ measuring^characteristic^assay^of^the^sample^itself. Against^this^background,^it^is^an^object^underlying^the^present^disclosure^to^provide^a^method^by^ means^of^which the^disadvantages^of^the^prior^art^methods^can^be^reduced^or^even^overcome. SUMMARY^OF^THE^DISCLOSURE In^a^ first^aspect^ the^disclosure^pertains^ to^monitoring^probes^comprising^at^ least^one^element^ having^a^nucleotide^sequence,^wherein^said^element^is^selected^from^the^group^consisting^of^ - a^ translator^ (c)^ element^ comprising^ a^ nucleotide^ sequence^ allowing^ the^ binding/hybridization^of^a^signal^oligonucleotide;^ - an^ identifier^ element^ (t)^ comprising^ a^ nucleotide^ sequence^ allowing^ the^ binding/hybridization^of^a^decoding^element; - a^target^nucleic^acid^sequence^allowing^the^binding/hybridization^of^a^binding^element. In^a^second aspect^the^disclosure^pertains^to^monitoring^probes comprising^at^least^one^identifier^ element (t)^comprising^a^nucleotide^sequence^which^is^essentially^complementary^to^at^least^a^ section^ of^ the^ unique^ set^ identifier^ sequence^ of^ an^ identifier^ element^ (T)^ of^ a^ corresponding^ decoding^element.^In^one^embodiment^the^nucleotide^acid^sequence of^the^monitoring^probe is^ attached^ to^ a^ particle^ selected^ from^ a^ non-magnetic^ bead^ particle,^ magnetic^ bead^ particle,^ a^ streptavidin^coated^particle,^a^biotin^coated^particle,^and/or^any^combination^thereof.^In^another^ embodiment^the^probe^further^comprises^a^binding^element complementary^to^at^least^a^section^ of^a^target^nucleotide^acid^sequence.^In^a^further^embodiment^the^nucleic^acid^of^the^monitoring^ probe is^ attached,^ preferably^ by^ a^ lasting^ attraction^ between^ atoms^ or^ ions^ that^ enable^ the^ RES-PA18-PCT^ formation^of^ a^molecule;^ the^bond^may^ result^ from^ the^ electrostatic^ force^between^oppositely^ charged^ions^as^in^ionic^bonds,^or^through^the^sharing^of^electrons^as^in^covalent^bonds.^Thus,^the^ attachment^is^preferably^by^covalent^bond,^hydrogen^bonds,^a^van-der-Waals^interaction^and/or^ ionic^bond^to^a^particle^selected^from^a^magnetic^bead^particle,^a^streptavidin^coated^particle,^a^ biotin^coated^particle,^and/or^any^combination^thereof. In^yet^a^further^embodiment^the^monitoring^probe is^designed^to^facilitate^a^fluorescent^signal^ substantially^equivalent^to^the^signal^intensity^obtained^for^the^target^biological^analytes.^This^can^ be^done^by^a^direct^labelling^of^the^monitoring^probe,^however,^preferably^by^interacting^with^a^ signal^oligonucleotide^which^either^binds^directly^to^the^monitoring^probe and/or^indirectly^via^ one^or^more^decoding^element(s).^The^use^of^decoding^elements^between^the^monitoring^probe^ and^the^signal^oligonucleotide^allows^for^a^multiplex^approach,^e.g., the^use^of^the^same^signal^for^ set^of^different^monitoring^probe(s),^and/or^the^use^of^different^signal^oligonucleotides^interacting^ with^the^same^monitoring^probe(s),^e.g.,^in^order^to^differentiate^between^different^reaction^steps^ and/or^different^reaction^conditions.^ In^yet^a^further^embodiment^the^nucleic^acid^sequence^of^the monitoring^probe contains^a^spacer^ element^between^the^portion^to^be^immobilized^onto^the^particle^and^the^nucleic^acid^identifier^ element^(t). In^yet^a^further^embodiment^the^monitoring^probe is^immobilized^on^a^slide^surface through^a^ poly-lysine^ coating^ of^ the^ slide^ surface.^ In^ yet^ a^ further^ embodiment^ the^monitoring^ probe is^ immobilized^to^the^poly-lysine^coated^slide^surface^using^homobifunctional^or^heterobifunctional^ cross-linking^reagents^via^a^reactive^group.^In^yet^a^further^embodiment^the^monitoring^probe or^ the^slide^surface comprise^a^reactive^group^consisting^of^an^alkyne^or^azide,^and^the^other^contact^ surface^comprises^the^partner^azide or^alkyne,^such^that^the^immobilization^can^occur^using^click^ chemistry. In^yet^a^further^embodiment^the^size^of^the^monitoring^probe is^250^nm^or^less;^225^nm^or^less;^ 210^nm^or^less;^but^at^least^90^nm,^at^least^130^nm^at^least^180^nm,^in^one^embodiment^the^size^of^ the^monitoring^probe is^about^200^nm. In^a^second^aspect^the^disclosure^pertains^to^method^to^monitor^a^in^situ hybridization^multiplex^ reaction^wherein^the^method^comprises^at^least^the^steps^of: RES-PA18-PCT^ 1.^contacting^the^analyte^nucleotide^sequence^with^at^least^one detecting nucleic^acid^sequence in^ order^to^conduct^an^in-situ^hybridization;^and 2.^adding^the^monitoring^probe^according^to^any^of^claims^1^to^9^before,^during^and/or^after^adding^ the^detecting nucleic^acid^sequence in^step^1. In^ a^ further^ embodiment^ the method^ to^ monitor^ an^ in^ situ hybridization^ multiplex^ reaction^ comprises^at^least^one^monitoring^probe as^described^herein,^wherein^the^ in^situ hybridization^ multiplex^reaction^is^characterized^by^comprising:^ a.) a^binding^element which^is^essentially^complementary^to^at^least^a^section^of^an^analyte^ nucleotide^ sequence^ to^ be^ detected^ and^ an^ identifier^ element^ (T)^ comprising^ a^ nucleotide^ sequence^which^is^unique^to^the^analyte^nucleotide^sequence;^and/or b.) a^decoding^element comprising^an^identifier^element^(t)^comprising^a^nucleotide^sequence^ which^is^essentially^complementary^to^at^least^a^section^of^the^unique^set^identifier^sequence^of^ the^ identifier^ element^ (T)^of^ the^ corresponding^binding^ element,^ and^a^ translator^ element^ (c)^ comprising^a^nucleotide^sequence^allowing^a^specific^hybridization^of^a^signal^oligonucleotide;^ and/or c.) a^ signal^ oligonucleotide comprising^ a^ translator^ connector^ element^ (C)^ comprising^ a^ nucleotide^sequence^which^ is^essentially^complementary^ to^at^ least^a^section^of^the^nucleotide^ sequence^of^a^translator^element^(c)^comprised^in^a^decoding^oligonucleotide,^and^a^signal^element^ facilitating^a^signal^which^is^specific^for^the^polymorphic^analyte;^and wherein^the^method^comprises^a^monitoring^probe^comprising^at^least^an^identifier^element^(t)^ comprising^a^nucleotide^sequence^which^is^essentially^complementary^to^at^least^a^section^of^the^ unique^set^identifier^sequence^of^the^identifier^element^(T)^of^the^corresponding^binding^element;^ and^ wherein^the method^comprises^at^least^the^steps^of: 1.^contacting^the^binding^element with^the^analyte^nucleotide^sequence; 2.^contacting^the^binding^element with^the^decoding^element;^and 3.^contacting^the^signal^oligonucleotide with^the^decoding^element;^and wherein^the^monitoring^probe^is^added before,^during^and/or^after^adding^the^decoding^element^ in^step^2. The in-situ hybridization^ multiplex^ reaction^ is^ described^ in^ more^ details^ in^ this^ description,^ however,^ it^ should^be^mentioned^ in^a^ first^overview,^ that^ the^monitoring^probe^of^ the^present^ RES-PA18-PCT^ invention^ is^ designed^ to^ allow^ the^ monitoring^ of^ the^ correct^ in^ situ hybridization^ multiplex^ reaction^by^being^part^of^the^reaction,^thereby^being^exposed^to^the^same^reactants^and^reactions^ steps^as^used^in^the^in^situ hybridization^multiplex^reaction.^ In^one^embodiment^the^method/^monitoring^probe comprises^a^set^of^monitoring^probes,^wherein^ for^each^of^ the^different^ identifier^elements^(t)^of^ the^decoding^elements the set^of^monitoring^ probes^comprises^at^least^one^monitoring^probe with^the^same^identifier^elements^(t). In^a^third^aspect^the^disclosure^pertains^to^a detection^mix comprising^the^monitoring^probe as^ described^herein,^ and^ further^ comprising^ at^ least^ one^ element^ selected^ from^a^decoy^element consisting^ either^ of^ a^ magnetic^ bead^ particle,^ a^ streptavidin^ coated^ particle,^ a^ biotin^ coated^ particle,^ and/or^ any^ combination^ thereof;^ optionally^ bound^ to^ a^ nucleic^ acid^ oligonucleotide,^ which^ does^ not^ correspond^ to^ the^ identifier^ element^ (t);^ a^ decoding^ element comprising^ an^ identifier^element^(t)^comprising^a^nucleotide^sequence^which^is^essentially^complementary^to^at^ least^ a^ section^ of^ the^ unique^ set^ identifier^ sequence^ of^ the^ identifier^ element^ (T)^ of^ the^ corresponding^binding^element,^and^a^translator^element^(c)^comprising^a^nucleotide^sequence^ allowing^a^specific^hybridization^of^a^signal^oligonucleotide;^a^binding^elementwhich^is^essentially^ complementary^ to^ at^ least^ a^ section^ of^ an^ analyte^nucleotide^ sequence^ to^ be^ detected^ and^ an^ identifier^element^(T)^comprising^a^nucleotide^sequence^which^is^unique^to^the^analyte^nucleotide^ sequence;^and^a^signal^oligonucleotide comprising^a^translator^connector^element^(C)^comprising^ a^nucleotide^sequence^which^is^essentially^complementary^to^at^least^a^section^of^the^nucleotide^ sequence^of^a^translator^element^(c)^comprised^in^a^decoding^oligonucleotide,^and^a^signal^element^ facilitating^ a^ signal^which^ is^ specific^ for^ the^ polymorphic^ analyte;^ as^well^ as^ any^ combination^ thereof. In^ a^ fourth^ aspect^ the^ disclosure^ encompasses^ a method,^ wherein^ the monitoring^ probe as^ disclosed^herein,^can^be^detected^by^a^signal^element^with^a^fluorophore^being^the^same^or^being^ different^ from^ the^ fluorophore^ used^ in^ the^ detection^ of^ the^ target^ nucleic^ acid^ sequence^ that^ permits^normalizing^the^signal^of^the^immobilized^monitoring^probe. In^yet^a^further^embodiment^ a^first^signal^element^with^a^first^fluorophore allows^for^measurement^of^hybridization^efficiency^ of^the^decoder^and^readout^probe^labelledwith^a^second^signal^element^with^a^second^fluorophore by^measuring^the^ratio^between^the^first^and^the^second^fluorophore. In^yet^a^further^embodiment^ the^ first^ signal^ element with^ a^ fluorophore^ enables^ the^ detection^ of^ general^ nonspecific^ hybridization^by^identifying^cycles^in^which^the^particle^is^labelled with^a^second^signal^element with^a^fluorophore^that^is^inconsistent^with^the^decoder^element. RES-PA18-PCT^ In^a^fifth^aspect^the^disclosure^pertains^to^a^method^for^generating^a^detection^mix as^disclosed^ herein. In^ a^ sixth^ aspect^ the^disclosure^pertains^method^ for^ controlling^ the^density^of^ the^monitoring^ probes disclosed^herein on^a^slide^surface by^attaching^azide^or^alkyne^partner^to^a^poly-lysine^ before^coating^it. In^yet^a^further^embodiment^unreactive^azide^or^alkyne^is^removed,^followed^by^ a^reaction^step^with^detection^mix as^disclosed^herein. In^a^seventh^aspect^the^disclosure^encompasses a^monitoring^probe as^described^herein placed^ underneath^ a^ tissue^ in^ order^ to^ assess^ the^ efficacy^ of^ a^ tissue^ sample^ preparation^ process^ comprising^ in^ situ oligonucleotides^which^ penetrate^ through^ the^ tissue^ and^ hybridize^ to^ their^ intended^targets. In^an^eighth^aspect^the^disclosure^encompasses^a^method^to^monitor^temperature^ranges^of^an^in- situ^environment^in^which^monitoring^probe(s) as^described^herein have^different^delta^G^values^ corresponding^ to^ different^ melting^ points^ of^ the^ secondary^ structure^ in^ order^ to^ monitor^ temperature^ fluctuations^ that^ occur^ during^ the^ hybridization^ process. In^ yet^ a^ further^ embodiment^ the^delta^G^value^of^ the^monitoring^probes is^adjusted^by^ the^sequence^ length^or^ sequence^composition.^In^yet^a^further^embodiment^the^delta^G^value^of^the^monitoring^probes is^ adjusted^by^using^modified^bases^which^have^higher^stability^than^standard^bases.^In^yet^a^further^ embodiment^ temperature^ fluctuation^ can^ be^ read-out^ by^ specific^ change^ in^ the^ fluorescence^ and/or^color^facilitated^by^the^direct^and/or^indirect^interaction^of^the^monitoring^probe^with^the signal^oligonucleotide. The^“delta^G^value”^is^defined^herein^as^is^the^difference^in^free^energy^between^the^products^and^ the^reactants.^If^this^value^is^negative,^the^reaction^is^spontaneous,^if^it^is^positive,^the^reaction^is^ non-spontaneous,^and^if^it^is^zero,^the^reaction^is^at^equilibrium.^In^in-situ^hybridization^the^delta^ G^values^corresponds^to^different^melting^points^of^the^secondary^structure^and^is^a^measure^for^ the^hybridization^strength. Thus,^in^a^nineth^aspect^the^disclosure^encompasses^a set^of^monitoring^probes adapted^to^any^of^ the^methods^disclosed^herein. RES-PA18-PCT^ In^a^tenth^aspect^the^disclosure^pertains^also^to^a^kit of^parts^which enable^the^optimization^of in^ situ^hybridization^methods, comprising^at^least^slides^containing^immobilized^monitoring^probes and/or^ binding^ sites^ for monitoring^probes. In^ yet^ a^ further^ embodiment^ the^ kit^ according^ is^ adapted for^monitoring^a^tissue^sample^preparation.^ In^an^eleventh^aspect^the^disclosure^pertains^to^a^control^slide^for^quality^control^comprising^a^set^ of^ immobilized^ monitoring^ probes and/or^ binding^ sites^ for monitoring^ probes as^ described^ hereinunder.^ In^ yet^ a^ further^ embodiment^ the control^ slide^ can^ be^ used for^ assessing^ in-situ^ hybridization^instrument installations. In^a^ twelfth^embodiment^uses^of^ the^monitoring^probe for^monitoring^ in^situ^hybridizations^ is^ used^in^an^analytical,^laboratorial,^diagnostic,^pharmaceutical^or^medical^context. IN-SITU^HYBRIDIZATION^MULTIPLEX^REACTION The^ present^ disclosure pertains^ to a^ reagent^ (monitoring^ probe^ or^monitoring^ probe^ set)^ for^ methods^and^kits^for^detection^of^a^polymorphic analyte^in^a^sample^by^specific^signal-encoding^ of^said^analyte by^in-situ^hybridization.^ In^one^embodiment,^a^method^for^detecting^a^polymorphic^analyte^in^a^sample^by^specific^signal- encoding^of^said^polymorphic^analyte^is^disclosed,^comprises the^steps^of:^ (A) contacting^the^sample^with^a^set^of^polymorphic^analyte-specific^probes^for^encoding^different^ variations^ of^ the^ same^ polymorphic^ analyte,^ each^ analyte-specific^ probe^ interacting^ with^ a^ different^variation^and/or^sub-structure^of^ the^polymorphic^analyte,^wherein^ the^polymorphic^ analyte^is^present^in^the^sample^in^at^least^as^two^or^more^variant^forms^of^a^specific^nucleic^acid^ sequence^and^wherein^the^sequences^of^each^polymorphic^analyte^are^related^to^each^other^with^ a^sequence^identity^of^between^85%^to^99%,^each^polymorphic^analyte-specific^probe^comprising^ (aa)^a^binding^element^(S)^that^specifically^interacts^with^one^of^the^at^least^one or^more^variants^ and/or^sub-structure^of^the^polymorphic^analyte^to^be^encoded,^and (bb)^ an^ identifier^ element^ (T)^ comprising^ a^ nucleotide^ sequence^ which^ is^ unique^ to^ the^ polymorphic^analyte to^be^encoded^(unique^set^identifier^sequence),^ wherein^each^set^of^polymorphic^analyte-specific^probes^differ^from^another^set^of^polymorphic^ analyte-specific^probes^in^the^nucleotide^sequence^of^the^identifier^element^(T), and RES-PA18-PCT^ (B) contacting^the^sample^with^at^least^a^first^set^of^decoding^oligonucleotides^per^polymorphic^ analyte,^wherein^in^each^set^of^decoding^oligonucleotides^for^an^individual^analyte^each^decoding^ oligonucleotide^of^the^for^the^first^set^of^analyte-specific^probes^according^to^step^A1^comprises: (aa)^an^ identifier^connector^element (t)^comprising^a^nucleotide^sequence^which^ is^essentially^ complementary^to^at^least^a^section^of^the^unique^set^identifier^sequence^of^the^identifier^element^ (T)^of^the^corresponding^analyte-specific^probe^set^A1,^and (bb)^a^translator^element^(c)^comprising^a^nucleotide^sequence^allowing^a^specific^hybridization^ of^a^signal^oligonucleotide; wherein^the^decoding^oligonucleotides^of^a^set^for^an^individual^analyte^differ^from^the^decoding^ oligonucleotides^of^another^set^for^a^different^analyte^in^the^first^connect^element^(t);^and (C) contacting^the^sample^with^at^least^a^set^of^signal^oligonucleotides,^each^signal^oligonucleotide^ comprising: (aa)^a^ translator^connector^element^(C)^comprising^a^nucleotide^sequence^which^ is^essentially^ complementary^ to^ at^ least^ a^ section^ of^ the^ nucleotide^ sequence^ of^ a^ translator^ element^ (c)^ comprised^in^a^decoding^oligonucleotide,^and (bb)^a^signal^element^facilitating^a^signal^which^is^specific^for^the^polymorphic^analyte;^and (D) Detecting^the^signal^caused^by^the^signal^element; (E) Optionally^selectively^removing^the^decoding^oligonucleotides^and^signal^oligonucleotides^ from^ the^ sample,^ thereby^ essentially^ maintaining^ the^ specific^ binding^ of^ the^ analyte-specific^ probes^to^the^analytes^to^be^encoded; (F) Performing^further^cycles^comprising^steps^A)^to^E)^for^each^further^polymorphic^analyte^ to^generate^an^encoding^scheme^with^a^specific^signal^per^set^of^polymorphic^analytes,^wherein^in^ particular^the^last^cycle^may^stop^with^step^(D). Depending^on^the^design^of^the^probe^set,^the^detected^signal^may^label^a^gene^family,^a^single^ gene,^a^specific^genetic^locus,^a^benign^or^malign^genetic^variation,^genetic^specificity^or^biomarker^ in^a^cluster^of^patients,^a^virus-genome,^a^certain^subset^of^nucleic^acid^variation^(e.g.^a^group^of^ SNPs,^a^mutation,^a^virus-variant,^a^gene-variant,^a^transcript,^isoforms,^splice-variants,^etc.).^ As^ such,^ the^ technology^ allows^ the^ high-throughput^ detection^ of^ nucleic^ acids^ with^ variable^ sequences^of^at^least^60%,^at^least^65%,^at^least^70%,^at^least^75%,^at^least^80%,^at^least^85%,^at^ least^90%,^and^up^to^92%,^up^to^95%,^up^to^98%,^up^to^99%,^up^to^99.9%^sequence^identity.^In^ some^embodiments^between^65%^to^99%,^between^70%^to^99%,^between^75%^to^99%,^between^ 80^%^to^99%,^between^85%^to^99%,^between^85%^to^98%,^between^85%^to^95%^,^between^90%^ to^95%^sequence^identity^in^a^sample. RES-PA18-PCT^ The^technology^is^also^more^robust^than^conventional^methods,^since^the^signal^is^still^detectable^ even^if^just^a^subset^of^probes^bind^the^target.^As^such^the^paradigm^“the^label^identifies^the^probe”^ is^overcome^and^the rule^“the^label^identifies^the^target” was^applied^instead.^ In^a^ further embodiment,^ this^disclosure^relates to^ in^vitro methods for^diagnosis^of^a^disease^ selected^ from^ the^ group^ comprising^ cancer,^ neuronal^ diseases,^ cardiovascular^ diseases,^ inflammatory^diseases,^autoimmune^diseases,^diseases^due^to^a^viral^or^bacterial^infection,^skin^ diseases,^skeletal^muscle^diseases,^dental^diseases^and^prenatal^diseases^comprising^the^use^of^the^ multiplex^method^according^to^the^present^disclosure. In^yet^another^embodiment, this^disclosure^provides in^vitromethods for^diagnosis^of^a^disease^in^ plants^ selected^ from^ the^ group^ comprising:^ diseases^ caused^ by^ biotic^ stress,^ preferably^ by^ infectious^ and/or^ parasitic^ origin,^ or^ diseases^ caused^ by^ abiotic^ stress,^ preferably^ caused^ by^ nutritional^deficiencies^and/or^unfavourable environment,^said^method^comprising^the^use^of^the^ multiplex^method^according^to^the^present^disclosure. In^yet^another^embodiment,^this^disclosure^relates to^optical^multiplexing^systems^suitable^for^the^ method^according^to^the present^disclosure,^comprising^at^least: - at^least^one^reaction^vessel^for^containing^the^kits^or^part^of^the^kits^according^to^the^ present^disclosure; - a^detection^unit^comprising^a^microscope,^in^particular^a^fluorescence^microscope - a^camera - a^liquid^handling^device. In^yet another^embodiment,^this^embodiment^provides in^vitromethods^for^screening,^identifying^ and/or^testing^a^substance^and/or^drug^comprising: (a) contacting^a^test-sample^comprising^a^sample^with^a^substance^and/or^drug^ (b) detecting^different^analytes^in^a^sample^by^sequential^signal-encoding^of^said^analytes^with^ a^method^according^to^the^present^disclosure. According^to^the^present^disclosure,^unique^tags^(identifier)^are^used^per^target^(e.g., mRNA^of^one^ single^gene)^or^for^a^target^group.^Groups^can^be^formed^to^be^indicative^for^a^certain^identity,^ process,^ biological^ function^ or^ disease^ (examples:^ cell^ type,^ inflammation,^ signal^ processing,^ cancer). RES-PA18-PCT^ Surprisingly,^ the^methods^ and^kits^ according^ to^ the^present^disclosure^ led to^ the^ reduction^of^ complexity.^Many^ different^ probes^with^ different^binding^ sequences^ share^ the^ same^ (one^ per^ target)^unique^tag.^These^tags^have^reduced^the^sequence^complexity^(to^one^per^target)^and^also^ have^predetermined^constant^properties^(e.g., thermodynamic^stability). Advantages of^the^methods^and^kits^according^to^the^present^disclosure^are:^ a) Full^ flexibility^of^ the^process^ to^determine^the^ identity^of^ the^ tag,^e.g.^use^more^or^ less^ signals^and/or^rounds,^varying^numbers^of^fluorophores,^number^of^total^signals^per^tag^ lower^numbers^of^targets(e.g.^20)^can^be^identified^with^high^confidence^in^less^rounds^ (e.g.^4)^than^a^large^number^of^targets^(e.g.^100,^these^need^8^rounds^for^the^same^level^of^ confidence),^even^if^in^both^cases^the^exact^same^unique^tags^are^used. b) All^unique^tags^are^used^(recycled)^in^many^consecutive^rounds^of^hybridization^and^all^ primary^probes^contribute^(provide^information^about^their^identity)^in^every^round^of^ identification. c) As^ all tags^ share^ the^ same^ predefined^ properties^ (e.g.^ thermodynamic^ stability^ which^ allows^for^selective^denaturing). In^some^advantageous^embodiments,^the^unique^tags are^design^as^follow: - No^ cross-hybridization^ between^ all^ oligonucleotides^ of^ the^ process^ (probes,^ decoders,^ readout),^so^that^all^tag^sequences^are^usable^together^(compatible) - No^cross-hybridization^between^connector^elements^(bridges)^of^different^unique^tags - Stability^ of^ hybridization^of^ the^unique^ tags^ should^be^ in^ a^narrow^ range:^ as^ stable^ as^ possible^(fast^hybridization,^i.e., short^cycle^times)^but^significantly^different^(in^this^case^ less^ stable)^ than^ the^ primary^ probe^ (for^ differential^ denaturation,^ without^ removing^ primary^probes). Therefore,^ the^ present^ description^ pertains^ in^ particular^ to^ the^ usage^ of^ a^ set^ of^ labeled^ and^ unlabeled^nucleic^acid^sequences^ for^specific^quantitative^and/or^spatial^detection^of^different^ analytes^in^parallel^via^specific^hybridization.^The^technology^allows^the^discrimination^of^more^ different^analytes^than^different^detection^signals^are^available.^The^discrimination^is^realized^via^ sequential^ signal-coding^ of^ the^ analytes^ achieved^ by^ several^ cycles^ of^ specific^ hybridization,^ detection^of^signals^and^selective^elution^of^the^hybridized^nucleic^acid^sequences.^In^contrast^to^ other^ state-of-the-art^ methods,^ the^ oligonucleotides^ providing^ the^ detectable^ signal^ are^ not^ directly^ interacting^with^ sample-specific^nucleic^ acid^ sequences^but^ are^mediated^by^ so^ called^ “decoding-oligonucleotides”.^This^mechanism^decouples^ the^dependency^between^ the^ analyte- RES-PA18-PCT^ specific^oligonucleotides^and^the^signal^oligonucleotides.^The^use^of^decoding-oligonucleotides^ allows^ a^much^ higher^ flexibility^while^ dramatically^ decreasing^ the^ number^ of^ different^ signal^ oligonucleotides^ needed^which^ in^ turn^ increases^ the^ coding^ capacity^ achieved^with^ a^ certain^ number^of^detection^rounds.^The^utilization^of^decoding-oligonucleotides^ leads^ to^a^sequential^ signal-coding^technology^that^is^e.g., more^flexible,^cheaper,^simpler,^faster^and/or^more^accurate^ than^other^methods. Furthermore,^the^present^disclosure^pertains^to^the^use^of^improved^decoding-oligonucleotides^ to^ increase^ the^ efficiency^ of^ the^ encoding^ scheme.^ The^ so^ called^ “multi-decoders”^ allows^ the^ recruiting^of^more^ than^ just^ one^signal^ oligonucleotide^ and^therefore^ can^ generate^new^ signal^ types^ by^ utilizing^ the^ combination^ of^ two^ or^ more^ different^ signal-oligonucleotides^ without^ decreasing^the^brightness^of^the^signals.^ Furthermore, due^to^the^use^of^a^first^set^of^analyte-specific^probes^according^to^step^A1 (i.e.^the^ transcript^plexity^of^A1)^which^is^at^least^10^times^higher in^numbers than^the^number^of^probes^ and/or^targets^of^the^second^set^of^analyte-specific^probes^according^to^step^A2^(i.e.^the^transcript^ plexity^of^A2) as^well^as^the^use^of^at^least^one,^preferably^at^least^two different^sets^of^decoding^ oligonucleotides^ for^set^A1 and^set^A2,^ spatially^overlapping^targets^ inside^a^ tissue^and^or^cell^ culture^sample^which^distance^is^beyond^the^diffraction^limit can^be^detected.^Further^advantages^ pertain^ to^an^ improvement^of the^overall^ signal^ to^noise^ratio,^ the^signal^spread^(i.e^signals^of^ transcripts^with^higher^expression^levels^can^be^detected^together^with^lowly^expressed^genes) and^the^multiplexing^capability, without^increasing^optical^crowding. Before^the^disclosure is^described^in^detail,^it^is^to^be^understood^that^this^disclosure is^not^limited^ to^the^particular^component^parts^of^the^steps^of^the^methods^described.^It^is^also^to^be^understood^ that^the^terminology^used^herein^is^for^purposes^of^describing^particular^embodiments^only^and is^not^intended^to^be^limiting.^It^must^be^noted^that,^as^used^in^the^specification^and^the^appended^ claims,^the^singular^forms^"a,"^"an"^and^"the"^include^singular^and/or^plural^referents^unless^the^ context^clearly^dictates^otherwise.^It^is^moreover^to^be^understood^that,^in^case^parameter^ranges^ are^ given^ which^ are^ delimited^ by^ numeric^ values,^ the^ ranges^ are^ deemed^ to^ include^ these^ limitation^values.^ RES-PA18-PCT^ BRIEF^DESCRIPTION^OF^THE^DRAWINGS Fig.^1: Embodiment^ where^ the^ analyte^ is^ a^ nucleic^ acid, and^ the^ probe^ set^ comprises^ oligonucleotides^ specifically^ binding^ to^ the^ analyte.^ The^ probes^ comprise^ a^ unique^ identifier^ sequence^allowing^hybridization^of^decoding^oligonucleotides. Fig.^2: Embodiment^where^the^analyte^is^a^protein, and^the^probe^set^comprises^proteins^(here:^ antibodies)^specifically^binding^to^the^analyte.^The^probes^comprise^a^unique^identifier^sequence^ allowing^hybridization^of^decoding^oligonucleotides. Fig.^3: Flowchart^of^the^method^according^to^the^disclosure. Fig.^4: Alternative^options^for^the^application^of^decoding^and^signal^oligonucleotides. Fig.^5: Example^ for^ signal^ encoding^of^ three^different^nucleic^ acid^ sequences^by^ two^different^ signal^ types^ and^ three^detection^ rounds;^ in^ this^ example,^ the^ encoding^ scheme^ includes^ error^ detection. Fig.^6: Number^of^generated^code^words^(logarithmic^scale)^against^number^of^detection^cycles. Fig.^7: Calculated^total^efficiency^of^a^5-round^encoding^scheme^based^on^single^step^efficiencies. Fig.^8: Comparison^of^relative^transcript^abundances^between^different^experiments. Fig.^9: Correlation^of^relative^transcript^abundances^between^different^experiments. Fig.^10:Comparison^of^intercellular^distribution^of^signals. Fig.^11:Comparison^of^intracellular^distribution^of^signals. Fig.^12:Distribution^pattern^of^different^cell^cycle^dependent^transcripts. Fig.^13:Detection^of^multiple^targets^using^an 8^round^code^with^2^labels^(A^and^B)^and^no^label^ (-).^The^targets^1,^2,^3,^4,^5,^20,^and^n^are^represented.^ ^The^rounds^1,^2,^3,^and^8^of^the^coding^ scheme^are^represented. Herein,^the^blank^is^part^of^the^code. RES-PA18-PCT^ Fig.^14:Detection^of^multiple^targets^can^be^performed^by^an^encoding^scheme^using^a^detectable^ marker.^The^ending^scheme^may^comprise^also^the^„0“^as^a^marker.^That^means^that^at^a^specific^ position^the^transcript^is^not^detected.^Consequently,^the^endocing^scheme^may^be^represented^ by^the^following^constructs^using^only^two^gene^specific^probes:^ 1) With^detectable^label^F:^detectable^during^imaging 2) With^detectable^label^F^and^quencher^Q:^not^detectable^during^imaging 3) With^quencher^Q:^not^detectable^during imaging 4) Without^label^F:^not^detectable^during^imaging 5) Without^signalling^oligonucelotide:^not^detectable^during^imaging 6) With^a^decoder^oligonucleotide^that^cannot^recruit^a^signalling^oligonucleotide 7) Without^decoder^oligonucelotide:^not^detectable^during^imaging Fig.^15:Possible^structures^of^a^multi-decoder.^The^numbers^depict^the^examples.^(A)^is^the^unique^ identifier^sequence,^(a)^is^the^corresponding^sequence^of^the^decoding^oligonucleotide^or^multi- decoder^and^(c1)^to^(c3)^are^different sequence^elements,^that^specifically^bind^to^different^signal^ oligonucleotides.^Examples^2^ to^5^ show^different^ versions^of^multi^decoders.^The^order^of^ the^ different^sequence^elements^as^well^as^the^number^of^signal^oligonucleotide^binding^elements^is^ not fixed.^ Example^ 1^ shows^ a^ normal^ decoding^ oligonucleotide^ since^ there^ is^ only^ one^ signal^ oligonucleotide^binding^element^(c1). Fig.^16:Example^ for^ signal^ encoding^ of^ three^ different^ nucleic^ acid^ sequences^ by^ using^multi- decoders^and^two^different^signal^oligonucleotides^creating^three^different^signal^types^and^three^ detection^rounds.^In^this^example,^the^encoding^scheme^includes error^detection^and^correction. Fig.^17:Number^of^generated^codewords^(logarithmic^scale)^against^number^of^detection^cycles. The^ number^ of^ codewords^ for^merFISH^ does^ not^ exponentially^ increase^ with^ the^ number^ of^ detection^ cycles^ but^ gets^ less^ effective^ with^ each^ added^ round.^ In^ contrast,^ the^ number^ of^ codewords^for^intronSeqFISH,^the^method^of^the^present^disclosure^without^using^multi-decoders,^ the method^with^multi-decoders^increases^exponentially.^The^slope^of^the^curve^for^the^method^ using^multi-decoders^is^much^higher^than^that^of^the^prior^invention,^leading^to^more^than^20^000^ 000^times^more^code^words^usable^after^20^rounds^of^detection. RES-PA18-PCT^ Fig.^ 18:^ Embodiment^ of^ a^monitoring^ probe^ according^ to^ the^ present^ disclosure,^wherein^ the^ monitoring^probe^is^bound^to^a^bead,^wherein^the^monitoring^probe^comprises^a^target^nucleic^ acid^sequence^allowing^the^binding/hybridization^of^a binding^element^(primary^probe),^wherein^ a^decoder^element^(decoder)^binds^to^the^binding^element^and^wherein^a^signal^oligonucleotide^ binds^to^the^decoder^element^for^readout. Fig.^19:^Visual^representation^of^mRNA^and^control^particle. Fig^20:^Particle^attachment^to^microscope^slights. Fig.^21:^Assay^for^parameter^optimization. Fig.^22:^Example^for^the^use^of^ZEN’s^DFIs. Fig.^23:^Results^^of^MC^Run^of^1-plex^particle Fig.^24:^Results^^of^MC^Run^of^1-plex^particle,^6-plex^particle,^12-plex^particle Fig.^25:^MC^Run^of^19-plex^particles Fig.26:^Embodiments^of^the^monitoring^probe^according^to^the^present^disclosure.^A)^Monitoring^ probes^(1)^attached^to^a^particle^(3)^like^a^magnetic^bead,^which^is^immobilized^to^a^slide^surface^ (2).^On^the^right^a^tissue^sample^(4)^is^place^on^top^of^the^particles.^B)^shows^a^specific^embodiment^ of^the^particle,^wherein^the^monitoring^probe^comprises^a^translator^(c)^element^(1a)^comprising^ a^nucleotide^sequence^allowing^the^binding/hybridization^of^a signal^oligonucleotide.^On^the^right,^ the^binding^of^the^signal^oligonucleotides^(5)^is^shown.^C)^shows^a^specific^embodiment^of^the^ particle,^wherein^ the^monitoring^probe^ comprises^ an^ identifier^ element^ (t)^ (1b)^ comprising^ a^ nucleotide^sequence^allowing^the^binding/hybridization^of^a^decoding^element (6).^On^the^right,^ the^binding^of^the^decoding^elements (6)^to^the^monitoring^probe^and^the^binding^of^the^signal^ oligonucleotides^ to the^ decoding^ elements is^ shown. D)^ shows^ a^ specific^ embodiment^ of^ the^ particle,^wherein^the^monitoring^probe^comprises^a^target^nucleic^acid^sequence^(1c)^allowing^the^ binding/hybridization^of^a^binding^element^(7).^On^the^right,^the^binding^of^the^decoding^elements^ (6)^to^the^monitoring^probe,^the^binding^of^the^decoding^element^(6)^and^the^binding^of^the^signal^ oligonucleotides^(5)^to^the^decoding^elements^(6)^is^shown. RES-PA18-PCT^ DETAILED^DESCRIPTION^OF^THE DISCLOSURE Disclosed^herein^are^novel^reagents^(monitoring^probe^and/or^a^set^of^monitoring^probes)^ for^ monitoring^ in^situ hybridization^multiplex^methods.^One^advantage^of^the^present^disclosure^is^ that^the^at^least^one^monitoring^probe^is^part^of^the^reaction^and^therefore^is^exposed^to^the^same^ reactants^and^reactions^steps^as^the^in-situhybridization^multiplex^analysis.^Furthermore,^another^ advantage^is^that^the^monitoring^probe^itself^has^“multiplexing-ability”,^i.e.,^interacts^directly^or,^ preferably,^indirectly^with^one^or^more^signal^oligonucleotides^via^decoding^elements.^This^allows^ a^ combinatorial^ variation^which^ allows^ to^ generate^ a^ readout^which^ is^ far^more^ complex^ and^ carries^ much^ more^ information,^ by^ the^ use^ of^ still^ only^ a^ few^ elements,^ than^ conventional^ monitoring^methods.^This^“multiplexing-ability”^allows^for^example^to^adjust^the^signal^intensity^ to^the^respective^in^situ^hybridization^multiplex^analysis;^allows^to^monitor^intermediate^steps^of^ the^ reaction^ in^ different^ colours^ and/or^ allows^ fluctuations^ in^ the^ reaction^ process,^ e.g.,^ temperature^fluctuations.^The^at^least^one^monitoring^probe^can^be^present in^solution^and/or^ immobilized^on^a^surface,^for^example^a^reaction^matrix,^a column,^a slide,^and^the^like.^ In^particular,^the^present^disclosure^pertains^to^methods^of^ground^truth^generation^for^detection^ optimization^and^transcript^classification. DNA,^ RNA^ or^ other^molecules^ within^ a^ biological^ tissue^ sample^ can^ be^ detected^ within^ their^ spatially^oriented^environment,^using^fluorescence^imaging^techniques,^such^as^smFISH.^^Recent^ development^in^combinatorial^smFISH^chemistry,^e.g.^the^spatial^biology^methods^of^the^present^ disclosure enable^parallel^ detection^of^ large^number^of^ such^molecules^within^ cells^ or^ tissues.^^ Such^detection^system^has^been^demonstrated^to^identify^biologically^relevant^transcripts^(RNAs)^ with^high^sensitivity^and^low^error^rate.^^However,^the^actual^quantities^of^the^target^molecules^ remain^unknown,^as^no^prior^tools^exist^to^do^absolute^quantification^of^molecules^within^cells^or^ tissue^samples^while^preserving^the^spatial^orientation^of^such^samples.^^Due^to^the^lack^of^actual^ quantities^of^targets^under^measurement,^the^sensitivity^(true^positive^#^/^(true^positive^#^+^false^ negative^#))^of^such^measurement^has^only^been^estimated^using^detected^events^using^traditional^ methods.^ ^ For^ example,^ the^ number^ of^ a^ RNA^ target^ detected^ by^ smFISH^ has^ been^ used^ as^ a^ reference^for^100%^detectable^targets^within^cells^or^tissue^samples.^^However,^multiple^MC^data^ sets^ have^ shown^ higher^ number^ of^ detectable^ RNA^ targets^ than^what^ smFISH^ quantifies.^ ^ In^ addition,^ the^smFISH^quantification^methods^rely^on^subjective^settings^of^ intensity^ threshold,^ making^ the^ ground^ truth^ reference^ a^moving^ target.^ ^ In^ this^document,^multiple^methods^ are^ RES-PA18-PCT^ described^to^generate^the^actual^number^of^targets^or^ground^truth^information,^which^can^be^used^ for^the optimization^of^detection^and^classification. In^particular,^the^detection^methods^according^to^the^present^disclosure^uses^oligo^hybridization^ and^de-hybridization^to^RNA^targets^to^create^on^states^(fluorophores^attached)^and^off^states^in^ multiple^ cycles.^ ^ Within^ each^ cycle,^ fluorescence^ images^ of^ the^ cells^ and^ tissues^ samples^ are^ collected.^ ^ Image^analysis^ is^performed^to^ identify^ the^on^and^off^states^of^measured^ locations^ within^the^samples.^^The^specific^sequence^of^the^on^and^off^states^associated^with^a^target^can^be^ considered^as^a^barcode^for^unique^identification^of^the^target.^ ^The^deviation^of^the^on^and^off^ states^for^any^location^under^measurement^is^the^results^of^many^factors,^such^as^integrity^of^RNA^ targets^within^the^sample,^oligos’ hybridization^reaction^completeness,^none-specific^binding^of^ fluorophores,^image^quality^and^uniformity^from^the^optics^used,^camera^pixels’^temporal^noise,^ etc.^^Due^to^the^complex^mechanism^to^generate^the^on^and^off^states,^it^is^critical^to^generate^the^ ground^truth^with^reduced^complexity. In^ some^ advantageous^ embodiments,^ the^ present^ disclosure^ pertains^ to^ oligo^ attached^ beads^ (monitoring^probes)^and^uses^thereof^to^create^ground^truth.^^The^oligos^attached^to^the^beads^can^ be^ a) same^oligos^as^the^decoder^tails (translator^element^(c))^that^binds^to^signal^oligonucleotides;^ b)^same^oligos^as^‘tails’^on^gene^specific^probes (identifier^element^(t)) for^binding^with^decoder elements; c)^combination^of^the^designed^target^sequences^on^a^RNA.^^ The^different^configurations^allow^the^oligo^beads^to^generate^on^and^off^states^that^associated^ with^ different^ complexity^ of^ the^ hybridization^ chemistry.^ The^ quantity^ of^ such^ beads^ can^ be^ controlled^by^the^concentration^of^the^bead^solution.^ ^The^beads^can^be^deposited^onto^a^glass^ slide.^^The^beads^can^be quantified^by^other^measurement^techniques,^e.g.^bright^field^or^dark^field^ microscope^imaging,^to^set^the^ground^truth.^^Tissue^samples^can^be^placed^on^top^of^these^beads.^^ Some^or^all^of^these^species^with^or^without^tissue^samples^on^top^can^be^measured^and^quantified^ against^the^ground^truth^information^obtained.^^The^information^can^be^used^to^identify^each^step^ of^the^assay,^e.g.^by^measuring^counts^of^gene^specific^oligo^combo^beads^without^tissue^on^top^ and^ then^ with^ tissue^ on^ top,^ one^ can^ quantify^ oligo^ penetration^ and^ hybridization^ reaction^ completeness. RES-PA18-PCT^ Some^further^embodiments^of^the^present^disclosure^pertain to^the^use^of^computer^simulated^ signal^values^to^create^ground^truth.^^Measured^signal^values^from^actual^known^targets^can^be^ used^to^simulate^the^ground^truth.^^The^simulated^signal^values^can^be^infused^within^a^data^set^ with^certain^spatial^distribution^or^at^specific^locations^of^interest.^^The^on^and^off^state^can^be^set^ to^match^barcodes^in^use^or^not^in^use.^^The^signal^values^can^be^adjusted^to^low^and^high^levels^ for^optimization.^ ^The^signals^can^also^be^generated^in^low^and^high^spatial^density^levels^with^ known^ separations.^ ^ The^ flexible^ parameter^ setting^ of^ the^ ground^ truth^ information^ provides^ image^ analysis^ development^ absolute^ reference^ for^ event^ classification,^ model^ training^ and^ optimization. Some^further^embodiments^of^the^present^disclosure^pertain^to^the^use^of^known^quantity^targets^ within^the^biological^sample,^e.g.^DNA^targets.^^DNA^FISH^targets^of^known^species^can^be^used^as^ the^ ground^ truth,^ as^ the^number^of^ copies^ is^ known.^ ^Detecting^such^ targets^within^ the^ tissue^ sample^provides^absolute^calibration^of^the^detection^system. In^ some^ advantageous^ embodiments,^ the monitoring^ probe^ comprises^ at^ least^ one^ identifier^ element^(“tail”)^which^unique^oligonucleotide-sequence^allows^the^specific^recognition^by^at^least^ one^decoding^element^via^selective^hybridization.^The^decoding^element^then^allows^the^detection^ of^ a^ signal^ oligonucleotide,^ either^directly,^ or^by^ a^ chain^of^ two,^ three,^ four^or^more^decoding^ elements.^The^signal^oligonucleotide^then^provides^an^optical^signal,^which^could^be^enzymatic,^a^ dye^or, preferably^a^fluorescent^signal.^ The^monitoring^probe^can^be^present^either^in^solution^or^is^attached^to^a^surface,^either^directly^ or^via^a^particle,^such^as^a^magnetic^bead,^a^biotin-tag,^a^strep-tag,^or^the^like.^In^one^embodiment^ the^oligonucleotide^comprises^a^spacer^sequence^between^the^identifier^element^(“tail”)^and^the^ part^of^the^oligonucleotide^which^is^attached^to^the^surface^and/or^bead.^In^one^embodiment^the^ monitoring^probe^is^immobilized^on^the^same^slide^on^which^the^in-situ^reaction^takes^place.^ As^such^the^disclosure^describes^a^monitoring^probe^(“control^particle”) that^can^be^used^for^the^ following^purposes: 1. Quality^control^(QC) and^release^of^kit^components^and^final^kit 2. Evaluation^of^workflows^and^validation^of^software^and^hardware^updates. 3. Performance^of^instrument^qualification^during^installation^or^instrument^repair. 4. Internal^standard^for^customer^runs 5. Training^of^customers^new^to^the^in-situ hybridization^workflow. RES-PA18-PCT^ 6. Validating^that^a^customer^can^perform^the^protocol^prior^to^running^clinical^samples^in^a^ diagnostic^test. 7. Evaluation^of^the^efficacy^of^tissue^preparation^to^enable^diffusion^of^detection^sequences^ into^(and^through)^the^tissue. In^one^ embodiment,^ the^ final^monitoring reagent^consists^ of^ a^ set of^monitoring^probes,^ each^ monitoring^probe^having^ a^different^ attached^ identifier^ element.^ In^ the^ examples^used^ in^ this^ disclosure,^ the^ identifier^elements attached^ to^ the^particles^are^referred^to^as^ “tail”^ sequences.^ Each^of^the^tail^sequences^is^complementary^to^one^of^the^decoder^sequences.^The^combination^of^ different^decoder^pools^and^cycles^of^detection^allows^ for^multiplexing^of^ the^different^unique^ elements^into^a^control^particle^pool.^At^the^end^of^the^run,^the^identity^of^each^particle^can^be^ determined^by^ the^sequence^ and^colour^of^ fluorescence^ at^each^ cycle.^Alternative^ to^using^ tail^ sequences,^the^attached^sequence^could^be^a^unique^sequence^analogous^to^a^targeted^RNA.^This^ would^enable^monitoring^the^entire^workflow,^including^the^overnight^steps^needed^to^hybridize^ the^ probe^ sets^ to^ the^ tissue.^ Alternatively,^ the^ sequence^ attached^ to^ the^ particle^ could^ be^ complementary^to^the^readout^probe.^This^would^enable^the^particle^to^serve^as^a control^to^the^ final^steps^involved^in^binding^and^washing^the^readout^probe. The^use^of^magnetic^particles^as^shown^in^the examples^hereinunder^facilitate^manufacturing^and^ washing^of^the^control^beads^using^a^magnetic^ field^ for^separation.^Alternatively,^nonmagnetic^ particles^can^be^used.^When^using^nonmagnetic^particles,^the^binding^and^washing^steps^can^be^ accomplished^using^standard^batch^or^chromatographical^processes.^Alternatively,^the^sequences^ could^be^attached^to^the^slide^by^spotting^directly. To^serve^as^a^relevant^control,^the^number^of^attached^oligonucleotides^needs^to^be^comparable^ to^ the^ number^ of^ tails^ that^ would^ be^ present^ on^ a^ targeted^mRNA^ analyte.^ Having^ too^many^ oligonucleotides^ per^ particle^ would result^ in^ fluorescent^ signals^ that generates^ images^ of^ inappropriate^size^and/or^ intensity.^Although^the^number^of^ tails^ attached^per^particle^ can^be^ achieved^ by^ titrating^ the^ concentration^ of^ tails^ during^ attachment,^ a^more^ controlled^method^ would^ be^ using^ a^ decoy.^ When^ using^ streptavidin-coated^ particles,^ decoys^ can^ consist^ of^ biotinylated^oligonucleotide,^free^biotin,^or^any^moiety^able^to^bind^to^the^biotin^binding^site.^Using^ a^ decoy^ to^ control^ the^ final^ number^ of^ tails^ attached^ to^ the^ particle^ results^ in^ a^more^ robust^ manufacturing^ process^ and^ allows^ saturation^ of^ the^ particle^ binding^ sites^ independent^ of^ the^ number^of^particles^in^the^preparation. RES-PA18-PCT^ Each^element^in^the^set^of^control^particles^will^have^a^unique^sequence^attached^to^the^surface.^ Each^element^can^be^manufactured, and^the^quality^control^can^take^place in^bulk.^Once^validated,^ the^individual^elements^can^be^pooled^to^generate^the^final^control^particle^pool.^Production^of^the^ particles^in^solution^facilitates^QC^of^the^individual^elements^as^well^as^the^final^pool^of^control^ particles.^ The^ attachment^ of^ the^ oligonucleotide^ to^ the^ particle^ can^ be^ accomplished^ by^ a^ variety^ of^ chemistries,^ as^ is^ standard^ in^ the^ art.^ Attachment^ using^ biotinylated^ oligonucleotide^ and^ streptavidin-coated^ particles^ was^ the^ method^ selected^ in^ this^ invention.^ To^ ensure^ that^ the^ particle^remains^attached^to^the^slide^during^the^steps^required^to^perform^molecular^cartography,^ the^ beads^ can^ be^ attached^ to^ the^ slide^ using^ a^ variety^ of^ chemistries.^ For^ example,^ when^ streptavidin-coated^particles^are^used,^the^biotin^moieties^can^be^attached^to^the^slide^surface^that^ will^bind^to^biotin-binding^sites^on^the^particle's^surface.^Alternatively,^reactive^moieties^can^be^ added^ to^ the^ surface^ of^ the^ slide^ either^ directly^ or^ by^ attaching^ to^ the^ particle^ additional^ oligonucleotides^that^contain^reactive^groups^such^as^alkyne^or^azides^that^enable^click^chemistry^ for^covalent^attachment. One^approach^to^manufacturing^the^control^slides^is^to^activate^the^particle^with^reactive^group,^ such^as^an^azide^or^alkyne.^A^second^reactive^group,^such^as^the^alkyne^or^azide,^can^be^attached^ to^a^coating^polymer^(Poly^lysine)^in^solution.^Activating^a^coating^polymer^in^solution^allows^for^ more^ controlled^manufacturing^ and^ the^ ability^ to^ QC^ and^ characterize^ the^ bulk^material.^ One^ approach^ would^ be^ to^ coat^ the^ slide^ surface^ with^ the^ bulk^ activated^ polymer^ followed^ by^ attachment^of^the^particle^to^the^coated^slide^surface.^Alternatively,^the^activated^bulk^polymer^ can^react^with^the^activated^particle^followed^by^coating^the^particle-polymer^complex^to^the^slide^ surface. The^tails^and^decoders^can^be^designed^to^monitor^environmental^conditions^that^may^influence^ the^performance^of^ the^platform.^By^designing^a^series^of^sequences^covering^different^delta^G^ values,^the^control^particles^will^be^able^to^detect^instrument^or^environmental^temperatures^that^ fall^outside^of^the^desired^range^of^the^assay.^By^designing^decoder^sequences^with^different^delta^ G^values^ at^either^ end,^ the^ environmental^ temperatures^at^different^steps^of^ the^hybridization^ workflow^ can^ be^monitored^ by^ the^workflow.^ Altered^ delta^ G^ values^ can^ be^ accomplished^ by^ selecting^the^base^composition^of^the^sequence,^altering^the^sequence^length^or^the^use^of^modified^ bases^ such^ as^ LNA,^ PNA,^ Methyl^ phosphonates,^ C-5^ propynyl^ derivatives,^ or^ incorporation^ of^ minor-group^binding^dyes. RES-PA18-PCT^ Quantification^of^hybridization^efficiencies^can^be^done^in^many^ways.^The^attached^tail^can^be^ fluorescently^labeled^with^an^orthogonal^fluorophore^(fluorophore^2)^to^quantify^the^amount^of^ tail^ attached^ to^ each^ individual^ particle.^ Comparing^ the^ amount^ of^ fluorescent^ readout^ probe^ (fluorophore^ 1)^ to^ the^ amount^ of^ attached^ tail^ (fluorophore^ 2),^ enables^ quantification^ of^ hybridization^efficiency.^The^orthogonal^fluorophore^can^be^used^to^label^the^particle^directly,^the^ attached^ tail^ or^ the^ decoder.^ Depending^ on^ which^ moiety^ is^ labeled,^ various^ stages^ of^ the^ hybridization^process^can^be^quantified. Slides^coated^with^particles^can^be^used^directly^without^requiring^any^biological^sample.^Such^ control^slides^can^be^used^for: 1. QC^and^release^of kit^reagents 2. Evaluation^of^workflows^and^validation^of^software^and^hardware^updates. 3. Performance^of^instrument^qualification^during^installation^or^instrument^repair. 4. Training^of^customers^new^to^the^in-situ hybridization^workflow. 5. Validating that^a^customer^can^perform^the^protocol^prior^to^running^clinical^samples^in^a^ diagnostic^test. Alternatively,^ customer^ samples^ can^ be^ applied^ directly^ to^ slides^ containing^ attached^ control^ beads.^In^these^cases,^the^control^bead^will: 1. Serve^ as^ an^ internal^ control^ to^monitor^ the^ entire^ workflow^ used^ by^ the^ customer^ in^ running^their^sample. 2. Monitor^ the^ efficiency^ of^ the^ tissue^ preparation^ process^ in^ permeabilizing^ the^ tissue^ sufficiently^to^allow^diffusion^of^the^assay^oligonucleotides^into^and^through^the^tissues. The^present^disclosure^also^describes^the^usage^of^at^least^one set^of^nucleic^acid^sequences^for^ specific^ quantitative^ and/or^ spatial^ detection^ of^ different^ analytes^ in^ parallel^ via^ specific^ hybridization.^The^technology^allows^the^discrimination^of^more^different^analytes^than^different^ detection^signals^are^available.^The^discrimination^may^be^realized^via^sequential^signal-coding^of^ the^analytes^achieved^by^several^cycles^of^specific^hybridization,^detection^of^signals^and^selective^ elution^of^the^hybridized^nucleic^acid^sequences. In^contrast^to^other^state-of-the-art^methods,^the^oligonucleotides^providing^the^detectable^signal^ are^not^directly^interacting^with^sample-specific^nucleic^acid^sequences^but^are^mediated^by^so^ RES-PA18-PCT^ called^ “decoding-oligonucleotides”.^ This^ mechanism^ decouples^ the^ dependency^ between^ the^ analyte-specific^ oligonucleotides^ and^ the^ signal^ oligonucleotides.^ The^ use^ of^ decoding- oligonucleotides^ allows^ a^much^higher^ flexibility^while^dramatically^decreasing^ the^number^of^ different^signal^oligonucleotides^needed^which^in^turn^increases^the^coding^capacity^achieved^with^ a^certain^number^of^detection^rounds. Furthermore,^ in^ contrast^ to^ other^ state-of-the-art^ methods^ using^ already^ “decoding- oligonucleotides”,^ at^ least^ one,^ preferably^ at^ least^ two different^ sets^ of^ “decoding- oligonucleotides”^are^used^directed^to^each^of^the^at^least^one,^preferably^at^least^two^different^ analytic^sets,^in^order^to^allow^the^detection^of^different^subgroups^of^targets^within^one^analytical^ run and^that^any^spatial^resolving^limitation^no^longer^applies.^ In^one^embodiment,^the^analytical^rounds^can^be^arranged^consecutively,^meaning^the^detection^ round(s)^of^a^first^analytical^set^is^finished^before^the^detection^round(s)^of^a^second^analytical^set^ start.^ In^a^second^embodiment^of^ the^method^of^ the^present^ invention,^ the^analytical^ rounds^can^be^ arranged^ interleaved,^ meaning^ the^ detection^ rounds^ of^ a^ first^ analytical^ set^ and^ a^ second^ analytical^set^alternate^in^a^certain^pattern,^e.g.: ^ Detection^round^1^=^1st^round^of^detection^of^first^analytical^set ^ Detection^round^2^=^1st^round^of^detection^of^second^analytical^set ^ Detection^round^3^=^2nd^round^of^detection^of^first^analytical^set ^ Detection^round^4^=^2nd^round^of^detection^of^second^analytical^set ^ Etc. The^utilization^of^decoding-oligonucleotides^leads^to^a^sequential^signal-coding^technology^that^is^ more^ flexible,^ cheaper,^simpler,^ faster^and/or^more^accurate^ than^other^methods and^allows^a^ resolution^beyond^the^diffraction^limit^of^optical^microscopes. DEFINITIONS According^ to^ the^ present^ disclosure^ an^ “slide”^ is^ any^ surface^ which^ can^ be^ used^ for^ in-situ^ hybridization.^In^some^embodiments^the^slide^is^a^microscope^slide,^in^some^embodiments^made^ out^of^glass. According^to^the^present^disclosure^an^“analytical^set”^is^a^set^of^probes^specific^for^a^subgroup^of^ targets.^Preferably^the^targets^within^a^first^analytical^set^directed^to^a^subgroup^of^targets^which^ RES-PA18-PCT^ do^not^usually^show^a^high^spatial^overlap,^whereas^a^second^analytical^set^directed^to^a^subgroup^ of^ targets^ which^ may^ spatially^ overlap^ with^ the^ first^ subgroup^ and/or^ may^ add^ additional^ information^which^allows^a^further^differentiation^of^the^signals^found^with^the^first^analytical^set.^ For^example,^a^first^analytical^set^may^be^directed^to^known^activating^mutations^in^promotor- structures^with^a^high^prevalence^for^cancer.^The^second^analytical^set^may^be^directed^to^genes^ associated^ with^ cancer^ development^ when^ over-expressed.^ Thus,^ a^ colocalization^ of^ signals^ detected^with^the^first^and^the^second^analytical^set^may^indicate^activated^promotors^in^genes^ associated^with^cancer.^Of^course^also^other^combinations^can^be^envisaged,^for^example^tissue- type-specific^probes^with^cancer^markers,^proto-oncogenic^targets^in^different^combinations,^etc.. According^to^the^present^disclosure^an^"analyte"^is^the^subject^to^be^specifically^detected^as^being^ present^or^absent^in^a^sample^and,^in^case^of^its^presence,^to^encode^it.^It^can^be^any^kind of^entity,^ including^a^protein,^polypeptide,^protein, or^a^nucleic^acid^molecule^(e.g.^RNA,^PNA^or^DNA)^of^ interest.^The^analyte^provides^at^least^one^site^for^specific^binding^with^analyte-specific^probes.^ Sometimes^ herein^ the^ term^ "analyte"^ is^ replaced^ by^ "target".^ An^ "analyte"^ according^ to^ the^ disclosure^ incudes^ a^ complex^ of^ subjects,^ e.g., at^ least^ one,^ preferably^ at^ least^ two^ individual^ nucleic^ acid,^ protein^ or^ peptides^molecules.^ In^ an^ embodiment^ of^ the^ disclosure^ an^ "analyte"^ excludes^a^chromosome.^In^another^embodiment^of^the^disclosure an^"analyte"^excludes^DNA. In^some^embodiments,^an^analyte^may^be^a^"coding^sequence",^“encoding^sequence”,^"structural^ nucleotide^sequence"^or^"structural^nucleic^acid^molecule"^which^refers^to^a^nucleotide^sequence^ that^ is^ translated^ into^ a^ polypeptide,^ usually^ via^ mRNA,^ when^ placed^ under^ the^ control^ of^ appropriate^regulatory^sequences.^The boundaries^of^the^coding^sequence^are^determined^by^a^ translation^start^codon^at^the^5'-terminus^and^a^translation^stop^codon^at^the^3'-terminus.^A^coding^ sequence^can^include,^but^is^not^limited^to,^genomic^DNA,^cDNA,^EST^and^recombinant^nucleotide^ sequences. A^"sample"^as^referred^to^herein^is^a^composition^in^liquid^or^solid^form^suspected^of^comprising^ the^analytes^to^be^encoded. In^particular,^the^sample^is^a^biological^sample,^preferably^comprising^ biological^ tissue,^ further^preferably^ comprising^biological^ cells^ and/or^ extracts^ and/or^part^ of^ cells. For^example,^thee^cell^is^a^prokaryotic^cells^or^a^eukaryotic^cell,^in^particular^a^mammalian^ cell,^ in^ particular^ a^ human^ cell.^ In^ some^ embodiments,^ the^ biological^ tissue,^ biological^ cells,^ extracts^ and/or^part^ of^ cells^ are^ fixed.^ In^particular,^ the^ analytes^ are^ fixed^ in^ a^permeabilized^ sample,^such^as^a^cell-containing^sample. RES-PA18-PCT^ As^used^in^the^present^disclosure,^"cell",^"cell^line",^and^"cell^culture"^can^be^used^interchangeably^ and^all^such^designations include^progeny.^Thus,^the^words^"transformants"^or^"transformed^cells"^ include^the^primary^subject^cell^and^cultures^derived^therefrom^without^regard^for^the^number^of^ transfers.^It^is^also^understood^that^all^progeny^may^not^be^precisely^identical^in^DNA^content,^due^ to^ deliberate^ or^ inadvertent^ mutations.^ Mutant^ progeny^ that^ have^ the^ same^ functionality^ as^ screened^for^in^the^originally^transformed^cell^are^included.^ An^“encoding^scheme”^may^describe^a^set^of^code^words^that^are^associated^with^the^analytes^to^ be^detected.^Each^code^word^refers^to^one^of^the^analytes^and^can^be^distinguished^from^all^other^ code^words.^A^code^word^hereby^is^a^sequence^of^signs^provided^by^the^detection^cycles^of^the^ method.^A^sign^within^a^code^word^is^a^detectable^signal^or^the^absence^of^a^signal.^A^code^word^ does^not^need^to^comprise^of^all^different^signals^used^in^the^method.^The^number^of^signs^in^a^ code^word^is^defined^by^the^number^of^detection^cycles. An^"oligonucleotide"^as^used^herein,^refers^to^s^short^nucleic^acid^molecule,^such^as^DNA,^PNA,^ LNA^ or^ RNA.^ The^ length^ of^ the^ oligonucleotides^ is^ within^ the^ range^ 4-200^ nucleotides^ (nt),^ preferably^6-80^nt,^more^preferably^8-60^nt,^more^preferably^10-50^nt,^more^preferably^12^to^35^ depending^on^the^number^of^consecutive^sequence^elements.^The^nucleic^acid^molecule^can^be^ fully^or^partially^single-stranded.^The^oligonucleotides^may^be^linear^or^may^comprise^hairpin^or^ loop^ structures.^ The^ oligonucleotides^ may^ comprise^ modifications^ such^ as^ biotin,^ labeling^ moieties,^blocking^moieties,^or^other^modifications. The^"analyte-specific^probe"^consists^of^at^least^one,^preferably^at^least^two^elements,^namely^the^ so-called^binding^element^(S)^which^specifically^interacts^with^one^of^the^analytes,^and^a^so-called^ identifier^element^(T)^comprising^the^'unique^identifier^sequence'.^The^binding^element^(S)^may^ be^a^nucleic^acid^such^as^a^hybridization^sequence^or^an^aptamer,^or^a^peptidic^structure^such^as^ an^antibody. The^"monitoring^probe"^comprises^at^least^one^element,^wherein^this^element^may^be^ - a^translator^(c)^element^according^to^the^present^disclosure,^wherein^the^translator^(c)^ comprises a^ nucleotide^ sequence^ allowing^ the^ binding/hybridization^ of^ a^ signal^ oligonucleotide;^ - an^ identifier^ element^ (t)^ comprising^ a^ nucleotide^ sequence^ allowing^ the^ binding/hybridization^of^a^decoding^element; RES-PA18-PCT^ - a^target^sequence^allowing^the^binding/hybridization^of^a^binding^element^according^to^ the^present^disclosure. In^ particular,^ the^ "monitoring^ probe"^ consists^ of^ at^ least^ one^ element,^ namely^ the^ so-called^ identifier^ element^ (T)^ comprising^ the^ 'unique^ identifier^ sequence'.^ However,^ in^ further^ embodiments^ the^ monitoring^ probe^ is^ attached^ to^ a^ surface,^ selected^ from^ a^ particle^ (e.g.,^ a^ magnetic^ or^ nonmagnetic^ bead),^ a^ functional^ group^ (e.g., streptavidin,^ biotin,^ a^ hybridization^ sequence^or^an^aptamer,^or^a^peptidic^structure^such^as^an^antibody)^and^a^surface^(e.g.,^a^slide),^ as^well^ as^ any^ combination^ thereof.^Thus,^ in^ one^ embodiment^ the^monitoring^probe^may^also^ comprise^a^so-called^binding^element^(S)^which^specifically^interacts^with^a^target^sequence. In^particular,^in^some^embodiments^the^binding^element^(S)^comprises^moieties^which^are^affinity^ moieties^from^affinity^substances^or^affinity^substances^in^their^entirety^selected^from^the^group^ consisting^ of^ antibodies,^ antibody^ fragments,^ receptor^ ligands,^ enzyme^ substrates,^ lectins,^ cytokines,^ lymphokines,^ interleukins,^ angiogenic^ or^ virulence^ factors,^ allergens,^ peptidic^ allergens,^ recombinant^ allergens,^ allergen-idiotypical^ antibodies,^ autoimmune-provoking^ structures,^ tissue-rejection-inducing^ structures,^ immunoglobulin^ constant^ regions^ and^ their^ derivatives,^ mutants^ or^ combinations^ thereof.^ In^ further^ advantageous^ embodiments,^ the^ antibody^fragment^is^a^Fab,^an^scFv;^a^single^domain,^or^a^fragment^thereof,^a^bis^scFv,^Fab2,^Fab3,^ minibody,^maxibody,^diabody,^triabody,^tetrabody^or^tandab,^in^particular a^single-chain^variable^ fragment^(scFv). The^"unique^identifier^sequence"^as^comprised^by^the^analyte-specific^probe^and^the^monitoring^ probe^ is^unique^ in^ its^sequence^compared^to^other^unique^ identifiers.^ "Unique"^ in^this^context^ means^that^it^specifically^identifies^only^one^analyte,^such^as^Cyclin^A,^Cyclin^D,^Cyclin^E^etc.,^or,^ alternatively,^it^specifically^identifies^only^a^group^of^analytes,^independently^whether^the^group^ of^analytes^comprises^a^gene^family^or^not.^Therefore,^the^analyte^or^a^group^of^analytes^to^be^ encoded^ by^ this^ unique^ identifier^ can^ be^ distinguished^ from^ all^ other^ analytes^ or^ groups^ of^ analytes^that^are^to^be^encoded^based^on^the^unique^identifier^sequence^of^the^identifier^element^ (T).^ Or,^ in^ other^ words,^ there^ is^ only^ one^ 'unique^ identifier^ sequence'^ for^ a^ particular^ target^ sequence,^analyte^or^a^group^of^analytes,^but^not^more^ than^one,^ i.e., not^even^two.^Due^ to^ the^ uniqueness^of^the^unique^identifier^sequence^the^identifier^element^(T)^hybridizes^to^exactly^one^ type^of^decoding^oligonucleotides.^The^length^of^the^unique^identifier^sequence^is^within^the^range^ 8-60^nt,^ preferably^ 12-40^nt,^more^preferably^14-20^nt,^ depending^on^ the^number^of^ analytes^ encoded^in^parallel^and^the^stability^of^interaction^needed.^A^unique^identifier^may^be^a^sequence^ RES-PA18-PCT^ element^of^ the^analyte-specific^probe,^attached^directly^or^by^a^ linker,^a^covalent^bond^or^high^ affinity^binding^modes,^e.g., antibody-antigen^interaction,^streptavidin-biotin^interaction^etc.^It^is^ understood that^the^term^"analyte^specific^probe"^includes^a^plurality^of^probes^which^may^differ^ in^their^binding^elements^(S)^in^a^way^that^each^probe^binds^to^the^same^analyte^but^possibly^to^ different^parts^thereof,^for^instance^to^different^(e.g.^neighboring)^or^overlapping^sections^of^the^ nucleotide^sequence^comprised^by^the^nucleic^acid^molecule^to^be^encoded.^However,^each^of^the^ plurality^of^the^probes^comprises^the^same identifier^element^(T). It^is^understood that^the^term^"^monitoring^probe^"^includes^a^plurality^of^probes^which^may^differ^ in^their^identifier^element^(T)^in^a^way^that^each^probe^binds^to^the^same^decoder^element^and/or^ signal^ oligonucleotide,^ but^ possibly^ to^ different^ parts^ thereof,^ for^ instance^ to^ different^ (e.g.^ neighbouring)^or^overlapping^sections^of^the^nucleotide^sequence^comprised^by^the^nucleic^acid^ molecule^to^be^encoded.^However,^in^at^least^one^embodiment,^each^of^the^plurality^of^the^probes comprises^the^same^binding^element^(S). A^“bipartite^labeling^probe^“^comprise^a^binding^sequence^capable^of^hybridizing^the^analyte^and^ a^binding^probe^sequence^capable^of^binding^a^detectable^signal^molecule like^a^fluorophore^or^a^ nucleic^acid^sequence^comprising^a^fluorophore. A^ "decoding^ oligonucleotide"^ or^ an^ “adapter”^ or^ a^ /adapter^ segment”^ consists^ of^ at^ least^ one,^ preferably^at^least^two^sequence^elements.^One^sequence^element^that^can^specifically^bind^to^a^ unique^identifier^sequence,^referred^to^as an^“identifier^connector^element^“(t)^or "first^connector^ element"^ (t),^ and^ a^ second^ sequence^ element^ specifically^ binding^ to^ a^ signal^ oligonucleotide,^ referred^to^as^"translator^element"^(c).^The^length^of^the^sequence^elements^is^within^the^range^8- 60^nt,^preferably^12-40^nt,^more^preferably^14-20^nt,^depending^on^the^number^of^analytes^to^be^ encoded^ in^ parallel,^ the^ stability^ of^ interaction^ needed, and^ the^ number^ of^ different^ signal^ oligonucleotides^used.^The^length^of^the^two^sequence^elements^may^or^may^not^be^the^same. In^some^advantageous embodiments,^the^decoding^oligonucleotide^in^the^kits^and/or^methods^of^ the^present^disclosure^may^be^a^“multi-decoder”.^A^“multi-decoder”^is^a^decoding^oligonucleotide^ that^consists^of^at^least^three^sequence^elements.^One^sequence^element^(the^identifier^connector^ element^(t))^can^specifically^bind^to^a^unique^identifier^sequence^(identifier^element^(T))^and^at^ least^one,^preferably^at^least^two other^sequence^elements^(translator^elements^(c))^specifically^ bind^different^signal^oligonucleotides^(each^of these^sequence^elements^specifically^binds^a^signal^ oligonucleotide^that^differs^to^all^other^signal^oligonucleotides^recruited^by^other^elements^of^the^ RES-PA18-PCT^ multi-decoder).^The^length^of^the^sequence^elements^is^within^the^range^8-60^nt,^preferably^12-40^ nt,^ more^ preferably^ 14-20^ nt,^ depending^ on^ the^ number^ of^ analytes^ detected^ in^ parallel,^ the^ stability^ needed^ and^ the^ number^ of^ different^ signal^ oligonucleotides^ used.^ The^ length^ of^ the^ sequence^elements^may^or^may^not^be^the^same. Therefore,^in^some^advantageous^embodiments,^the^decoding^oligonucleotide^is^a^multi-decoder^ comprising^ - an^identifier^connector^element^(t)^comprising^a^nucleotide^sequence^which^is^essentially^ complementary^ to^ at^ least^ a^ section^of^ the^unique^ identifier^ sequence^of^ the^ identifier^ element^(T)^of^the^corresponding^analyte-specific^probe^set,^and - at^least^one,^preferably^at^least^two translator^elements^(c)^comprising^each^a^nucleotide^ sequence^allowing^a^specific^hybridization^of^a^different^signal^oligonucleotide. - Therefore,^ the^ first^ translator^ element^ binds^ a^ different^ signal^ oligonucleotide^ as^ the^ second^ translator^ element.^ In^ particular,^ the^ signal^ oligonucleotides^ differ^ in^ the^ signal^ element^ comprised^in^the^signal^oligonucleotide,^e.g.^in^the^kind^of^the^fluorophore. A^"signal^oligonucleotide"^or^a^“reporter”^as^used^herein^comprises^at^least^one,^preferably^at^least^ two elements,^a^so-called^“translator^connector^element” (C)^or^"second^connector^element"^(C)^ having^ a^ nucleotide^ sequence^ specifically^ hybridizable^ to^ at^ least^ a^ section^ of^ the^ nucleotide^ sequence^of^ the^ translator^element^(c)^of^ the^decoding^oligonucleotide,^and^a^"signal^element"^ which^provides^a^detectable^signal.^This^element^can^either^actively^generate^a^detectable^signal^ or^provide^such^a^signal^via^manipulation,^e.g.^fluorescent^excitation.^Typical^signal^elements^are,^ for^example,^enzymes^that^catalyze^a^detectable^reaction,^fluorophores,^radioactive^elements^or^ dyes.^ A^"set"^refers^to^a^plurality^of^moieties^or^subjects,^e.g., analyte-specific^probes,^monitoring^probes, or^decoding^oligonucleotides,^whether^the^individual^members^of^said^plurality^are^identical or^ different^from^each^other.^In^an^analyte^specific^probe^set,^the^analyte^specific^probes^are^identical^ in^ the^ identifier^ element^ (T)^but^may^ comprise^ a^different^binding^ element^ (S)^ for^ specifically^ interacting^with^the^same^analyte^but^for^specifically^interacting^with^different^sub-structures^of^ the^same^analyte^to^be^encoded. In^a^monitoring^probe^set,^the^monitoring^probes^are^identical^in^ the^ binding^ element^ (S)^ but^ may^ comprise^ a^ different^ identifier^ element^ (T)^ for^ specifically^ interacting^with^the^surface^on^which^they^are^immobilized,^but^for^specifically^interacting^with^ different^decoding^elements.^In^some^embodiments,^the^set^may^further^comprise^elements,^such^ RES-PA18-PCT^ as^a^decoy,^which^may^have^a^similar^structure^as^the^monitoring^probe^but^may^not^comprise^the^ same^identifier^element^(T)^in^order^to^improve^the^signal-to-noise^ratio. “Selective^denaturation” may^be^the^process^of^eliminating^bound^decoding^oligonucleotides^and^ signal^oligonucleotides^with^highest^efficiency^while^at^the^same^time^the^target^specific^probes^ have^ to^stay^hybridized^with^ the^highest^efficiency.^The^ total^efficiency^of^ these^ two^combined^ events^may^to^be^at^least^0.22^for^two^detection^cycles,^0.37^for^three^detection^cycles,^0.47^for^ four^detection^cycles,^0.55^for^five^detection^cycles,^0.61^for^six^detection^cycles,^0.65^for^seven^ detection^cycles,^0.69^ for^eight^detection^cycles,^ 0.72^ for^nine^detection^cycles^and^0.74^ for^10^ detection^cycles,^0.76^for^11^detection^cycles^and^0.78^for^12^detection^cycles. In^an^embodiment^of^the^disclosure a^single^set^refers^to^a^plurality^of^oligonucleotides. An^"analyte^specific^probe^set" refers^to^a^plurality^of^moieties^or^sub-jects,^e.g., analyte-specific^ probes^that^are^different^from^each^other^and^bind^to^independent^regions^of^the^analyte.^A^single^ analyte^specific^probe^set^is^further^characterized^by^the^same^unique^identifier. A "decoding^oligonucleotide^set"^refers^to^a^plurality^of^decoding^oligonucleotides^specific^for^a^ certain^unique^ identifier^needed^to^realize^ the^encoding^independent^of^the^ length^of^ the^code^ word.^Each^and^all^of^the^decoding^oligonucleotides^included^in a^"decoding^oligonucleotide^set"^ bind^to^the^same^unique^identifier^element^(T)^of^the^analyte-specific^probe. In^certain^embodiments,^this^pattern^of^binding^or^hybridization^of^the^decoding^oligonucleotides^ may^be^converted^into^a^"codeword."^For^example,^the^codewords^could^be^also^"101"^and^"110"^ for^an^analyte,^where^a^value^of^1^represents^binding^and^a^value of^0^represents^no^binding.^The^ codewords^may^also^have^longer^lengths^in^other^embodiments^(see^Fig.^13).^A^codeword^can^be^ directly^related^to^a^specific^unique^identifier^sequence^of^a^analyte-specific^probe.^Accordingly,^ different^analyte-specific^probemay^match^certain^codewords,^which^can^then^be^used^to^identify^ the^different^analytes of^the^analyte-specific^probe^based^on^the^binding^patterns^of^the^decoding^ oligonucleotide.^However,^ if^ no^binding^ is^ evident,^ then^ the^ codeword^would^be^ "000"^ in^ this^ example. The^values^in^each^codeword^can^also^be^assigned^in^different^fashions^in^some^embodiments.^For^ example,^a^value^of^0^could^represent^binding^while^a^value^of^1^represents^no^binding.^Similarly,^ a^value^of^1^could^represent^binding^of^a^secondary^nucleic^acid^probe^with^one^type^of^signaling^ RES-PA18-PCT^ entity^while^a^value^of^0^could^represent^binding^of^a^secondary^nucleic^acid^probe^with^another^ type^ of^ distinguishable^ signaling^ entity.^ These^ signaling^ entities^ could^ be^ distinguished,^ for^ example,^ via^different^ colors^of^ fluorescence.^ In^ some^ cases,^ values^ in^ codewords^need^not^be^ confined^to^0^and^1.^The^values^could^also^be^drawn^from^larger^alphabets,^such^as^ternary^(e.g.,^0,^ 1,^and^2)^or^quaternary^(e.g.,^0,^1,^2,^and^3)^systems.^Each^different^value^could,^for^example,^be^ represented^by^a^different^distinguishable^signaling^entity,^ including^(in^some^cases)^one^value^ that^may^be^represented^by^the^absence^of^signal. The^codewords^for^each^analyte may^be^assigned^sequentially,^or^may^be^assigned^at^random.^For^ instance,^a^first^analytemay^be^assigned^to^101,^while^a^second^nucleic^acid^target^may^be^assigned^ to^ 110.^ In^ addition,^ in^ some^ embodiments,^ the^ codewords^ may^ be^ assigned^ using^ an^ error- detection^system^or^an^error- correcting^system,^such^as a^Hamming^system,^a^Golay^code,^or^an^ extended^ Hamming^ system^ (or^ a^ SECDED^ system,^ i.e.,^ single^ error^ correction,^ double^ error^ detection).^ Generally^speaking,^such^systems^can^be^used^to^identify^where^errors^have^occurred,^and^in^some^ cases,^ such^ systems^ can^ also^ be^ used^ to^ correct^ the^ errors^ and^ determine^ what^ the^ correct^ codeword^should^have^been.^For^example,^a^codeword^such^as^001^may^be^detected^as^invalid^and^ corrected^using^such^a^system^to^101,^e.g.,^if^001^is^not^previously^assigned^to^a^different^target^ sequence.^ A^ variety^ of^ different^ error-correcting^ codes^ can^ be^ used,^ many^ of^ which^ have^ previously^been^developed^for^use^within^the^computer^industry;^however,^such^error-correcting^ systems^ have^ not^ typically^ been^ used^within^ biological^ systems.^ Additional^ examples^ of^ such^ error-correcting^codes^are^discussed^in^more^detail^below. "Essentially^ complementary"^ means,^ when^ referring^ to^ two^ nucleotide^ sequences,^ that^ both^ sequences^can^specifically^hybridize^to^each^other^under^stringent^conditions,^thereby^forming^a^ hybrid^nucleic^acid^molecule^with^a^sense^and^an^antisense^strand^connected^to^each^other^via^ hydrogen^bonds^(Watson-and-Crick^base^pairs).^"Essentially^complementary"^includes^not^only^ perfect^ base-pairing^ along^ the^ entire^ strands,^ i.e., perfect^ complementary^ sequences^ but^ also^ imperfect^complementary^sequences^which,^however,^still^have^the^capability^to^hybridize^to^each^ other^ under^ stringent^ conditions.^ Among^ experts^ it^ is^ well^ accepted^ that^ an^ "essentially^ complementary"^ sequence has^ at^ least^ 88%^ sequence^ identity^ to^ a^ fully^ or^ perfectly^ complementary^sequence. RES-PA18-PCT^ "Percent^sequence^identity"^or^"percent^identity"^in^turn^means^that^a^sequence^is^compared^to^a^ claimed^or^described^sequence^after^alignment^of^the^sequence^to^be^compared^(the^"Compared^ Sequence")^with^ the^ described^ or^ claimed^ sequence^ (the^ "Reference^ Sequence").^ The^ percent^ identity^is^then^determined^according^to^the^following^formula:^percent^identity^=^100^[1^-(C/R)] wherein^ C^ is^ the^ number^ of^ differences^ between^ the Reference^ Sequence^ and^ the^ Compared^ Sequence^ over^ the^ length^ of^ alignment^ between^ the^ Reference^ Sequence^ and^ the^ Compared^ Sequence,^wherein^ (i)^each^base^or^amino^acid^in^the^Reference^Sequence^that^does^not^have^a^corresponding^ aligned^base^or^amino^acid^in^the^Compared^Sequence^and (ii)^each^gap^in^the^Reference^Sequence^and (iii)^each^aligned^base^or^amino^acid^in^the^Reference^Sequence^that^is^different^from^an^ aligned^base^or^amino^acid^in^the^Compared^Sequence,^constitutes^a^difference^and^(iiii)^ the^alignment^has^to^start^at^position^1^of^the^aligned^sequences; and^R^is^the^number^of^bases^or^amino^acids^in^the^Reference^Sequence^over^the^length^of^ the^ alignment^ with^ the^ Compared^ Sequence^ with^ any^ gap^ created^ in^ the^ Reference^ Sequence^also^being^counted^as^a^base^or^amino^acid. If^an^alignment^exists^between^the^Compared^Sequence^and^the^Reference^Sequence^for^which^the^ percent^identity^as^calculated^above^is^about^equal^to^or^greater^than^a^specified^minimum^Percent^ Identity^ then^ the^ Compared^ Sequence^ has^ the^ specified^ minimum^ percent^ identity^ to^ the^ Reference^ Sequence^ even^ though^ alignments^may^ exist^ in^ which^ the^ herein^ above^ calculated^ percent^identity^is^less^than^the^specified^percent^identity. In^the^"incubation"^steps^as^understood^herein^the^respective^moieties^or^subjects^such^as^probes^ or^oligonucleotide,^are^brought^into^contact^with^each^other^under^conditions^well^known^to^the^ skilled^ person^ allowing^ a^ specific^ binding^ or^ hybridization^ reaction,^ e.g.^ pH,^ temperature,^ salt^ conditions etc.^Such^steps^may^therefore,^be^preferably^carried^out^in^a^liquid^environment^such^ as^a^buffer^system^which^is^well^known^in^the^art.^ The^"removing"^steps^according^to^the^disclosure may^include^the^washing^away^of^the^moieties^ or^subjects^to^be^removed^such^as^the^probes^or^oligonucleotides^by^certain^conditions,^e.g.^pH,^ temperature,^salt^conditions^etc.,^as^known^in^the^art. It^ is^ understood^ that^ in^ an^ embodiment^ of^ the^method^ according^ to^ the^ present^ disclosure a^ plurality^of^analytes^can^be^encoded^in^parallel.^This^requires^the^use^of^different^sets^of^analyte- RES-PA18-PCT^ specific^probes^in^step^(1).^The^analyte-specific^probes^of^a^particular^set^differ^from^the^analyte- specific^probes^of^another^set.^This^means^that^the^analyte-specific^probes^of^set^1^bind^to^analyte^ 1,^the^analyte-specific^probes^of^set^2^binds to^analyte^2,^the^analyte-specific^probes^of^set^3^binds to^analyte^3,^etc.^In^this^embodiment^also^the^use^of^different^sets^of^decoding^oligonucleotides^is^ required^in^the^methods^according^to^the^present^disclosure. The^decoding^oligonucleotides^of^ a^particular^ set^ differ^ from^ the^decoding^oligonucleotides^of^ another^set.^This^means,^the^decoding^oligonucleotides^of^set^1^bind^to^the^analyte-specific^probes^ of^ above^ set^ 1^ of^ analyte-specific^ probes,^ the^ decoding^ oligonucleotides^ of^ set^ 2^ binds^ to^ the^ analyte-specific^probes^of^above^set^2^of^analyte-specific^probes,^the^decoding^oligonucleotides^of^ set^3^bind^to^the^analyte-specific^probes^of^above^set^3^of^analyte-specific^probes,^etc.^ It^is^also^understood^that^in^an^embodiment^of^the^method^according^to^the^present^disclosure^a^ plurality^of^monitoring^probes^can^be^encoded^in^parallel.^This^requires^the^use^of^different^sets^ of^ monitoring^ probes^ in^ step^ (2).^ The^ monitoring^ probes^ of^ a^ particular^ set^ differ^ from^ the monitoring^probes^of^another^set.^This^means^that^the^monitoring^probes^of^set^1^bind^to^decoding^ element^1,^the^monitoring^probes^of^set^2^binds^to^decoding^element^2,^the^monitoring^probes^of^ set^3^binds^to^decoding^element^3,^etc.^In^this^embodiment^also^the^use^of^different^sets^of^decoding^ oligonucleotides^is^required^in^the^methods^according^to^the^present^disclosure. The^decoding^oligonucleotides^of^ a^particular^ set^ differ^ from^ the^decoding^oligonucleotides^of^ another^set.^This^means,^the^decoding^oligonucleotides^of^set^1^bind^to^the^monitoring^probes^of^ above^set^1^of^monitoring^probes,^the^decoding^oligonucleotides^of^set^2^binds^to^the^monitoring^ probes^of^above^set^2^of^monitoring^probes,^ the^decoding^oligonucleotides^of^set^3^bind^to^ the^ monitoring^probes^of^above^set^3^of^monitoring^probes,^etc.^ In^this^embodiment, where^a^plurality^of^analytes^is^to^be^encoded^in^parallel, the^different^sets^of^ analyte-specific^probes^and/or^monitoring^probes^may^be^provided^as^a^premixture^of^different^ sets^of^analyte-specific^probes^and/or^monitoring^probes;^and/or^the^different^sets^of^decoding^ oligonucleotides^may^be^provided^as^a^premixture^of^different^sets^of^decoding^oligonucleotides.^ Each^mixture^may^be^contained^in^a^single^vial.^Alternatively,^the different^sets^of^analyte-specific^ probes^and/or^monitoring^probes;^and/or^the^different^sets^of^decoding^oligonucleotides^may^be^ provided^in^steps^singularly.^ RES-PA18-PCT^ A^"kit"^is^a^combination^of^individual^elements^useful^for^carrying^out^the^use^and/or^method of^ the^disclosure,^wherein^the^elements^are^optimized^for^use^together^in^the^methods.^The^kits^may^ also^contain^additional^reagents,^chemicals,^buffers,^reaction^vials^etc.^which^may^be^useful^for^ carrying^ out^ the^ method^ according^ to^ the^ disclosure.^ Such kits^ unify^ all^ essential^ elements^ required^ to^work^ the^method^ according^ to^ the^ disclosure,^ thus^minimizing^ the^ risk^ of^ errors.^ Therefore,^such^kits^also^allow^semi-skilled^laboratory^staff^to^perform^the^method^according^to^ the^present^disclosure. The^term^"quencher"^or^"quencher^dye"^or^"quencher^molecule"^refers^to^a^dye^or^an^equivalent^ molecule,^ such^ as^ nucleoside^ guanosine^ (G)^ or^ 2'-deoxyguanosine^ (dG),^ which^ is^ capable^ of^ reducing^the^fluorescence^of^a^fluorescent^reporter^dye^or^donor^dye.^A^quencher^dye^may^be^a^ fluorescent^dye^or^non-fluorescent^dye.^When^the^quencher^is^a^fluorescent^dye,^its^fluorescence^ wavelength^ is^ typically^ substantially^different^ from^ that^ of^ the^ reporter^dye^ and^the^quencher^ fluorescence^ is^ usually^ not^ monitored^ during^ an^ assay.^ Some^ embodiments^ of^ the^ present^ disclosure disclose^ signal^ oligonucleotides^ comprising^ a^ quencher^ and/or^ a^ quencher^ in^ combination^with^a^signal^element (see^Fig.^14),^and^therefore^the^signal^oligonucleotides^is^not^ detectable^during^imaging.^ In^ an^ embodiment^ of^ the^ disclosure the^ sample^ is^ a^ biological^ sample,^ preferably^ comprising^ biological^ tissue,^ further^ preferably^ comprising^ biological^ cells.^ A^ biological^ sample^ may^ be^ derived^from^an^organ,^organoids,^cell^cultures,^stem^cells,^cell^suspensions,^primary^cells,^samples^ infected^by^viruses,^bacteria^or^fungi,^eukaryotic^or^prokaryotic^samples,^smears,^disease^samples,^ a^tissue^section. The^method^is^particularly^qualified^to^monitor,^encode,^identify,^detect,^count, or^quantify^the^in^ situ^analysis^of^analytes^or^single^analyte^molecules^in^a^biological^sample,^i.e., such^as^a^sample^ which^ contains^ nucleic^ acids^ or^ proteins^ as^ said^ analytes.^ It^ is^ understood^ that^ the^ biological^ sample^may^be^in^a^form^as^it^is^in^its^natural^environment^(i.e., liquid,^semi-liquid,^solid^etc.),^or^ processed,^e.g., as^a^dried^ film^on^the^surface^of^a^device^which^may^be^re-liquefied^before^ the^ method^is^carried^out.^ In^another^embodiment^of^the^disclosure prior^to^step^(2)^the^biological^tissue^and/or^biological^ cells^ are^ fixed. For^ example,^ in^ some^embodiments,^ the^ cell^ and/or^ the^ tissue^ is^ fixed^prior^ to^ introducing^the^probes,^e.g.,^to^preserve^the^positions^of^the^analytes^like^nucleic^acids^within^the^ cell.^Techniques^ for^ fixing^cells^are^known^to^ those^of^ordinary^skill^ in^ the^art.^As^non-limiting^ RES-PA18-PCT^ examples,^ a^ cell^ may^ be^ fixed^ using^ chemicals^ such^ as^ formaldehyde,^ paraformaldehyde,^ glutaraldehyde,^ethanol,^methanol,^acetone,^acetic^acid,^or^the^like.^In^one^embodiment,^a^cell^may^ be^fixed^using^Hepes-glutamic^acid^buffer- mediated^organic^solvent^(HOPE). This^measure^has^the^advantage^that^the^analytes^to^be^encoded,^e.g., the^nuclei^acids^or^proteins,^ are^immobilized^and^cannot^escape.^In^doing^so,^the^analytes^then^prepared^for^a^better^detection^ or^encoding^by^the^method^according^to^the^disclosure.^ In^ yet^ a^ further^ embodiment^within^ the^ set^ of^ analyte-specific^ probes^ the^ individual^ analyte- specific^probes^ comprise^binding^ elements^(S1,^ S2,^ S3,^ S4,^ S5)^which^ specifically^ interact^with^ different^sub-structures^of^one^of^the^analytes^to^be^encoded. By^this^measure^the^method^becomes^even^more^robust^and^reliable^because^the^signal^intensity^ obtained^at^the^end^of^the^method^or^a^cycle,^respectively,^is^increased.^It^is^understood that^the^ individual^probes^of^a^set^while^binding^ to^the^same^analyte^differ^ in^their^binding^position^or^ binding^site^at^or^on^the^analyte.^The^binding^elements^S1,^S2,^S3,^S4,^S5^etc.^of^the^first,^second,^ third^fourth,^fifth^etc.^analyte-specific^probes^therefore^bind^to^or^at^a^different^position^which,^ however,^may^or^may^not^overlap. In^ an^ advantageous^ embodiment,^ the^ present^ disclosure^ pertains^ to^ kit^ for^multiplex^ analyte^ encoding,^comprising: (A1) at^least^a^first^set of^analyte-specific^probes^for^encoding^different^analytes,^each^set^ of^analyte-specific^probes^interacting^with^a^different^analyte,^wherein^if^the^analyte^is^a^ nucleic^acid^each^set^of^analyte-specific^probes^comprises^analyte-specific^probes^which^ specifically^ interact^ with^ different^ sub-structures^ of^ the^ same^ analyte,^ each^ analyte- specific^probe^comprising^ (aa)^ a^ binding^ element^ (S)^ that^ specifically^ interacts^ with^ one^ of^ the^ different^ analytes^to^be^encoded,^and (bb)^an^identifier^element^(T)^comprising^a^nucleotide^sequence^which^is^unique^ to^the^analyte^to^be^encoded^(unique^identifier^sequence),^ and RES-PA18-PCT^ (A2) at^least^a^second^set^of^analyte-specific^probes^for^encoding^different^analytes,^each^ set^of^analyte-specific^probes^interacting^with^a^different^analyte,^wherein^if^the^analyte^is^ a^nucleic^acid^each^set^of^analyte-specific^probes^comprises^analyte-specific^probes^which^ specifically^ interact^ with^ different^ sub-structures^ of^ the^ same^ analyte,^ each^ analyte- specific^probe^comprising^ (aa)^ a^ binding^ element^ (S)^ that^ specifically^ interacts^ with^ one^ of^ the^ different^ analytes^to^be^encoded,^and (bb)^an^identifier^element^(T)^comprising^a^nucleotide^sequence^which^is^unique^ to^the^analyte^to^be^encoded^(unique^identifier^sequence),^ wherein^ the^ analyte-specific^probes^of^ a^particular^ set^ of^ analyte-specific^probes^differ^ from^ the^ analyte-specific^ probes^ of^ another^ set^ of^ analyte-specific^ probes^ in^ the^ nucleotide^ sequence^of^the^identifier^element^(T),^and wherein^ the^ analyte-specific^probes^ in^ each^ set^ of^ analyte-specific^probes^binds^ to^ the^ same^analyte^and^comprises^the^same^nucleotide^sequence^of^the^identifier^element^(T)^which^is^ unique^to^said^analyte;^and wherein^(optionally)^the^number^of^probes^and/or^targets^of^first^set^of^analyte-specific^ probes according^to^step^A1 (i.e.^the^transcript^plexity^of^A1)^is^at^least^10^times^higher^ than^ the^number^of^probes^ and/or^ targets^ of^ the^ second^ set^ of^ analyte-specific^probes^ according^to^step^A2^(i.e.^the^transcript^plexity^of^A2);^and (B1) at^least^a^first set^of^decoding^oligonucleotides^per^analyte,^wherein^in^each^set^of^ decoding^ oligonucleotides^ for^ an^ individual^ analyte^ each^ decoding^ oligonucleotide^ comprises: (aa)^an^identifier^connector^element^(t)^comprising^a^nucleotide^sequence^which^ is^essentially^complementary^to^at^least^a^section^of^the^unique^identifier^sequence^ of^the^identifier^element^(T)^of^the^corresponding^analyte-specific^probe^set,^and RES-PA18-PCT^ (bb)^a^translator^element^(c)^comprising^a^nucleotide^sequence^allowing^a^specific^ hybridization^of^a signal^oligonucleotide; and (B2) at^least^a^second^set^of^decoding^oligonucleotides^per^analyte,^wherein^in^each^set^of^ decoding^ oligonucleotides^ for^ an^ individual^ analyte^ each^ decoding^ oligonucleotide^ comprises: (aa)^an^identifier^connector^element^(t)^comprising^a^nucleotide^sequence^which^ is^essentially^complementary^to^at^least^a^section^of^the^unique^identifier^sequence^ of^the^identifier^element^(T)^of^the^corresponding^analyte-specific^probe^set,^and (bb)^a^translator^element^(c)^comprising^a^nucleotide^sequence^allowing^a^specific^ hybridization^of^a^signal^oligonucleotide; wherein^the^decoding^oligonucleotides^of^a^set^for^an^individual^analyte^differ^from^the^ decoding^oligonucleotides^of^another^set^for^a^different^analyte^in^the^identifier^connect^element^ (t);^and (C) a^set^of^signal^oligonucleotides,^each^signal^oligonucleotide^comprising: (aa)^a^translator^connector^element^(C)^comprising^a^nucleotide^sequence^which^is^ essentially^ complementary^ to^ at^ least^ a^ section^of^ the^nucleotide^ sequence^of^ a translator^element^(c)^comprised^in^a^decoding^oligonucleotide,^and (bb)^a^signal^element;^ And further^comprising^a^monitoring^probe^comprising^at^least^an^identifier^element^ (t)^ comprising^ a^nucleotide^ sequence^which^ is^ essentially^ complementary^ to^ at^ least^a^section^of^the^unique^set^identifier^sequence^of^the^identifier^element^(T)^of^ the^corresponding^binding^element. RES-PA18-PCT^ A^ multiplex^ method^ or^ assay^ allow^ the^ simultaneously^ measurement^ of^ multiple^ analytes^ according^ to^ the^present^disclosure it^may^be^used^to^determine^the^presence^or^absence^of^ a^ plurality^of^predetermined^(known)^analytes^like^nucleic^acid^target^sequences^in^a^sample.^An analyte^may^be “predetermined”^in^that^its^sequence^is known^to^design^a^probe^that^binds^to^the that^target. In^some^advantageous embodiments^according^to^the^present^disclosure^at^least^2,^at^least^5,^at^ least^10,^at^least^15,^at^least^20,^in^particular^at^least^25,^in^particular^at^least^30^different^analytes^ are^detected and/or^quantified^in^a^sample^in^parallel. For^example,^there^may^be^at^least^5,^at^ least^10,^at^least^20,^at^least^50,^at^least^75,^at^least^100,^at^least^300,^at^least^1,000,^at^least^3,000,^ at^ least^10,000,^or^ at^ least^30,000^distinguishable^ analyte-specific^probes^ that^are^applied^ to^a^ sample,^e.g.,^simultaneously^or^sequentially. In^the^multiplexing^methods^of^the^present^disclosure,^in^particular^at^least 2^different^subgroups^ of^ analytes^ (e.g., mRNA^molecules)^ i.e., tags^with^ spatially^ overlap^ (i.e.^ a^ distance^ beyond^ the^ diffraction^limit^of^the^respective^microscope)^are^targeted. In^some^advantageous^embodiments,^at^least^4^rounds^to^collect^information for^identification^of^ the^analyte^are^carried^out,^wherein^multiple readout^increases^the^accuracy^of^identification^and^ avoids^ false^ positives.^ The^ unique^ tag^ can^ be^ identified^ by^ various^ techniques,^ including^ hybridization,^ e.g., with^ labelled probes,^ directly^ or^ indirectly^ or^by^ sequencing^ (by^ synthesis,^ ligation).^In^particular,^the^identity^of^the^tag^can^be^encoded^with^one^single^signal^(binary^code),^ two^ or^ more^ signals,^ wherein^ the^ signal^ can^ be^ a^ fluorescent^ label^ (e.g.^ attached^ to^ an^ oligonucleotide). In^ some^ advantageous^ embodiments according^ to^ the^ present^ disclosure, the^ kit^ does^ not^ comprise^sets^of^analyte-specific^probes^as^defined^under^item^A1) and^A2). Preferably,^if^the^analyte^in^the^kits^or^methods^according^to^the^present disclosure^is^a^nucleic^ acid,^each^set^of^analyte-specific^probes^comprises^at^ least^ five^(10)^analyte-specific^probes,^ in^ particular^at^least^fifteen^(15)^analyte-specific^probes,^in^particular^at^least^twenty^(20)^analyte- specific^ probes^ which^ specifically^ interact^ with^ different^ sub-structures^ of^ the^ same^ analyte.^ Nucleic^ acid^ analyte^ includes^ specific^ DNA^ molecules,^ e.g.^ genomic^ DNA,^ nuclear^ DNA,^ RES-PA18-PCT^ mitochondrial^DNA,^viral^DNA,^bacterial^DNA,^extra- or^intracellular^DNA^etc.,^and^specific^mRNA^ molecules, e.g.^hnRNA,^miRNA,^viral^RNA,^bacterial^RNA,^extra- or^intracellular^RNA,^etc.. Preferably,^if^the^analyte^in^the^kits^or^methods^according^to^the^present^disclosure^is^a^peptide,^a^ polypeptide^or^a^protein,^each^set^of^analyte-specific^probes^comprises^at^least^one,^preferably^at^ least^ two (2)^analyte-specific^probes,^ in^particular^at^ least^ three^(3)^analyte-specific^probes,^ in^ particular^at^least^four^(4)^analyte-specific^probes^which^specifically^interact^with^different^sub- structures^of^the^same^analyte.^ In^some^advantageous^embodiments according^to^the^present^disclosure^the^kit^comprises^at^least^ one,^ preferably^ at^ least^ two different^ sets^ of^ signal^ oligonucleotides,^ wherein^ the^ signal^ oligonucleotides^ in^ each^ set^ comprise^ a^ different^ signal^ element^ and^ comprise^ a^ different^ connector^element^(C).^ In^particular,^the^kit^may^comprise^at^least^one,^preferably^at^least^two different^sets^of^decoding^ oligonucleotides^per^analyte, wherein^the^decoding^oligonucleotides^comprised^in^these^different^ sets^comprise^the^same^identifier^connector^element^(t)^comprising^a^nucleotide^sequence^which^ is^essentially^complementary^to^at^least^a^section^of^the^unique^identifier^sequence^of^the^identifier^ element^ (T)^ of^ the^ corresponding^ analyte-specific^ probe^ set,^ and^ wherein^ the^ decoding^ oligonucleotides^of^the^different^sets^per^analyte^differ^in^the^translator^element^(c)^comprising^a^ nucleotide^sequence^allowing^a^specific^hybridization^of^a^signal^oligonucleotide.^ In^ some^ embodiments^ the^ kit^ comprises^ at^ least^ one,^ preferably^ at^ least^ two different^ sets^ of^ decoding^oligonucleotides^per^analyte,^wherein^the^decoding^oligonucleotides^comprised^in^these^ different^ sets^ comprise^ the^ same^ identifier^ connector^ element^ (t)^ comprising^ a^ nucleotide^ sequence^ which^ is^ essentially^ complementary^ to^ at^ least^ a^ section^ of^ the^ unique^ identifier^ sequence^ of^ the^ identifier^ element^ (T)^ of^ the^ corresponding^ analyte-specific^ probe^ set,^ and^ wherein^the^decoding^oligonucleotides^of^the^different^sets^for^at^least^one^analyte^differ^in^the^ translator^element^(c)^comprising^a^nucleotide^sequence^ allowing^a^specific^hybridization^of^a^ signal^oligonucleotide.^ In^some^advantageous^embodiments,^the^number^of^different^sets^of^decoding^oligonucleotides^ per^analyte^comprising^different^translator^elements^(c)^corresponds^to^the^number^of^different^ sets^ of^ signal^ oligonucleotides^ comprising^ different^ connector^ elements^ (C).^ However,^ the^ decoding^ oligonucleotides^ in^ a^ particular^ set^ of^ decoding^ oligonucleotides^may^ interacts^with^ RES-PA18-PCT^ identical^identifier^elements^(T)^which^are^unique^to^a^particular^analyte.^In^particular,^all^sets^of^ decoding^oligonucleotides^for^the^different^analytes^may^comprise^the^same^type(s)^of^translator^ element(s)^(c). In^another^embodiment,^the^present^disclosure is^generally^directed^to^a^methods^including^acts^ of^ exposing^ a^ sample^ to^ a^ plurality^ of^ analyte-specific^ probes;^ for^ each^ of^ the^ analyte-specific^ probes,^determining^binding^of^the^analyte-specific^probes^within^the^sample;^creating^codewords^ based^on^the binding^of^the^analyte-specific^probes,^the^decoding^oligonucleotides^and^the^signal^ oligonucleotides;^ and^ for^ at^ least^ some^ of^ the^ codewords,^ matching^ the^ codeword^ to^ a^ valid^ codeword.^In^certain^embodiments,^this^pattern^of^binding^or^hybridization^of^the^analyte-specific^ probes,^ the^decoding^oligonucleotides^and^the^signal^oligonucleotides^may^be^converted^ into^a^ "codeword."^For^example,^for^instance,^the^codewords^may^be^"101"^and^"110"^for^a^first^analyte^ and^ a^ second^ analyte,^ respectively,^ where^ a^ value^ of^ 1^ represents^ binding^ and^ a^ value^ of^ 0^ represents^no^binding^of^decoding^oligonucleotides^and/or^the^binding^of^signal^oligonucleotides without^and/or^quenched^signal^element. The^analyte^in^the^detection^round/cycle^is^therefore^ not^detectable^during^imaging. To^create^such^a^zero^(0)^in^a^codeword^for^an^individual^analyte^the^kit^may^comprise:^ (D) at^ least^ a^set^ of^non-signal^decoding^oligonucleotides^ for^binding^ to^ a^particular^ identifier^ element^(T)^of^analyte-specific^probes,^wherein^the^decoding^oligonucleotides^in^the^same^set^of^ non-signal^decoding^oligonucleotides^interacting^with^the^same^different^identifier^element^(T), wherein^each^non-signal^decoding^oligonucleotide^comprises^an^identifier^connector^element^(t)^ comprising^a^nucleotide^sequence^which^is^essentially^complementary^to^at^least^a^section^of^a^ unique^ identifier^ sequence,^ and^ does^ not^ comprise^ a^ translator^ element^ (c)^ comprising^ a^ nucleotide^sequence^allowing^a^specific^hybridization^of^a^signal^oligonucleotide.^ To^create^such^a^zero^(0)^in^a^codeword^for^an^individual^analyte^the^kit^may^comprise:^ (D) at^ least^ a^set^ of^non-signal^decoding^oligonucleotides^ for^binding^ to^ a^particular^ identifier^ element^(T)^of^analyte-specific^probes,^wherein^the^decoding^oligonucleotides^in^the^same^set^of^ non-signal^decoding^oligonucleotides^interacting^with^the^same^different^identifier^element^(T), wherein^each^non-signal^decoding^oligonucleotide^comprises^an^identifier^connector^element^(t)^ comprising^a^nucleotide^sequence^which^is^essentially^complementary^to^at^least^a^section^of^a^ unique^identifier^sequence,^and^comprise^a^translator^element^that^does^not^interact/bind^to^a^ signal^oligonucleotide^due^to^an^instabil^binding^sequence^and/or^due^to^the^translator^element^is^ RES-PA18-PCT^ to^ short^ (c)^ comprising^ a^ nucleotide^ sequence^ allowing^ a^ specific^ hybridization^ of^ a^ signal^ oligonucleotide.^ In^some^advantageous^embodiments,^the^kit^comprises:^ (D) at^least^one,^preferably^at^least^two (2)^different^sets^of^non-signal^decoding^oligonucleotides^ for^binding^ to^at^ least^one,^preferably^at^ least^ two different^ identifier^elements^(T)^of^analyte- specific^ probes,^ each^ set^ of^ non-signal^ decoding^ oligonucleotides^ interacting^ with^ a^ different^ identifier^element^(T), wherein^each^non-signal^decoding^oligonucleotide^comprises^an^identifier^connector^element^(t)^ comprising^a^nucleotide^sequence^which^is^essentially^complementary^to^at^least^a^section^of^a^ unique^ identifier^ sequence,^ and^ does^ not^ comprise^ a^ translator^ element^ (c)^ comprising^ a^ nucleotide^sequence^allowing^a^specific^hybridization^of^a^signal^oligonucleotide.^ In^some^advantageous^embodiments,^the^different^sets^of^non-signal^decoding^oligonucleotides^ may^be^comprised^in^a^pre-mixture^of^different^sets^of^non-signal^decoding^oligonucleotides^or^ exist^separately.^ Furthermore,^in^some^advantageous^embodiments^the^kit^may comprise:^ (E) a^set^of^non-signal^oligonucleotides,^each^non-signal^oligonucleotide^comprising: (aa)^a^ translator^connector^element^(C)^comprising^a^nucleotide^sequence^which^ is^essentially^ complementary^to^at^least^a^section^of^the^nucleotide^sequence^of the^translator^element^(c),^and (bb)^a^quencher^(Q),^a^signal^element^and^a^quencher^(Q),^or^does^not^comprise^a^signal^element.^ In^some^advantageous^embodiments,^the^kit^comprises:^ (E) at^ least^ one,^ preferably^ at^ least^ two sets^ of^ non-signal^ oligonucleotides,^ each^ non-signal^ oligonucleotide^comprising: (aa)^a^ translator^connector^element^(C)^comprising^a^nucleotide^sequence^which^ is^essentially^ complementary^to^at^least^a^section^of^the^nucleotide^sequence^of^the^translator^element^(c),^and^ (bb)^a^quencher^(Q),^a^signal^element^and^a^quencher^(Q),^or^does^not^comprise^a^signal^element.^ In^ some^ advantageous^ embodiments,^ the^ different^ sets^ of^ non-signal^ oligonucleotides^may^ be^ comprised^in^a^pre-mixture^of^different^sets^of^non-signal^oligonucleotides^or^exist^separately.^ RES-PA18-PCT^ Further,^ in^ some^ embodiments^ the^ decoding^ oligonucleotides^ in^ a^ particular^ set^ of^ decoding^ oligonucleotides^interacts^with^identical^identifier^elements^(T)^which^are^unique^to^a^particular^ analyte. In^ some^ advantageous^ embodiments,^ the^ different^ sets^ of^ decoding^ oligonucleotides^ may^ be^ comprised^in^a^pre-mixture^of^different^sets^of^decoding^oligonucleotides^or^exist^separately.^In^ some^advantageous^embodiments,^the^different^sets^of^analyte-specific^probes^may^be^comprised^ in^ a^ pre-mixture^ of^ different^ sets^ of^ analyte-specific^ probes^ or^ exist^ separately. In^ some^ advantageous^embodiments,^the^different^sets^of^signal^oligonucleotides^may^be^comprised^in^a^ pre-mixture^of^different^sets^of^signal^oligonucleotides^or^exist^separately. In^ some^ advantageous^ embodiments,^ a^ mixture^ of^ decoding^ oligonucleotides^ and/or^ multi- decoders^is^provided^that^specifically^hybridize^to^the^unique^identifier^sequences^of^the^probe^ sets.^In^some^embodiments,^the^decoding^oligonucleotides^comprise^of^at^least^one,^preferably^at^ least^ two sequence^ elements,^ a^ first^ element^ that^ is^ complementary^ to^ the^ unique^ identifier^ sequences^of^the^corresponding^probe^set^and^a^second^sequence^element^(translator^element)^ that^provides^a^sequence^for^the^specific^hybridization^of^a^signal^oligonucleotide,^the^translator^ element^ defines^ the^ type^ of^ signal^ that^ is^ recruited^ to^ the^ decoding^ oligonucleotide.^ In^ some^ embodiments^ multi-decoders^ comprising^ at^ least^ three^ sequence^ elements^ are^ used,^ a^ first^ element^that is^complementary^to^the^unique^identifier^sequences^of^the^corresponding^probe^set^ and^at^least^to^additional^sequence^elements^(translator^elements)^that^provide^sequences^for^the^ specific^hybridization^of^at^least^one,^preferably^at^least^two different^signal^oligonucleotides.^The^ translator^elements^define^the^type^of^signals^that^are^recruited^to^the^multi-decoder.^Different^ possible^structures^of^a^multi-decoder^can^be^seen^in^Fig.^15. Since^a^multi-decoder^does^recruit^a^ full^signal^oligonucleotide^per^translator^element,^the^brightness^of^the^signals^in^each^channel^is^ not^lower^than^the^brightness^of^signals^with^decoding^oligonucleotides. The usage^of^multi-decoders^increases^further^the^efficiency^of^the^encoding^scheme.^Figure^16 shows^a^possible^encoding^scheme^using^multi-decoders^based^upon^the^same^conditions^used^for^ the^examples with^the^decoding^oligonucleotide^with^two^sequence^elements.^One^can^clearly^see^ that^the^multi-decoder^based^encoding^scheme^can^create^a^higher^hamming^distance,^with^the^ same^number^of^ rounds^ and^the^ same^number^of^different^ signal^ oligonucleotides^used^ in^ the^ example^of^Fig.^5. As^mentioned^above^the^analyte^to^be^encoded^may^be a^nucleic^acid,^preferably^DNA,^PNA^or^RNA,^ in^particular mRNA,^a peptide,^polypeptide,^a^protein,^and/or^mixures thereof. RES-PA18-PCT^ In^some^advantageous^embodiments,^the^binding^element^(S)^comprises^an^amino^acid^sequence^ allowing^a^specific^binding^to^the^analyte^to^be^encoded. The binding^element^(S)^may^comprise^ moieties^ which^ are^ affinity^ moieties^ from^ affinity^ substances^ or^ affinity^ substances^ in^ their^ entirety^selected^from^the^group^consisting^of^antibodies,^antibody^fragments,^anticalin^proteins,^ receptor^ligands, enzyme^substrates,^lectins,^cytokines,^lymphokines,^interleukins,^angiogenic^or^ virulence^ factors,^ allergens,^ peptidic^ allergens,^ recombinant^ allergens,^ allergen-idiotypical^ antibodies,^ autoimmune-provoking^ structures,^ tissue-rejection-inducing^ structures,^ immunoglobulin^constant^regions^and^combinations^thereof. In^some^advantageous^embodiments,^the^binding^element^(S)^may^comprise^or^is^an^antibody^or^ an^antibody^fragment^selected^from^the^group^consisting^of^Fab,^scFv;^single^domain,^or^a^fragment^ thereof,^bis^scFv,^F(ab)2,^F(ab)3,^minibody,^diabody,^triabody,^tetrabody^and^tandab. As^ mentioned^ above,^ the^ method^ according^ to^ the^ present^ disclosure^ comprises^ selectively^ removing^ the^decoding^oligonucleotides^ and^ signal^ oligonucleotides^ from^ the^ sample,^ thereby^ essentially^maintaining^ the^ specific^binding^of^ the^analyte-specific^probes^ to^ the^ analyte^ to^be^ encoded. In^ particular^ all^ steps^ are^ performed^ sequentially.^ However, some^ steps^ may^ be^ performed^simultaneously,^in^particular^the^contacting^steps^A)^to^C),^in^particular^B)^and^C). By^ this^ measure^ the^ requirements^ for^ another^ round/cycle^ of^ binding^ further^ decoding^ oligonucleotides^ to^ the^same^analyte-specific^probes^are^established,^ thus^ finally^resulting^ in^a^ code^ or^ encoding^ scheme^ comprising^more^ than^ one^ signal.^ This^ step^ is^ realized^ by^ applying^ conditions^and^ factors^well^known^to^ the^skilled^person,^e.g., pH,^ temperature,^salt^conditions,^ oligonucleotide^concentration,^polymers^etc. In^some^advantageous^embodiments,^all^ steps^are^automated,^ in^particular^by^using^a^robotic^ system^ and/or^ an^ optical^ multiplexing^ system^ according^ to^ the^ present^ disclosure.^ In^ some^ examples,^the^steps^may^be performed^in^a^fluidic^system. As^mentioned^above,^with^the^methods^according^to^the^present^disclosure^an^encoding^scheme^ with^a^code^word^per^analyte^set^is^generated.^Therefore,^each^analyte^set^may^be^associated^with^ a^specific^code^word,^wherein^said^code^word^comprise^a^number^of^positions,^and^wherein^each^ position^corresponds^ to^one^cycle^resulting^ in^a^plurality^of^distinguishable^ encoding^schemes^ RES-PA18-PCT^ with^the^plurality^of^code^words. In^particular, said^encoding^scheme^may^be predetermined^and^ allocated^to^the^analyte^to^be^encoded. In^some^advantageous^embodiments,^the^code^words^obtained^for^the^individual^analytes^in^the^ performed^ cycles^ comprise^ the^ detected^ signals^ and^ additionally^ at^ least^ one^ element^ corresponding^to^no^detected^signal like^0,1^or^0,1,2^etc.^(see^also^Fig.^13^and^Fig.^14). In^particular,^ no^signal^is^detected^for^at^least^one^analyte^within^at^least^one^cycle if^using^the^non-signal^probe^ according^to^Figure^14,^Nr.^2^to^4,^or^a^non-signal^decoding^oligonucleotide^as^shown^in^Figure^14^ Nr.^5,^or^if^in^one^cycle^no^decoding^oligonucleotide^is^contacted^with^the^corresponding^identifier^ sequence^comprised^on^analyte-specific^probe^interacting^with^the^corresponding^analyte^in^the^ sample. In^this^cycle^the^position^has^the^value^zero^(0). In^some^advantageous^ embodiments,^at^ least^ for^one^ individual^analyte^a^position^of^ the^code^ word^ is^ zero^ (0). In^ particular, the^ ^ code^ word^ zero^ (0)^ is^ generated^ ^ by^ using^ no^ decoding^ oligonucleotides^ ^having^an^ identifier^connector^element^(t)^comprising^a^nucleotide^sequence^ which^is^essentially^complementary^to^at^least^a^section^of^the^unique^identifier^sequence^of^the^ identifier^ element^ (T)^ of^ a^ corresponding^ analyte-specific^ probe^ for^ an^ individual^ analyte. As^ mentioned^above,^in^some^embodiments, if^at^ least^for^one^individual^analyte^a^position^of^the^ code^ word^ is^ zero^ (0)^ in^ this^ cycle^ no^ corresponding^ decoding^ oligonucleotides^ having^ an^ identifier^ connector^ element^ (t)^ comprising^ a^ nucleotide^ sequence^ which^ is^ essentially^ complementary^to^at^least^a^section^of^the^unique^identifier^sequence^of^the^identifier^element^(T)^ of^a^corresponding^analyte-specific^probe^for^an^individual^analyte^are^used. Furthermore, in^ some^ advantageous^ embodiments the^ sample^ is^ contacted^with^ at^ least^ one,^ preferably^ at^ least^ two different^ sets^ of^ signal^ oligonucleotides,^ wherein^ the^ signal^ oligonucleotides^ in^ each^ set^ comprise^ a^ different^ signal^ element^ and^ comprise^ a^ different^ connector^element^(C). In^more^particular^embodiments,^the^sample^is^contacted^with^at^least^one,^preferably^at^least^one,^ preferably^ at^ least^ two different^ sets^ of^ decoding^ oligonucleotides^ per^ analyte,^ wherein^ the^ decoding^ oligonucleotides^ comprised^ in^ these^ different^ sets^ comprise^ the^ same^ identifier^ connector^element^(t)^comprising^a^nucleotide^sequence^which^is^essentially^complementary^to^at^ least^a^section^of^the^unique^identifier^sequence^of^the^identifier^element^(T)^of^the^corresponding^ analyte-specific^probe^set,^and^ wherein^the^decoding^oligonucleotides^of^the^different^sets^per^ analyte^differ^in^the^translator^element^(c)^comprising^a^nucleotide^sequence^allowing^a^specific^ RES-PA18-PCT^ hybridization^of^a^signal^oligonucleotide and^a^monitoring^probe^comprising^at^least^an^identifier^ element^(t)^comprising^a^nucleotide^sequence^which^is^essentially^complementary^to^at^least^a^ section^of^the^unique^set^identifier^sequence^of^the^identifier^element^(T)^of^the^corresponding^ binding^element;^wherein^the^monitoring^probe^ is^added before,^during^and/or^after^adding^ the^decoding^element. As^mentioned^above,^the^different^sets^of^non-signal^oligonucleotides^may^be^comprised^in^a^pre- mixture^of^different^sets^of^non-signal^oligonucleotides^or^exist^separately.^ In^ further^ embodiments,^ the^ decoding^ oligonucleotides^ in^ a^ particular^ set^ of^ decoding^ oligonucleotides^interacts^with^identical^identifier^elements^(T)^which^are^unique^to^a^particular^ analyte. As^mentioned^above,^the^different^sets^of^decoding^oligonucleotides^may^be^comprised^in^a^pre- mixture^of^different^sets^of^decoding^oligonucleotides^or^exist^separately as^well^as^the^different^ sets^of^analyte-specific^probes^may^be^comprised^ in^a^pre-mixture^of^different^sets^of^analyte- specific^probes^or^ exist^separately as^well the^different^ sets^ of^ signal^ oligonucleotides^may^be^ comprised^in^a^pre-mixture^of^different^sets^of^signal^oligonucleotides^or^exist^separately. In^ some^ advantageous^ embodiments of^ the^ method^ according^ to^ the^ present^ disclosure,^ the^ binding^element^(S)^comprise^a^nucleic^acid^comprising^a^nucleotide^sequence^allowing^a^specific^ binding^ to^ the^ analyte^ to^ be^ encoded,^ preferably^ a^ specific^ hybridization^ to^ the^ analyte^ to^ be^ encoded.^ In^some^advantageous^embodiments of^the^method^according^to^the^present^disclosure,^after^step^ A)^and^before^step^^B)^the^non-bound^analyte-specific^probes^may^be removed,^in^particular^by^ washing,^further after^step^B)^and^before^step^^C)^the^non-bound^decoding^oligonucleotides^may^ be^removed,^ in^particular^by^washing further, after^step^C)^and^before^step^ ^D)^ the^non-bound^ signal^oligonucleotides^may^be removed,^in^particular^by^washing. In^ some^ advantageous^ embodiments of^ the^ method^ according^ to^ the^ present^ disclosure,^ the^ analyte^specific^probes^may^be incubated^with^the^sample,^thereby^allowing^a^specific^binding^of^ the^analyte^specific^probes^to^the^analytes^to^be^encoded,^further^the^decoding^oligonucleotides^ may^ be incubated^with^ the^ sample,^ thereby^ allowing^ a^ specific^ hybridization^ of^ the^ decoding^ oligonucleotides^to^identifier^elements^(T)^of^the^respective^analyte-specific^probes,^further the^ signal oligonucleotides^ may^ be incubated^ with^ the^ sample,^ thereby^ allowing^ a^ specific^ RES-PA18-PCT^ hybridization^of^the^signal^oligonucleotides^to^translator^elements^(T)^of^the^respective^decoding^ oligonucleotides. As^mentioned^above,^the^analyte^to^be^encoded^may^be a^nucleic^acid,^preferably^DNA,^PNA,^RNA,^ in^particular^mRNA,^a^peptide,^polypeptide,^ a^protein or^combinations^ thereof. Therefore,^ the^ binding^ element^ (S)^may^ comprise^ an^ amino^ acid^ sequence^ allowing^ a^ specific^ binding^ to^ the^ analyte^to^be^encoded. Examples for^a^binding^element^(S)^aremoieties^which^are^affinity^moieties^ from^affinity^substances^or^affinity^substances^in^their^entirety^selected^from^the^group^consisting^ of^antibodies,^antibody^fragments,^anticalin^proteins,^receptor^ligands,^enzyme^substrates,^lectins,^ cytokines,^ lymphokines,^ interleukins,^ angiogenic^ or^ virulence^ factors,^ allergens,^ peptidic^ allergens,^ recombinant^ allergens,^ allergen-idiotypical^ antibodies,^ autoimmune-provoking^ structures,^ tissue-rejection-inducing^ structures,^ immunoglobulin constant^ regions^ and^ combinations^ thereof. In^ particular,^ the^ binding^ element^ (S)^ is^ an^ antibody^ or^ an^ antibody^ fragment^selected^from^the^group^consisting^of^Fab,^scFv;^single^domain,^or^a^fragment^thereof,^bis^ scFv,^Fab^2,^Fab^3,^minibody,^diabody,^triabody,^tetrabody^and^tandab. By^this^measure^the^method^is^further^developed^to^such^an^extent^that^the^encoded^analytes^can^ be^detected^by^any^means^which^is^adapted^to^visualize^the^signal^element.^Examples^of^detectable^ physical^features^include^e.g., light,^chemical^reactions,^molecular^mass,^radioactivity,^etc. In^ some^ advantageous^ embodiments,^ the^ signal^ caused^ by^ the^ signal^ element,^ therefore^ in^ particular^the^binding^of^the^signal^oligonucleotides^to^the^decoding^oligonucleotides,^interacting^ with^the^corresponding^analyte^probes,^bound^to^the^respective^analyte^is^determined^by: (a) Imaging^at^least^a^portion^of^the^sample;^and/or (b) Using^an^optical^imaging^technique;^and/or (c) Using^a^fluorescence^imaging^technique;^and/or (d) Multi-color^fluorescence^imaging^technique;^and/or^ (e) Super-resolution^fluorescence^imaging^technique. The^kits^and^method^according to^the^present^disclosure^may^be^used^ideally^for^in^vitromethods^ for^ diagnosis^ of^ a^ disease^ selected^ from^ the^ group^ comprising^ cancer,^ neuronal^ diseases,^ cardiovascular^diseases,^inflammatory^diseases,^autoimmune^diseases,^diseases^due^to^a^viral^or^ bacterial^infection,^skin^diseases,^skeletal^muscle^diseases,^dental^diseases, and^prenatal^diseases. RES-PA18-PCT^ Further,^the^kits^and^method^according to^the^present^disclosure^may^be^used^also^ideally^for^in^ vitro methods^for^diagnosis^of^a^disease^in^plants^selected^from^the^group^comprising:^diseases^ caused^by^biotic^stress,^preferably^by^ infectious^and/or^parasitic^origin,^or^diseases^caused^by^ abiotic^stress,^preferably^caused^by^nutritional^deficiencies^and/or^unfavorable^environment. Further,^the^kits^and^method^according to^the^present^disclosure^may^be^used^also^ideally^for^in^ vitromethods^for^screening,^identifying^and/or^testing^a^substance^and/or^drug^comprising: (a) contacting^a^test^sample^comprising^a^sample^with^a^substance^and/or^drug^ (b) detecting^different^analytes^in^a^sample^by^sequential^signal-encoding^of^said^ analytes^with^a^method^according^to^the^present^disclosure. An^ optical^ multiplexing^ system^ suitable^ for^ the^ method^ according^ to^ the^ present^ disclosure,^ comprising^at^least: - a reaction^vessel^for^containing^the^kits^or^part^of^the^kits^according^to^the^present^ disclosure; - a^detection^unit^comprising^a^microscope,^in^particular^a^fluorescence^microscope; - a^camera; - a^liquid^handling^device. In^ some^ embodiments, optical^ multiplexing^ system^may^ comprise^ further^ a^ heat^ and^ cooling^ device and/or^a^robotic^system. In^ some^embodiments,^ the^method^according^ to^ the^present^disclosure^ encodes a^nucleic^ acid^ analyte,^such^as^an^mRNA,^e.g., such^an^mRNA^coding^for^a^particular^protein. In^some^advantageous^embodiments,^the^method^described^herein^is^used^for^specific^detection^ of^many^different^analytes^in^parallel.^The^technology^allows^to^distinguish^a^higher^number^of^ RES-PA18-PCT^ analytes^than^different^signals^are^available.^The^process^includes^at^least^four^consecutive^rounds^ of^ specific^ binding,^ signal^ detection^ and^ selective^ denaturation^ (if^ a^ next^ round^ is^ required),^ eventually^producing^ a^ signal^ code.^To^decouple^ the^dependency^between^ the^ analyte^ specific^ binding^ and^ the^ oligonucleotides^ providing^ the^ detectable^ signal,^ a^ so^ called^ “decoding”- oligonucleotide^ is^ introduced.^The^decoding^oligonucleotide^ transcribes^ the^ information^of^ the^ analyte^specific^probe^set^to^the^signal^oligonucleotides.^ In^a^specific^embodiment^the^method^may^comprise^the^steps^of^1.^providing^one^or^more^analyte^ specific^probe^sets,^the^set^of^analyte^specific^probes^consist^of^one^or^more^different^probes,^each^ differing^in^the^binding^moiety^that^specifically^interacts^with^the^analyte,^all^probes^of^a^single^ probe^set^are^tethered^to^a^sequence^element^(unique^identifier),^that^is^unique^to^a^single^probe^ set^and^allows^the^specific^hybridization^of^a^decoding^oligonucleotide,^2.^specific^binding^of^the^ probe^sets^to^their^target^binding^sites^of^the^analyte,^3.^eliminating^non-bound^probes^(e.g.^by^a^ wash^step),^4.^providing^a^mixture^of^decoding^oligonucleotides^that^specifically^hybridize^to^the^ unique^identifier^sequences^of^the^probe^sets,^the^decoding^oligonucleotides^comprise^of^at^least^ one,^ preferably^ at^ least^ two sequence^ elements,^ a^ first^ element^ that^ is^ complementary^ to^ the^ unique^ identifier^ sequences^ of^ the^ corresponding^ probe^ set^ and^ a^ second^ sequence^ element^ (translator^ element)^ that^ provides^ a^ sequence^ for^ the^ specific^ hybridization^ of^ a^ signal^ oligonucleotide,^the^translator^element^defines^the^type^of^signal^that^is^recruited^to^the^decoding^ oligonucleotide,^5.^specific^hybridization^of^the^decoding^oligonucleotides^to^the^unique^identifier^ sequences^provided^by^the^bound^probe^sets,^6.^eliminating^non-bound^decoding^oligonucleotides^ (e.g.^by^washing^step),^7. providing^ a^ mixture^ of^ signal^ oligonucleotides,^ comprising^ of^ a^ signal^ that^ can^ be^ detected^ and^ a^ nucleic^ acid^ sequence^ that^ specifically^ hybridizes^ to^ the^ translator^element^of^one^of^the^decoding^oligonucleotides^used^in^the^former^hybridization^step,^ 8.^ specific^ hybridization^ of^ the^ signal^ oligonucleotides,^ 9.^ eliminating^ non-bound^ signal^ oligonucleotides,^10.^detection^of^the^signals,^11. selective^ release^ of^ decoding^ oligonucleotides^and^signal^oligonucleotides^while^the^binding^of^specific^probe^sets^to^the^analyte^ is^almost^or^completely^unaffected,^12.^eliminating^released^decoding^oligonucleotide^and^signal^ oligonucleotides^(e.g.^by^a^washing^step)^while^the^binding^of^specific^probes^sets^to^the^analytes^ is^ almost^ or^ completely^unaffected^ ,^ repeating^ the^ steps^4^ to^12^ at^ least^ three^ times^until^ the^ detection^of^ a^ sufficient^number^of^ signals^ to^ generate^ an^ encoding^ scheme^ for^ each^different^ analyte^of^interest.^^ RES-PA18-PCT^ It^ is^ to^ be^ understood^ that^ the^ before-mentioned^ features^ and^ those^ to^ be^mentioned^ in^ the^ following^cannot^only^be^used^in^the^combination^indicated^in^the^respective^case,^but^also^in^other^ combinations^or^in^an^isolated^manner^without^departing^from^the^scope^of^the^disclosure. The^disclosure is^now^further^explained^by^means^of^embodiments^resulting^in^additional^features,^ characteristics^and^advantages^of^the^disclosure.^The^embodiments^are^of^pure^illustrative^nature^ and^ do^ not^ limit^ the^ scope^ or^ range^ of^ the^ disclosure.^ The^ features^mentioned^ in^ the^ specific^ embodiments^are^general^features^of^the^disclosure which^are^not^only^applicable^in^the^specific^ embodiment^but^also^in^an^isolated^manner^in^the^context^of^any^embodiment^of^the^disclosure. The^method^disclosed^herein^is^used^for^specific^detection^of^many^different^analytes^in^parallel.^ The^ technology^ allows^ distinguishing^ a^ higher^ number^ of^ analytes^ than^ different^ signals^ are^ available.^The^process^preferably^includes^at^least^one,^preferably^at^least^two consecutive^rounds^ of^ specific^ binding,^ signal^ detection^ and^ selective^ denaturation^ (if^ a^ next^ round^ is^ required),^ eventually^producing^ a^ signal^ code.^To^decouple^ the^dependency^between^ the^ analyte^ specific^ binding^ and^ the^ oligonucleotides^ providing^ the^ detectable^ signal,^ a^ so^ called^ “decoding”^ oligonucleotide^ is^ introduced.^The^decoding^oligonucleotide^ transcribes^ the^ information^of^ the^ respective^analyte^specific^probe^set^to^the^signal^oligonucleotides. The^present^disclosure^pertains^further^to^monitormethods^of^detecting^an^analyte,^comprising:^ - attaching^a^plurality^of^analyte-specific^probes^to^the^analyte,^wherein^the^analyte-specific^ probes^ independently^ attach^ to^ the^ analyte^ and^ wherein^ the^ analyte-specific^ probes^ share^ a^ common^identifier^segment^(T);^^ - annealing^ a^ plurality^ of^ first^ decoding^ oligonucleotides^ to^ the^ analyte-specific^ probes,^ wherein^ the^ first^ decoding^ oligonucleotides^ share^ a^ first^ common^ region^ that^ is^ reverse^ complementary^to^the^common^identifier^segment^and^a^second^common^region; - annealing^ a^ first^signal^ oligonucleotide^ to^ at^ least^one^of^ the^plurality^ of^ first^decoding^ oligonucleotides^ such^ that^ an^ oligo^ tethered^ to^ the^ first^ signal^ oligonucleotide^ is^ reverse^ complementary^to^the^second^common^region; - detecting^the^first^signal^oligonucleotide; RES-PA18-PCT^ - removing^the^plurality^of^first^decoding^oligonucleotides; - annealing^a^plurality^of^second^decoding^oligonucleotides^to^the^analyte-specific^probes,^ wherein^ the^ second^ decoding^ oligonucleotides^ share^ a^ first^ common^ region^ that^ is^ reverse^ complementary^to^the^common^identifier^segment^and^a^second^decoding^oligonucleotides^second^ common^region^that^differs^from^the^second^common^region^of^the^first^decoding; - annealing^ a^ second^ signal^ oligonucleotide^ to^ at least^ one^ of^ the^ plurality^ of^ second^ decoding^ oligonucleotides^ such^ that^ an^ oligo^ tethered^ to^ the^ signal^ oligonucleotide^ is^ reverse^ complementary^to^the^second^decoding^oligonucleotide^second^common^region;^and^detecting^the^ second^signal^oligonucleotide. In^particular,^in^the^above^mentioned^embodiment^a^second^aliquot^of^a^plurality^of^first^decoding^ oligonucleotides^ is^ annealed^ to^ the^ analyte-specific^ probes. Furthermore,^ a^ first^ aliquot^ of^ a^ plurality^of^first^decoding^oligonucleotides^is^annealed^to the^analyte-specific^probes. In^ some^ further^ advantageous^ embodiments,^ the^ present^ disclosure^ pertains^ to^ a^ method^ of^ assigning^an^analyte^to^a^position^in^an^image,^comprising^assigning^a^fluorescence^pattern^to^the^ analyte,^observing^the^fluorescence^pattern^at^the^position^in^the^image,^and^assigning^the^analyte^ to^ the^position,^ in^particular^wherein^observing^ the^ fluorescence^pattern^ comprises^ repeating^ steps^of^labeling^the^position^using^a^fluorophore^tagged^oligo^drawn^from^a^re-accessible pool,^ performing^ a^ single^ excitation^ at^ the^ position^ in^ the^ image,^ and^ contacting^ the^ analyte^ to^ a^ denaturant,^in^particular^wherein^observing^the^fluorescence^pattern^comprises^repeating^steps^ of^ labeling^ the^ position^ using^ a^ fluorophore^ tag-recruiting^ bridging^ oligo^ drawn^ from^ a^ re- accessible pool,^performing^a^single^excitation^at^ the^position^ in^ the^ image,^and^contacting^ the^ analyte^to^a^denaturant. In^ some^ further^ advantageous^ embodiments,^ the^present^disclosure^pertains^ to^ a^ composition^ comprising^a^cell^having^nucleic^acids^distributed^therein,^wherein^a^first^nucleic^acid^is^tagged^by^ a^first^plurality^of^probes^that^target^adjacent^segments^of^the^first^nucleic^acid^and^that^share^a^ common^first^tether^segment;^a^second^nucleic^acid^is^tagged^by^a^second^plurality^of^probes^that^ target^ adjacent^ segments^ of^ the^ second^ nucleic^ acid^ and^ that^ share^ a^ common^ second^ tether^ segment;^ and^a^ third^nucleic^ acid^ is^ tagged^by^ a^ third^plurality^ of^probes^ that^ target^ adjacent^ segments^of^the^third^nucleic^acid^and^that^share^a^common^third^tether^segment;^a^first^adapter^ population^comprising^molecules^having^a^first^tether^reverse^complementary^region^and^a^first^ RES-PA18-PCT^ fluorophore^adapter^tether;^a^second^adapter^population^comprising^molecules^having^a^second^ tether^reverse^complementary^region^and^a^second^fluorophore^adapter^tether;^a^third^adapter^ population^comprising^molecules^having^a^third^tether^reverse^complementary^region^and^a^first^ fluorophore^ adapter^ tether;^ a^ population^ of^ first^ fluorophores^ having^ a^ first^ tether^ reverse^ complementary^region;^and^a^population^of^second^fluorophores^having^a^second^tether^reverse^ complementary^region. In^ some^ further^ advantageous^ embodiments,^ the^ present^ disclosure^ pertains^ to^ a^ method^ of^ assigning^ coded^ fluorescence^ patterns^ to^ a^ plurality^ of^ target^ analytes^ in^ a^ cell,^ comprising:^ subjecting^the^cell^to^a^plurality^of^detection^rounds,^each^detection^round^comprising:^contacting^ the^cell^to^representatives^of^the^same^at^least^one,^preferably^at^least^two populations^of^tagged^ fluorescence^moieties,^and^removing^the^fluorescent^moieties^after^a^single^excitation^event,^ in^ particular - wherein^the^number^of^patterns^detectable^increases^exponentially^with^the^number^of^ detection^rounds,^ - wherein^the^fluorescence^moieties^are^not^tagged with^nucleic^acid^tags^that^are^specific^ to^the^target^nucleic^acids,^and^ - wherein^ separate^ aliquots^ of^ common^ tagged^ fluorescence^ moieties^ are^ used^ across^ multiple^detection^rounds. In^particular,^with^the^above^notified^method^a^total^decoding^efficiency^of^at^least^30%^may^be^ achieved. In^ some^ further^ advantageous^ embodiments,^ the^ present^ disclosure^ pertains^ to^ a^ method^ of^ assigning^ coded^ fluorescence^ patterns^ to^ a^ plurality^ of^ target^ analytes^ in^ a^ cell,^ comprising:^ contacting^a^target^to^a^bipartite^labeling^probe,^the^bipartite^labeling^probe^comprising^a^target- specific^moiety^and^a^fluorophore-specifying^moiety;^contacting^the^bipartite^labeling^probe^to^a^ first^aliquot^of^a^fluorophore^reservoir^comprising^no^more^than^two^populations^of^fluorophores;^ replacing^the^fluorophore^specifying^moiety^in^the^bipartite^probe,^and^contacting^the^bipartite^ labeling^probe^to^a^second^aliquot^of^the^fluorophore^reservoir^comprising^the^same^no^more^than^ two^populations. In^some^embodiments^of^the^above^notified^method,^replacing^the^fluorophore^specifying^moiety^ in^ the^bipartite^probe^ comprises^denaturing^ a^binding^between^a^ target-specific^moiety^ and^a^ fluorophore-specifying^ moiety^ after^ subjecting^ the^ bipartite^ labeling^ probe^ bound^ to^ a^ RES-PA18-PCT^ fluorophore^ of^ the^ fluorophore^ to^ excitation^ energy.^ In^ particular,^ replacing^ the^ fluorophore^ specifying^ moiety^ in^ the^ bipartite^ probe^ comprises^ drawing^ from^ one^ of^ no^ more^ than^ two^ fluorophore^specifying^moiety^reservoirs. In^ some^ further^ advantageous^ embodiments,^ the^present^disclosure^pertains^ to^monitoring a^ method^of^detecting^an^analyte,^comprising:^ - attaching^a^plurality^of^probes^to^the^analyte,^in^particular^a^nucleic^acid,^wherein^the^ probes^independently^attach/anneal^to^the^analyte^and^wherein^the^probes^share^a^ common^identifier^segment;^ - annealing^a^plurality^of^first^adapter^segments^to^the^probes,^wherein^the^first^adapter^ segments^share^a^first^common^region^that^is^reverse^complementary^to^the^common^ identifier segment^ and^ a^ second^ common^ region,^ in^ particular^ configured^ to^ accommodate^a^single^reporter^/^selected^from^no^more^than^two^reporter^categories;^ - annealing^a^first^reporter^to^at^least^one^of^the^plurality^of^first^adapter^segments^such^ that^an^oligo^ tethered^to^the^ first^reporter^ is^reverse^complementary^ to^ the^second^ common^region;^detecting^the^first^reporter; - removing^ the^plurality^ of^ first^ adapter^ segments,^ in^particular^without^ annealing^ a^ second^reporter^to^the^at^least^one^of^the^plurality^of^first^adapter^segments; - annealing^a^plurality^of^second^adapter^segments to^the^probes,^wherein^the^second^ adapter^segments^share^a^first^common^region^that^is^reverse^complementary^to^the^ common^identifier^segment^and^a^second^adapter^second^common^region^that^differs^ from^the^second^common^region^of^the^first^adapter^segments,^in^particular^configured^ to^ accommodate^ a^ single^ reporter^ /^ selected^ from^ no^ more^ than^ two^ reporter^ categories;^^ - annealing^a^second^reporter^to^at^least^one^of^the^plurality^of^second^adapter^segments^ such^that^an^oligo^tethered^to^the^second^reporter^is^reverse^complementary^to^the^ second^ adapter^ second^ common^ region;^ and^ detecting^ the^ second^ reporter,^ in^ particular^without^annealing^a^third^reporter^to^the^at^least^one^of^the^plurality^of^first^ adapter^segments.^ METHODS^AND^EXAMPLES In^an application^variant,^the^analyte^or^target^is^nucleic^acid,^e.g., DNA^or^RNA,^and^the^probe^set^ comprises^oligonucleotides^that^are^partially^or^completely^complementary^to^the^whole^sequence^ or^a subsequence^of^the^nucleic^acid^sequence^to^be^detected^(Figure^1).^The^nucleic^acid^sequence^ RES-PA18-PCT^ specific^ oligonucleotide^ probe^ sets^ comprising^ analyte-specific^ probes^ (1)^ including^ a^ binding^ element^(S)^that^specifically^hybridizes^to^the^target^nucleic^acid^sequence^to^be^detected,^and^an^ identifier^element^(T)^comprising^a^nucleotide^sequence^which^is^unique^to^said^set^of^analyte- specific^probes^(unique^identifier^sequence). In^a^further application^variant,^the^analyte^or^target^is^a^protein^and^the^probe^set^comprises^one^ or^more^proteins,^e.g.^antibodies^(Figure^2).^The^protein^specific^probe^set^comprising^analyte- specific^ probes^ (1)^ including^ a^ binding^ element^ (T)^ such^ as^ the^ (hyper-)variable^ region^ of^ an^ antibody,^ that^ specifically^ interacts^ with^ the target^ protein^ to^ be^ detected,^ and^ the^ identifier^ element^(T). In^a^further application^variant,^at^least^one^analyte^is^a^nucleic^acid^and^at^least^a^second^analyte^ is^a^protein^and^at^ least^ the^ first^probe^set^binds^ to^ the^nucleic^acid^sequence^and^at^ least^ the^ second^probe^set^binds^specifically^to^the^protein^analyte.^Other^combinations^are^possible^as^well. An^embodiment^of^the^general^method^of^the^present^disclosure^may^be: Step^1:^Applying^the^at^least^20^analyte- or^target-specific^probe^sets.^The^target^nucleic^ acid^sequence^is^incubated^with^a^probe^set^consisting^of^oligonucleotides^with^sequences^ complementary^to^the^target^nucleic^acid.^In^this^example,^a^probe^set^of^5^different^probes^ is^ shown,^ each^ comprising^ a^ sequence^ element^ complementary^ to^ an^ individual^ subsequence^of^the^target^nucleic^acid^sequence^(S1^to^S5).^In^this^example,^the^regions^do^ not^ overlap.^ Each^ of^ the^ oligonucleotides^ targeting^ the^ same^ nucleic^ acid^ sequence^ comprises^the^identifier^element^or^unique^identifier^sequence^(T),^respectively.^ Step^2:^Hybridization^of^the^probe^set.^The^probe^set^is^hybridized^to^the^target^nucleic^acid^ sequence^ under^ conditions^ allowing^ a^ specific^ hybridization.^ After^ the^ incubation,^ the^ probes^are^hybridized^to^their^corresponding^target^sequences^and^provide^the^identifier^ element^(T)^for^the^next^steps. Step^3:^Eliminating^non-bound^probes.^After^hybridization,^the^unbound^oligonucleotides^ are^eliminated,^e.g.^by^washing^steps. Step^4:^Applying^the^decoding^oligonucleotides.^The^decoding^oligonucleotides^consisting^ of^at^ least^one,^preferably^at^least^two sequence^elements^(t)^and^(c)^are^applied.^While^ RES-PA18-PCT^ sequence^ element^ (t)^ is^ complementary^ to^ the^ unique^ identifier^ sequence^ (T),^ the^ sequence^ element^ (c)^ provides^ a^ region^ for^ the^ subsequent^ hybridization^ of^ signal^ oligonucleotides^(translator^element). Step^ 5:^ Hybridization^ of^ decoding^ oligonucleotides.^ The^ decoding^ oligonucleotides^ are^ hybridized^with^the^unique^identifier^sequences^of^the^probes^(T)^via^their^complementary^ first^sequence^elements^(t).^After^incubation,^the^decoding^oligonucleotides^provide^the^ translator^sequence^element^(c)^for^a^subsequent^hybridization^step. Step^ 6:^ Eliminating^ the^ excess^ of^ decoding^ oligonucleotides.^ After^ hybridization,^ the^ unbound^decoding^oligonucleotides^are^eliminated,^e.g.^by^washing^steps. Step^7:^Applying^the^signal^oligonucleotide.^The^signal^oligonucleotides^are^applied.^The^ signal^ oligonucleotides^ comprise^ at^ least^ one^ second^ connector^ element^ (C)^ that^ is^ essentially^complementary^to^the^translator^sequence^element^(c)^and^at^least^one^signal^ element^that^provides^a^detectable^signal^(F). Step^ 8:^ Hybridization^ of^ the^ signal^ oligonucleotides.^ The^ signal^ oligonucleotides^ are^ hybridized^ via^ the^ complementary^ sequence^ connector^ element^ (C)^ to^ the^ translator^ element^(c)^of^decoding^oligonucleotide.^After^incubation,^the^signal^oligonucleotides^are^ hybridized^to^their^corresponding^decoding^oligonucleotides^and^provide^a^signal^(F)^that^ can^be^detected. Step^9:^Eliminating^the^excess^of^signal^oligonucleotides.^After^hybridization,^the^unbound^ signal^oligonucleotides^are^eliminated,^e.g.^by^washing^steps. Step^10:^Signal^detection.^The^signals^provided^by^the^signal^oligonucleotides^are^detected. The^following^steps^(steps^11^and^12)^are^unnecessary^for^the^last^detection^round. Step^11:^Selective^denaturation.^The^hybridization^between^the^unique^identifier^sequence^ (T)^and^the^first^sequence^element^(t)^of^the^decoding^oligonucleotides^is^dissolved.^The^ destabilization^ can^ be^ achieved^ via^ different^ mechanisms^ well^ known^ to^ the^ trained^ RES-PA18-PCT^ person^ like^ for^ example:^ increased^ temperature,^ denaturing^ agents,^ etc.^The^ target- or^ analyte-specific^probes^are^not^affected^by^this^step. Step^12:^Eliminating^the^denatured^decoding^oligonucleotides.^The^denatured^decoding^ oligonucleotides^and^signal^oligonucleotides^are^eliminated^(e.g.^by^washing^steps)^leaving^ the^specific^probe^sets^with^free^unique^identifier^sequences,^reusable^in^a^next^round^of^ hybridization^and^detection^(steps^4^to^10).^This^detection^cycle^(steps^4^to^12)^is^repeated^ at^least^four times^until^the^planed^encoding^scheme^is^completed. Another^embodiment^of^the^general^method^of^the^present^disclosure^using^multi-decoders^may^ be (Fig.^16): Step^1: Target^nucleic^acids:^In^this^example^three^different^target^nucleic^acids^(A),^(B)^ and^(C)^have^to^be^detected^and^differentiated^by^using^only^two^different^types^of^signal^ oligonucleotides.^Before^starting^the^experiment,^a^certain^encoding^scheme^is^set.^In^this^ example,^ the^ three^ different^ nucleic^ acid^ sequences^ are^ encoded^ by^ three^ rounds^ of^ detection^with^ three^different^ signal^ types^ (1),^ (2)^ and^(1/2)^ and^a^ resulting^hamming^ distance^of^3^to^allow^for^error^detection.^The^planed^code^words^are: sequence^A:^(1)^– (1)^– (2) sequence^B:^(2)^– (2)^– (1/2) sequence^C:^(1/2)^– (1/2)^– (1) Step^2:^Hybridization^of^the^probe^sets:^For^each^target^nucleic^acid,^an^own^probe^set^is^ applied,^specifically^hybridizing^to^ the^corresponding^nucleic^acid^sequence^of^ interest.^ Each^probe^set^provides^a^unique^identifier^sequence^(T1),^(T2)^or^(T3).^This^way^each^ different^target^nucleic^acid^is^uniquely^labeled.^In^this^example^sequence^(A)^is^labeled^ with^(T1),^sequence^(B)^with^(T2)^and^sequence^(C)^with^(T3). The^illustration^in^Fig.^16^ summarizes^Steps^1^to^3^of^Fig.^3. Step^ 3:^ Hybridization^ of^ the^ decoding^ oligonucleotides^ and^ multi-decoders:^ For^ each^ unique^identifier^present,^a^certain^decoding^oligonucleotide^or^multi-decoder^is^applied^ specifically^ hybridizing^ to^ the^ corresponding^ unique^ identifier^ sequence^ by^ its^ first^ RES-PA18-PCT^ sequence^element^(here^(t1)^to^(T1),^(t2)^to^(T2)^and^(t3)^to^(T3)).^Each^of^the^decoding^ oligonucleotides^or^multi-decoders^provides^a^translator^or^two^translator^elements^that^ define^ the^ signals^ that^will^ be^ generated^ after^ hybridization^of^ signal^ oligonucleotides.^ Here^nucleic^ acid^ sequence^ (A)^ is^ labeled^with^ (c1),^ (B)^ is^ labeled^with^(c2)^ and^ (C)^ is^ labeled^with^ both^ translator^ elements^ (c1)^ and^ (c2)^ resulting^ in^ the^ signal^ (1/2).^ The^ illustration^in^Fig.^16^summarizes^steps^4^to^6^of^Fig.3. Step^4:^Hybridization^of^ signal^ oligonucleotides:^ For^ each^ type^of^ translator^ element,^ a^ signal^ oligonucleotide^with^ a^ certain^ signal,^ differentiable^ from^ signals^ of^ other^ signal^ oligonucleotides,^ is^applied.^This^signal^oligonucleotide^can^specifically^hybridize^to^the^ corresponding^translator^element.^The^illustration^in^Fig.^16^summarizes^steps^7^to^9^of^ Fig.^3 Step^5:^Signal^detection^for^the^encoding^scheme:^The^different^signals^are^detected.^Note^ that^in^this^example^the nucleic^acids^(A),^(B)^and^(C)^can^already^be^distinguished^after^ the^ first^ round^of^detection.^This^ is^ in^ contrast^ to^ the^ step^5^of^Fig.^ 5^ explained^by^ the^ additional^signal^type^(1/2)^that^can^be^realized^due^to^multi-decoders.^Although^nucleic^ acid^ sequences^ can^ already^ be^ distinguished,^ the^ additional^ rounds^ contribute^ to^ the^ planned^hamming^distance^of^3.^The^illustration^in^Fig.^16^corresponds^to^step^10^of^Fig.^3. Step^6:^Selective^denaturation:^The^decoding^(and^signal)^oligonucleotides^and/or^multi- decoders^ of^ all^ nucleic^ acid^ sequences^ to^ be^ detected^ are^ selectively^ denatured^ and^ eliminated^ as^ described^ in^ steps^ 11^ and^ 12^ of^ Fig.3. Afterwards^ the^ unique^ identifier^ sequences^of^the^different^probe^sets^can^be^used^for^the^next^round^of^hybridization^and^ detection. Step^7:^Second^round^of^detection:^A^next^round^of^hybridization^and^detection^is^done^as^ described^ in^ steps^ 3^ to^ 5.^ Note^ that^ in^ this^ new^ round^ the^ mix^ of^ different^ decoding^ oligonucleotides^and^multi-decoders^is^changed.^For^example,^decoding^oligonucleotide^of^ nucleic^acid^sequence^(A)^used^in^the^first^round^comprised^of^sequence^elements^(t1)^and^ (c1)^while^the^new^multi^decoder^of^round^2^comprises^of^the^sequence^elements^(t1),^(c1)^ and^(c2).^Note^that^now^a^hamming^distance^of^2^is^already^given^after^2^rounds,^which^is^ the^final^result^of^the^example^in^Fig.^3^after^3^rounds. RES-PA18-PCT^ Step^8:^Third^round^of^detection:^Again,^a^new^combination^of^decoding^oligonucleotides^ and/or^multi-decoders^is^used^leading^to^new^signal^combinations.^After signal^detection,^ the^resulting^code^words^for^the^three^different^nucleic^acid^sequences^are^not^only^unique^ and^therefore^distinguishable^but^comprise^a^hamming^distance^of^3^to^other^code^words.^ Due^to^the^hamming^distance,^an^error^in^the^detection^of the^signals^(signal^exchange)^ would^not^ result^ in^ a^ valid^ code^word^and^ therefore^ could^be^detected^ and^because^of^ hamming^distance^3^also^corrected,^in^contrast^to^the^encoding^scheme^of^Fig.^3. This^way^ three^ different^ nucleic^ acids^ can^ be^ distinguished in^ three^ detection^ rounds^ with^ two^ different^signals,^allowing^an^error^detection^and^correction. Note^that^in^every^round^of^detection,^the^type^of^signal^provided^by^a^certain^unique^identifier^is^ controlled^by^the^use^of^a^certain^decoding^oligonucleotide.^As^a^result,^the^sequence^of^decoding^ oligonucleotides^applied^in^the^detection^cycles^transcribes^the^binding^specificity^of^the^probe^set^ into^a^unique^signal^sequence. The^ steps^ of^ decoding^ oligonucleotide^ hybridization^ (steps^ 4^ to^ 6)^ and^ signal^ oligonucleotide^ hybridization^(steps^7^to^9)^can^also^be^combined^in^two^alternative^ways^as^shown^in^Figure^4. Opt.^1:^Simultaneous^hybridization.^Instead^of^the^steps^4^to^9^of^Figure^3,^specific^hybridization^ of^decoding^oligonucleotides^and^signal^oligonucleotides^can^also^be^done^simultaneously^leading^ to^the^same^result^as^shown^in^step^9^of^Figure^3,^after^eliminating^the^excess^decoding- and^signal^ oligonucleotides. Opt.^2:^Preincubation.^Additionally^to^option^1^of^Figure^3,^decoding- and^signal^oligonucleotides^ can^be^preincubated^in^a^separate^reaction^before^being^applied^to^the^target^nucleic^acid^with^the^ already^bound^specific^probe^set. 1.^Example^for^signal^encoding^of^three^different^nucleic^acid^sequences^by^two^different^signal^ types^and^three^detection^rounds Figure^3^shows^the^general^concept^of^generation^and^detection^of^specific^signals^mediated^by^ decoding^oligonucleotides.^It^does^not^show^the^general^concept^of^encoding^that^can^be^achieved^ by^this^procedure.^To^illustrate^the^use^of^the^process^shown^in^Figure^3^for^the^generation^of^an^ encoding^scheme,^Figure^5^shows^a^general^example^for^a^multiple^round^encoding^experiment^ with^three^different^nucleic^acid^sequences.^In^this^example,^the^encoding^scheme^includes^error^ detection. RES-PA18-PCT^ Step^1:^Target^nucleic^acids.^In^this^example^three^different^target^nucleic^acids^(A),^(B)^and^(C)^ have^to^be^detected^and^differentiated^by^using^only^two^different^types^of^signal.^Before^starting^ the^experiment,^a^certain^encoding^scheme^is^set.^In^this^example,^the^three^different^nucleic^acid^ sequences^are^encoded^by^three^rounds^of^detection^with^two^different^signals^(1)^and^(2)^and^a^ resulting^hamming^distance^of^2^to^allow^for^error^detection.^The^planed^code^words^are:^ sequence^A:^(1)^– (2)^– (2);^ sequence^B:^(1)^– (1)^– (1);^ sequence^C:^(2)^– (1)^– (2). Step^2:^Hybridization^of^the^probe^sets.^For^each^target^nucleic^acid,^an^own^probe^set^is^applied,^ specifically^hybridizing^ to^ the^corresponding^nucleic^acid^sequence^of^ interest.^Each^probe^set^ provides^a^unique^identifier^sequence^(T1),^(T2)^or^(T3).^This^way^each^different^target^nucleic^ acid^is^uniquely^labeled.^In^this^example^sequence^(T)^is^labeled^with^(T1),^sequence^(B)^with^(T2)^ and^sequence^(C)^with^(T3).^The^illustration^summarizes steps^1^to^3^of^Figure^3. Step^ 3:^ Hybridization^ of^ the^ decoding^ oligonucleotides.^ For^ each^ unique^ identifier^ present,^ a^ certain^decoding^oligonucleotide^is^applied^specifically^hybridizing^to^the^corresponding^unique^ identifier^sequence^by^its^first^sequence^element^(here^(t1)^to^(T1),^(t2)^to^(T2)^and^(t3)^to^(T3)).^ Each^of^the^decoding^oligonucleotides^provides^a^translator^element^that^defines^the^signal^that^ will^be^generated^after^hybridization^of^signal^oligonucleotides.^Here^nucleic^acid^sequences^(A)^ and^(B)^are^labeled^with^the^translator^element^(c1)^and^sequence^(C)^is^labeled^with^(c2).^The^ illustration^summarizes^steps^4^to^6^of^Figure^3. Step^ 4:^ Hybridization^ of^ signal^ oligonucleotides.^ For^ each^ type^ of^ translator^ element,^ a^ signal^ oligonucleotide^ with^ a^ certain^ signal^ (2),^ differentiable^ from^ signals^ of^ other^ signal^ oligonucleotides,^ is^ applied.^ This^ signal^ oligonucleotide^ can^ specifically^ hybridize^ to^ the^ corresponding^translator^element.^The^illustration^summarizes^steps^7^to^9^of^Figure^3. Step^5:^Signal^detection^for^the^encoding^scheme.^The^different^signals^are^detected.^Note^that^in^ this^example^the^nucleic^acid^sequence^(C)^can^be^distinguished^from^the^other^sequences^by^the^ unique^signal^(2)^it^provides,^while^sequences^(A)^and^(B)^provide^the^same^kind^of^signal^(1)^and^ cannot^be^distinguished^after^the^first^cycle^of^detection.^This^is^due^to^the^fact,^that^the^number^of^ RES-PA18-PCT^ different^nucleic^acid^sequences^to^be^detected^exceeds^the^number^of^different^signals^available.^ The^illustration^corresponds^to^step^10^of^Figure^3. Step^ 6:^ Selective^ denaturation.^ The^ decoding^ (and^ signal)^ oligonucleotides^ of^ all^ nucleic^ acid^ sequences^to^be^detected^are^selectively^denatured^and^eliminated^as^described^in^steps^11^and^ 12^of^Figure^3.^Afterwards^the^unique^identifier^sequences^of^the^different^probe^sets^can^be^used^ for^the^next^round^of^hybridization^and^detection.^ Step^ 7:^ Second^ round^ of^ detection.^ A^ next^ round^ of^ hybridization^ and^ detection^ is^ done^ as^ described^ in^ steps^ 3^ to^ 5.^ Note^ that^ in^ this^ new^ round^ the^ mix^ of^ different^ decoding^ oligonucleotides^is^changed.^For^example,^decoding^oligonucleotide^of^nucleic^acid^sequence^(A)^ used^ in^ the^ first^round^comprised^of^sequence^elements^(t1)^and^(c1)^while^ the^new^decoding^ oligonucleotide^ comprises^ of^ the^ sequence^ elements^ (t1)^ and^ (c2).^ Note^ that^ now^ all^ three^ sequences^can^clearly^be^distinguished^due^to^the^unique^combination^of^first^and^second^round^ signals. Step^8:^Third^round^of^detection.^Again,^a^new^combination^of^decoding^oligonucleotides^is^used^ leading^to^new^signal^combinations.^After^signal^detection,^the^resulting^code^words^for^the^three^ different^nucleic^acid^sequences^are^not^only^unique^and^therefore^distinguishable^but^comprise^ a^ hamming^ distance^ of^ 2^ to^ other^ code^words.^ Due^ to^ the^ hamming^ distance,^ an^ error^ in^ the^ detection^of^the^signals^ (signal^exchange)^would^not^result^ in^a^valid^code^word^and^therefore^ could^be^detected.^By^this^way^three^different^nucleic^acids^can^be^distinguished^in^three^detection^ rounds^with^two^different^signals,^allowing^error^detection. 2.^Advantages^over^prior^art^technologies Coding^strategy Compared^to^state-of-the-art^methods,^one^particular^advantage^of^the^method^according^to^the^ disclosure is^the^use^of^decoding^oligonucleotides^breaking^the^dependencies^between^the^target^ specific^probes^and^the^signal^oligonucleotides. Without^decoupling^target^specific^probes^and^signal^generation,^two^different^signals^can^only^be^ generated^for^a^certain^target^if^using^two^different^molecular^tags.^Each^of^these^molecular^tags^ can^ only^ be^ used^ once.^ Multiple^ readouts^ of^ the^ same^ molecular^ tag^ do^ not^ increase^ the^ information^about^the^target.^In^order^to^create^an^encoding^scheme,^a^change^of^the^target^specific^ RES-PA18-PCT^ probe^set^after^each^round^is^required^(SeqFISH)^or^multiple^molecular^tags^must^be^present^on^ the^same^probe^set^(like^merFISH,^intronSeqFISH). Following^the^method^according^to^the^disclosure,^different^signals^are^achieved^by^using^different^ decoding^oligonucleotides^reusing^the^same^unique^identifier^(molecular^tag)^and^a^small^number^ of^ different,^mostly^ cost-intensive^ signal^ oligonucleotides.^ This^ leads^ to^ several^ advantages^ in^ contrast^to^the^other^methods. (1) The^encoding^scheme^is^not^defined^by^the^target^specific^probe^set^as^it^is^the^case^for^all^ other^methods^ of^ prior^ art.^ Here^ the^ encoding^ scheme^ is^ transcribed^ by^ the^ decoding^ oligonucleotides.^This^leads^to^a^much^higher^flexibility^concerning^the^number^of^rounds^ and^the^freedom^in^signal^choice^for^the^codewords.^Looking^on^the^methods^of^prior^art^ (e.g.^merFISH^or^ intronSeqFISH),^ the^ encoding^ scheme^ (number,^ type^ and^ sequence^of^ detectable^signals)^for^all^target^sequences^is^predefined^by^the^presence^of^the^different^ tag^sequences^on^the^specific^probe^sets^(4^of^16^different^tags^per^probe^set^in^the^case^of^ merFISH^ and^ 5^ of^ 60^ different^ tags^ in^ the^ case^ of^ intron^ FISH).^ In^ order^ to^ produce^ a^ sufficient^ number^ of^ different^ tags^ per^ probe^ set,^ the^ methods^ use^ rather^ complex^ oligonucleotide^designs^with^several^tags^present^on^one^target^specific^oligonucleotide.^ In^order^to^change^the^encoding^scheme^for^a^certain^target^nucleic^acid,^the^specific^probe^ set^has^to^be^replaced.^The^method^according^to^the^disclosure describes^the^use^of^a^single^ unique^ tag^sequence^(unique^ identifier)^per^analyte,^because^ it^can^be^reused^ in^every^ detection^round^to^produce^a^new^information.^The^encoding^scheme^ is^defined^by^the^ order^of^decoding^oligonucleotides^that^are^used^in^the^detection^rounds.^Therefore,^the^ encoding^scheme^is^not^predefined^by^the^specific^probes^(or^the^unique^tag^sequence)^but^ can^be^adjusted^to^different^needs,^even^during^the^experiment.^This^is^achieved^by^simply^ changing^the^decoding^oligonucleotides^used^in^the^detection^rounds^or^adding^additional^ detection^rounds. (2) The^number^of^different^signal^oligonucleotides^must^match^the^number^of^different^tag^ sequences^with^methods^of^prior^ art^ (16^ in^ the^ case^of^merFISH^and^60^ in^ the^ case^of^ intronSeqFISH).^Using^ the^method^according^ to^ the^disclosure,^ the^number^of^different^ signal^ oligonucleotides^matches^ the^ number^ of^ different^ signals^ used.^ Due^ to^ this,^ the^ number^of^signal^oligonucleotides^stays^constant^for^the^method^described^here^and^never^ exceeds^the^number^of^different^signals^but^increases^with^the^complexity^of^the^encoding^ scheme^ in^ the^ methods^ of^ prior^ art^ (more^ detection^ rounds^ more^ different^ signal^ RES-PA18-PCT^ oligonucleotides^needed).^As^a^result,^the^method^described^here^leads^to^a^much^lower^ complexity^(unintended^interactions^of^signal^oligonucleotides^with^environment^or^with^ each^other)^and^dramatically^reduces^the^cost^of^the^assay^since^the^major^cost^factor^are^ the^signal^oligonucleotides. (3) In^the^methods^of^prior^art,^the^number^of^different^signals^generated^by^a^target^specific^ probe^set^is^restricted^by^the^number^of^different^tag^sequences^the^probe^set^can^provide.^ Since^each^ additional^ tag^sequence^ increases^ the^ total^size^of^ the^ target^specific^probe,^ there^ is^ a^ limitation^ to^ the^ number^ of^ different^ tags^ a^ single^ probe^ can^ provide.^ This^ limitation^is^given^by^the^size^dependent^increase^of^several^problems^(unintended^inter- and^ intramolecular^ interactions,^ costs,^ diffusion^ rate,^ stability,^ errors^ during^ synthesis^ etc.).^Additionally,^there^is^a^limitation^of^the^total^number^of^target^specific^probes^that^ can^be^applied^to^a^certain^analyte.^In^case^of^nucleic^acids,^this^limitation^is^given^by^the^ length^of^the^target^sequence^and^the^proportion^of^suitable^binding^sites.^These^factors^ lead^to^severe^ limitations^in^the^number^of^different^signals^a^probe^set^can^provide^(4^ signals^in^the^case^of^merFISH^and^5^signals^in^the^case^of^intronSeqFISH).^This^limitation^ substantially^ affects^ the^ number^ of^ different^ code^words^ that^ can^ be^ produced^with^ a^ certain^ number^ of^ detection^ rounds.^ In^ the^ approach^ of^ the^ disclosure only^ one^ tag^ is^ needed^ and^ can^ be^ freely^ reused^ in^ every^ detection^ round.^ This^ leads^ to^ a^ low^ oligonucleotide^ complexity/length^ and^ at^ the^ same^ time^ to^ the^ maximum^ encoding^ efficiency^possible^(number^of^colorsnumber^of^rounds).^The^vast^differences^of^coding^capacity^ of^our^method^compared^to^the^other^methods^is^shown^in^Figures^1^and^5.^Due^to^this^in^ approach^of^the^disclosure a^much^lower^number^of^detection^rounds^is^needed^to^produce^ the^ same^amount^of^ information.^A^ lower^number^of^detection^ rounds^ is^ connected^ to^ lower^cost,^lower^experimental^time,^lower^complexity,^higher^stability^and^success^rate,^ lower^amount^of^data^to^be^collected^and^analyzed^and^a^higher^accuracy^of^the^results. Coding^capacity^ All^three^methods^compared^in^the^Table^1^below^use^specific^probe^sets^that^are^not^denatured^ between^different^rounds^of^detection.^For^intronSeqFISH^there^are^four^detection^rounds^needed^ to^ produce^ the^pseudo^ colors^ of^ one^ coding^ round,^ therefore^ data^ is^ only^ given^ for^ rounds^ 4,^ 8,12,16^and^20.^The^merFISH-method^uses^ a^ constant^number^of^4^ signals,^ therefore^ the^data^ starts^with^the^smallest^number^of^rounds^possible.^After^8^detection^rounds^our^method^exceeds^ the^maximum^coding^capacity^reached^with^20^rounds^of^merFISH^(depicted^with^one^asterisk)^ and^ after^ 12^ rounds^ of^ detection^ the^ maximum^ coding^ capacity^ of^ intron^ FISH^ is^ exceeded^ RES-PA18-PCT^ (depicted^with^ two^asterisks).^For^ the^method^according^to^ the^disclosure usage^of^3^different^ signals^is^assumed^(as^is^with^intronSeqFISH). Coding^capacity Number^ of^ Method^ of^ the^ intron^ Rounds: present^ merFISH FISH disclosure 1 3 - - 2 9 - - 3 27 - - 4 81 12 1 5 243 - 5 6 729 - 15 7 2187 - 35 8* 6561 144 70 9 19683 - 126 10 59049 - 210 11 177147 - 330 12** 531441 1728 495 13 1594323 - 715 14 4782969 - 1001 15 14348907 - 1365 16 43046721 20736 1820 17 129140163 - 2380 18 387420489 - 3060 19 1162261467 - 3876 20 3486784401 248832 4845 ^^^^^^^^^^^^^^^^^^^^^^^^^^^^Table^1:^Comparison^of^coding^capacity As^shown^in^Figure^6^the^number^of^codewords^for^merFISH^does^not^exponentially^increase^with^ the^number^of^detection^ cycles^but^ gets^ less^ effective^with^ each^ added^ round.^ In^ contrast,^ the^ number^ of^ codewords^ for^ intronSeqFISH in^ the^method^ according^ to^ the^ disclosure increases^ RES-PA18-PCT^ exponentially.^The^slope^of^the^curve^for^the^proposed^method^is^much^higher^than^that^of^intron^ FISH,^leading^to^more^than^10,000^times^more^code^words^usable^after^20^rounds^of^detection. Note^that^this^maximum^efficiency^of^coding^capacity^is^also^reached^in^case^of^seqFISH,^where^ specific^probes^ are^denatured^after^ every^detection^ round^and^a^new^probe^ set^ is^ specifically^ hybridized^ to^ the^ target^ sequence^ for^ each^ detection^ round.^ However,^ this^method has^major^ downsides^to^technologies^using^only^one^specific^hybridization^for^their^encoding^scheme^(all^ other^methods): (1) For^the^efficient^denaturation^of^the^specific^probes,^rather^crude^conditions^must^be^used^ (high^ temperatures,^ high^ concentrations^ of denaturing^ agent,^ long^ incubation^ times)^ leading^to^much^higher^probability^for^the^loss^or^the^damage^of^the^analyte. (2) For^each^round^of^detection^an^own^probe^set^has^to^be^used^for^every^target^nucleic^acid^ sequence.^Therefore,^the^number^of^specific^probes^needed^for^the^experiment^scales^with^ the^ number^ of^ different^ signals^ needed^ for^ the^ encoding^ scheme.^ This^ dramatically^ increases^the^complexity^and^the^cost^of^the^assay. (3) Because^ the^hybridization^ efficiency^of^ every^ target^nucleic^ acid^molecule^ is^ subject^ to^ some^ probabilistic^ effects,^ the^ fluctuations^ of^ signal^ intensity^ between^ the^ different^ detection^rounds^is^much^higher^than^in^methods^using^only^one^specific^hybridization^ event,^reducing^the^proportion^of^complete^codes. (4) The^time^needed^for^the specific^hybridization^is^much^longer^than^for^the^hybridization^ of^signal^oligonucleotides^or^decoding^oligonucleotides^(as^can^be^seen^in^the^method^parts^ of^the^intronSeqFISH,^merFISH^and^seqFISH^publications),^which^dramatically^increases^ the^time^needed^to^complete^an^experiment. Due^to^these^reasons^all^other^methods^use^a^single^specific^hybridization^event^and^accept^the^ major^downside^of^lower^code^complexity^and^therefore^the^need^of^more^detection^rounds^and^a^ higher^oligonucleotide^design^complexity. The^method^according^to^the^disclosure combines^the^advantages^of^seqFISH (mainly^complete^ freedom^concerning^the^encoding^scheme)^with^all^advantages^of^methods^using^only^one^specific^ hybridization^event^while^eliminating^the^major^problems^of^such^methods. Note^that^the^high^numbers^of^code^words^produced^after^20^rounds^can also^be^used^to^introduce^ higher^hamming^distances^(differences)^between^different^codewords,^allowing^error^detection^ RES-PA18-PCT^ of^1,^2^or^even^more^errors^and^even^error^corrections.^Therefore,^even^very^high^coding^capacities^ are^still^practically^relevant. As^mentioned^above,^ the^ usage^of^multi-decoders^ further^ increases^ the^ coding^ capacity^of^ the^ encoding^ scheme.^ Instead^of^ being^ limited^ to^ the^having^ exactly^ the^ same^number^of^different^ signal^types^as^different^signal^oligonucleotides^and^corresponding^translator^elements,^the^use^of^ multi-decoders^increases^the^signal^types^that^can^be^used^to:^(N^x^(N+1))/2^(with^N^being^the^ number^of^different^signal^oligonucleotides^used).^For^the^code^used^in^table^1^with^3^different^ signal^ oligonucleotides^ this^would^mean^ the^ following^ 7^ different^ signal^ types^ could^ be^ used:^ (S1),(S2),(S3),(S1/S2),(S1/S3),(S2/S3),(S1/S2/S3).^The^effect^to^the^coding^efficiency^can^be^seen^ in^Table^1b^and^Figure^17. Coding^capacity Method^ of^ the^ Number^ of^ Method^ of^ the^ present^disclosure^ intron^ Rounds: present^ merFISH with^ multi- FISH disclosure decoders 1 3 7 - - 2 9 49 - - 3 27 343 - - 4 81 2401 12 1 5* 243 16807 - 5 6 729 117649 - 15 7** 2187 823543 - 35 8 6561 5764801 144 70 9 19683 40353607 - 126 10 59049 282475249 - 210 11 177147 1977326743 - 330 12*** 531441 1,3841^x^10^10 1728 495 13 1594323 9,6889^x^10^10 - 715 14 4782969 6,7822^x^10^11 - 1001 15 14348907 4,7476^x^10^12 - 1365 16 43046721 3,3233^x^10^13 20736 1820 RES-PA18-PCT^ 17 129140163 2,3263^x^10^14 - 2380 18 387420489 1,6284^x^10^15 - 3060 19 1162261467 1,1399^x^10^16 - 3876 20 3486784401 7,9792^x^10^16 248832 4845 Table^1b^shows^the^coding^capacity^of^the^four^methods.^All^four^methods^compared^in^the^table^ use^ specific^ probe^ sets^ that^ are^ not^ denatured^ between^ different^ rounds^ of^ detection.^ ^ For^ intronSeqFISH^there^are^four^detection^rounds^needed^to^produce^the^pseudo^colors^of^one^coding^ round,^ therefore^data^ is^only^given^ for^rounds^4,^8,12,16^and^20.^The^merFISH-method^uses^ a^ constant^ number^ of^ 4^ signals,^ therefore^ the^ data^ starts^ with^ the^ smallest^ number^ of^ rounds^ possible.^After^4^detection^rounds^the^method^with^multi-decoders^as^described^here^exceeds^the^ maximum^coding^capacity^reached^with^20^rounds^of^merFISH^(depicted^with^one^asterisk),^after^ 7^rounds^of^detection^the^maximum^coding^capacity^of^intron^FISH^is^exceeded^(depicted^with^two^ asterisks)^and^after^12^rounds^of^detection^the^maximum^coding^capacity^of^the^method^of^the^ present^disclosure is^ exceeded^ (depicted^with^ three^ asterisks).^The^usage^of^3^different^signal^ oligonucleotides^is^assumed^(as^is^with^intronSeqFISH). 3.^ Selective^ denaturation,^ oligonucleotide^ assembly^ and^ reuse^ of^ unique^ identifiers^ are^ surprisingly^efficient A^key^ factor^of^ the^method^according^ to^ the^disclosure is^ the^ consecutive^process^of^decoding^ oligonucleotide^ binding,^ signal^ oligonucleotide^ binding,^ signal^ detection^ and^ selective^ denaturation.^In^order^to^generate^an^encoding^scheme,^this^process^has^to^be^repeated^several^ times^(depending^on^the^length^of^the^code^word).^Because^the^same^unique^identifier^is^reused^in^ every^detection^cycle,^all^events^from^the^first^to^the^last^detection^cycle^are^depending^on^each^ other.^ Additionally,^ the^ selective^ denaturation^ depends^ on^ two^ different^ events:^ While^ the^ decoding^oligonucleotide^has^to^be^dissolved^from^the^unique^identifier^with^highest^efficiency,^ specific^probes^have^to stay^hybridized^with^highest^efficiency. Due^ to^ this^ the^ efficiency^ E^ of^ the^whole^ encoding^ process^ can^ be^ described^ by^ the^ following^ equation: RES-PA18-PCT^
Figure imgf000068_0001
Based^ on^ this^ equation^ the^ efficiency^ of^ each^ single^ step^ can^ be^ estimated^ for^ a^ given^ total^ efficiency^of^the^method.^The^calculation^is^hereby^based^on^the^assumption,^that^each^process^has^ the^same^efficiency.^The^total^efficiency^describes^the^portion^of^successfully^decodable^signals^of^ the^total^signals^present. The^total^efficiency^of^the^method^is^dependent^on^the^efficiency^of^each^single^step^of^the^different^ factors^described^by^the^equation.^Under^the^assumption^of^an^equally^distributed^efficiency^the^ total^efficiency^can^be^plotted^against^the^single^step^efficiency^as^shown^in^Figure^7.^As^can^be^ seen,^a^practically^relevant^total^efficiency^for^an^encoding^scheme^with^5^detection^cycles^can^only^ be^ achieved^ with^ single^ step^ efficiencies^ clearly^ above^ 90%.^ For^ example,^ to^ achieve^ a^ total^ efficiency^ of^ 50%^ an^ average^ efficiency^ within^ each^ single^ step^ of^ 97.8%^ is^ needed.^ These^ calculations^are^even^based^on^the^assumption^of^a^100%^signal^detection^and^analysis^efficiency.^ Due^ to^broad^DNA^melting^ curves^of^ oligonucleotides^of^ a^ variety^of^ sequences,^ the^ inventors^ assumed^ prior^ to^ experiments^ that^ the^ selective^ denaturation^ would^ work^ less^ efficient^ for^ denaturation^ of^ decoding^ oligonucleotides^ and^ that^ sequence^ specific^ binding^ probes^ are^ not^ stable^enough.^In^contrast^to^this^assumption,^we^found^a^surprising^effectiveness^of^all^steps^and^ a^high^stability^of^sequence^specific^probes^during^selective^denaturation. Experimentally,^the^inventors^achieved^a^total^decoding^efficiency^of^about^30%^to^65%^based^on^ 5^detection^cycles.^A^calculation^of^the^efficiency^of^each^single^step^(Bsp,^Bde,^Bsi,^Ede,^Ssp)^by^the^ formula^given^above^revealed^an^average^efficiency^of^about^94.4%^to^98%.^These^high^efficiencies^ are^very^surprising^and^cannot^easily^be^anticipated^by^a^well-trained^person^in^this^field.^ 4. Experimental^data 1.^Example^for^signal^encoding^of^two^subgroups^of^nucleic^acid^sequences^with^spatial^overlap The^ table^ below^ shows^ examples of^ implementations^ using^ two^ subgroups^ (analytic^ sets) in^ consecutive^runs: RES-PA18-PCT^ Run^1 Run^2 Transcript^ Total^ Fluidics^ Fluidics^ Plexity^(Set^ Plexity^1 Plexity^2 rounds rounds rounds 1^/^Set^2) 100^ 1.0 9 100^/^2 8 1 2 (129) 300^ 2.0 16 300^/^25 10 6 25^(29) (419) 2.1 Improved^ 500^ 16 500^/^50 10 6 50^(60) transcript^ (685) detection For^“run^1.0”^n=8^rounds^of^hybridization^and^imaging^was^used^to^detect^100^genes.^For^“run^2.0”^ the^number^of^rounds^is^increased^to^n=10^and^can^thereby^target^300^genes.^For^“run^2.1”^an^ enhanced^transcript^detection^probe^is^used^which^allows^to^target^500^transcripts^with^the^same^ number^of^rounds^(n=10).^^^ Consecutive^runs^are^run^on^the^same^tissue^sections^to^detect^transcripts^beyond^the^diffraction^ limit. The^main^steps^of^the^method^are i. Hybridization^of^target^probes^of^the^two^analytic^sets happen^simultaneously^before^the^ first^run.^ ii. Tails^of^the^target^probes^are^unique^for^both^runs.^e.g.,^100,^300 and^500 tails^(for^100,^ 300,^or^500 transcripts,^respectively)^are^used^for^the^first^run with^the^first^analytic^set and^2,^ 25,^ 50 tails^ (for^2,^ 25,^ 50 transcripts,^ respectively)^ for^ the^ second^ run with^ the^ second^analytic^set.^This^requires^also^different^decoder^sets^for^both^runs.^ iii. Both^runs^generate^independent^datasets^that^can^be^combined^in-silico^afterwards. RES-PA18-PCT^ Thereby,^ 1)^ enhancing^ the^multiplexing^ capability^without^ increasing^optical^ crowding^ and^2)^ detecting^ spatial^ overlapping^ transcripts^ which^ would^ be^ otherwise^ not^ possible^ due^ to^ the^ diffraction^limit^of^the^microscope. This^is^e.g.^allows^for^detection^of^fusion^genes^(cancer)^or^for^ detection^of^co-localization^of^different^transcript^types^(e.g.^transcriptional^hubs^in^the^nucleus).^ In^addition,^ transcripts^with^higher^expression^ levels^can^also^be^ analyzed^because^ the^“signal^ spread”^ of^ the^ high^ number^ of^ abundant^ signals^ vs.^ the^ detection^ of^ other^ (especially^ lowly^ expressed)^genes^is^improved. In^principle,^both^runs^can^be^also^interwoven,^e.g.: ^ Detection^round^1^=^1st^round^of^detection^of^first^analytical^set ^ Detection^round^2^=^1st^round^of^detection^of^second^analytical^set ^ Detection^round^3^=^2nd^round^of^detection^of^first^analytical^set ^ Detection^round^4^=^2nd^round^of^detection^of^second^analytical^set ^ Etc.
RES-PA18-PCT^ Sequences Sequence^of^Tails^attached^to^particle used^in^this^disclosure N Cat^ Spec Gene Target Tail Code Tail^Sequence o No. ies PDX1 ENSG000001 XYRYXR tagacAGATGTGGTTAGT 8 Hs EGFR 2 J 46648 YX CT 1 PQ02 ENSG000000 XRYYRX tagacCTAATCTTACTTA Hs BRCA1 12 5 S 12048 YX TAACG 1 ENSG000001 YRXRRX tagacGAAGGATCGGAAA PJ71C Hs APC 13 1 34982 YX G 1 PY02 ENSG000001 RRXXYY tagacAGAATCAAAAGTG Hs AOX1 24 8 L 38356 YX AAC 1 PPA1 ENSG000001 RRYXYX tagacATCTAGGCACTTT Hs E2F1 30 4 D 01412 RX AC 1 PZX1 ENSG000001 RYXXRY tagacAAGCTTCTATTAG Hs BCR 62 9 3 86716 XY CT P612 ENSG000001 YXXYRX 5 Hs CDK1 64 tagacGTTTTCGGCATTG 7 70312 YR PEX1 ENSG000001 RYXXYX tagacTGCTATAAGAGTC 9 Hs TP53 69 K 41510 RY CC 1 PJ31 ENSG000001 RYXYRX tagacGCTTACTCGAAGA Hs DDX5 70 0 G 08654 XR C P1A1 ENSG000000 XYRXYX tagacGCAAACATCTTTG 1 Hs BIRC5 72 S 89685 RR G P412 CDC25 ENSG000001 XYYRXY tagacAATACTAGAAAGT 3 Hs 82 5 C 58402 XY ATCTG P812 ENSG000001 RXXRYX tagacTACTGATACAAGT 6 Hs CENPF 94 9 17724 RR AGAT 1 PT71 ENSG000001 YXYYRX tagacAACCTAAAGTTGC Hs BCL2 106 7 M 71791 XY TA 1 PNT1 ENSG000001 XRXYYY tagacCTTAAGTCTTAAT Hs AKT1 110 3 X 42208 XY GAAAG RES-PA18-PCT^ PCX1 ENSG000001 RXXRRR tagacGAAACGTATATTT Hs KRAS 113 H 33703 XY TCC 1 PQ61 AURK ENSG000001 YXRXXR tagacCCGTATAGGATAC Hs 115 6 B 78999 RR AG P4X1 ENSG000001 XRRRXY tagacCATATTCGTAACA Hs HPRT1 117 9 65704 XR CG 1 PJX1 ENSG000001 YYYXYX tagacTTGCTACTAGTAA Hs ACO2 118 2 Q 00412 XR GTG P212 ENSG000001 RRRXXR tagacCCATAGCTTATTT Hs CCNE1 126 3 05173 XY AAGC Sequences^of^Decoders used^in^this^disclosure Tail C1^(Read^Out^- Yellow) C2^(Read^Out^- Red) ACCTTAAGAATGGAATAGACTAA GGCTAATTTATTACCTAGACTA T2 CCACATCT ACCACATCT ACCTTAAGAATGGAATCGTTATA GGCTAATTTATTACCTCGTTAT T12 AGTAAGATTAG AAGTAAGATTAG ACCTTAAGAATGGAATCTTTCCG GGCTAATTTATTACCTCTTTCCG T13 ATCCTTC ATCCTTC ACCTTAAGAATGGAATGTTCACT GGCTAATTTATTACCTGTTCACT T24 TTTGATTCT TTTGATTCT ACCTTAAGAATGGAATGTAAAGT GGCTAATTTATTACCTGTAAAG T30 GCCTAGAT TGCCTAGAT ACCTTAAGAATGGAATAGCTAAT GGCTAATTTATTACCTAGCTAA T62 AGAAGCTT TAGAAGCTT ACCTTAAGAATGGAATCAATGCC GGCTAATTTATTACCTCAATGCC T64 GAAAAC GAAAAC ACCTTAAGAATGGAATGGGACTC GGCTAATTTATTACCTGGGACTC T69 TTATAGCA TTATAGCA ACCTTAAGAATGGAATGTCTTCG GGCTAATTTATTACCTGTCTTCG T70 AGTAAGC AGTAAGC RES-PA18-PCT^ ACCTTAAGAATGGAATCCAAAGA GGCTAATTTATTACCTCCAAAG T72 TGTTTGC ATGTTTGC ACCTTAAGAATGGAATCAGATAC GGCTAATTTATTACCTCAGATA T82 TTTCTAGTATT CTTTCTAGTATT ACCTTAAGAATGGAATATCTACT GGCTAATTTATTACCTATCTACT T94 TGTATCAGTA TGTATCAGTA T10 ACCTTAAGAATGGAATTAGCAAC GGCTAATTTATTACCTTAGCAA 6 TTTAGGTT CTTTAGGTT T11 ACCTTAAGAATGGAATCTTTCAT GGCTAATTTATTACCTCTTTCAT 0 TAAGACTTAAG TAAGACTTAAG T11 ACCTTAAGAATGGAATGGAAAAT GGCTAATTTATTACCTGGAAAA 3 ATACGTTTC TATACGTTTC T11 ACCTTAAGAATGGAATCTGTATC GGCTAATTTATTACCTCTGTATC 5 CTATACGG CTATACGG T11 ACCTTAAGAATGGAATCGTGTTA GGCTAATTTATTACCTCGTGTTA 7 CGAATATG CGAATATG T11 ACCTTAAGAATGGAATCACTTAC GGCTAATTTATTACCTCACTTAC 8 TAGTAGCAA TAGTAGCAA T12 ACCTTAAGAATGGAATGCTTAAA GGCTAATTTATTACCTGCTTAA 6 TAAGCTATGG ATAAGCTATGG

Claims

RES-PA18-PCT^ CLAIMS 1. A^ monitoring^ probe comprising^ at^ least^ one^ element having^ a^ nucleotide^ sequence,^ wherein^said element^is^selected^from^the^group^consisting^of^ - a^ translator^ (c)^ element comprising a^ nucleotide^ sequence^ allowing^ the^ binding/hybridization^of^a^signal^oligonucleotide; - an^ identifier^ element^ (t)^ comprising^ a^ nucleotide^ sequence^ allowing^ the^ binding/hybridization^of^a^decoding^element,^wherein^in^particular^the^identifier^element (t)^ comprising^ a^nucleotide^ sequence^which^ is^ essentially^ complementary^ to^ at^ least^ a^ section^ of^ the^ unique^ set^ identifier^ sequence^ of^ an^ identifier^ element^ (T)^ of^ a^ corresponding^decoding^element; - a^target^nucleic^acid^sequence allowing^the^binding/hybridization^of^a^binding^element. 2.^ The monitoring^ probe according^ to^ claim^ 1^ comprising^ at^ least^ one^ identifier^ element (t)^ comprising^a^nucleotide^sequence^which^is^essentially^complementary^to^at^least^a^section^of^the^ unique^set^identifier^sequence^of^an^identifier^element^(T)^of^a^corresponding^decoding^element.^ 3. The^monitoring^probe according^to^any^one^of^claims 1 to^2,^wherein^the^nucleotide^acid^ sequence of^ the^monitoring^probe is^attached^ to^a^particle^selected^ from^a^non-magnetic^bead^ particle,^magnetic^bead^particle,^a^streptavidin^coated^particle,^a^biotin^coated^particle,^and/or^any^ combination^thereof. 4.^ The^monitoring^probe^according^to^any^one^of^claims^1^to^3,^wherein^the^probe^further^ comprises^ a^ binding^ element complementary^ to^ at^ least^ a^ section^ of^ a^ target^ nucleotide^ acid^ sequence. 5. The^monitoring^probe according^to^any^one^of^claims^1^to^3,^wherein^the^target^nucleotide^ acid^sequence is^attached^ to^a^particle^selected^ from^a^non-magnetic^bead^particle,^a^magnetic^ bead^particle,^ a^ streptavidin^ coated^particle,^ a^ biotin^ coated^particle,^ and/or^ any^ combination^ thereof. 6. The^monitoring^probe according^to^any^of^claims^4 or^5,^wherein^the^nucleic^acid^sequence^ contains^a^spacer^element^between^the^portion^to^be^immobilized^onto^the^particle^and^the^nucleic^ acid^binding^element and/or^identifier^element (t). RES-PA18-PCT^ 7. The^monitoring^probe according^to^any^of^claims^1^to^6,^wherein^the^monitoring^probe is^ immobilized^on^a^slide^surface through^a^poly-lysine^coating^of^the^slide^surface. 8. The^monitoring^probe according^to^claim^7,^wherein^the^monitoring^probe is^immobilized^ to^the^poly-lysine^coated^slide^surface^using^homobifunctional^or^heterobifunctional^cross-linking^ reagents^via^a^reactive^group. 9. The^monitoring^probe according^ to^claim^8,^wherein^ the^monitoring^probe or^ the^slide^ surface comprise^a^reactive^group^consisting^of^an^alkyne^or^azide,^and^the^other^contact^surface^ comprises^ the^ partner^ azide^ or^ alkyne,^ such^ that^ the^ immobilization^ can^ occur^ using^ click^ chemistry. 10. The^monitoring^probe according^to^any^of^claims^1^to^9,^wherein^the^size^of^the^monitoring^ probe is^250^nm^or^less. 11. A^ method^ to^ monitor^ a^ in^ situ hybridization^ multiplex^ reaction^ wherein^ the^ method^ comprises^at^least^the^steps^of: 1.^contacting^the^analyte^nucleotide^sequence^with^at^least^one detecting nucleic^acid^sequence in^ order^to^conduct^an^in-situ hybridization;^and 2.^adding the^monitoring^probe^according^to^any^of^claims^1^ to^10 before,^during^and/or^after^ adding^the^detecting nucleic^acid^sequence in^step^1. 12. The method^according^to^claim^11,^ wherein^the^in-situ hybridization^multiplex^reaction^is^characterized^by^comprising adding^a: a.) a^binding^element which^is^essentially^complementary^to^at^least^a^section^of^an^analyte^ nucleotide^ sequence^ to^ be^ detected^ and^ an^ identifier^ element^ (T)^ comprising^ a^ nucleotide^ sequence^which^is^unique^to^the^analyte^nucleotide^sequence;^and/or b.) a^decoding^element comprising^an^identifier^element^(t)^comprising^a^nucleotide^sequence^ which^is^essentially^complementary^to^at^least^a^section^of^the^unique^set^identifier^sequence^of^ the^ identifier^ element^ (T)^of^ the^ corresponding^binding^ element,^ and^a^ translator^ element^ (c)^ comprising^a^nucleotide^sequence^allowing^a^specific^hybridization^of^a^signal^oligonucleotide; and/or c.) a^ signal^ oligonucleotide comprising^ a^ translator^ connector^ element^ (C)^ comprising^ a^ nucleotide^sequence which^ is^essentially^complementary^ to^at^ least^a^section^of^the^nucleotide^ RES-PA18-PCT^ sequence^of^a^translator^element^(c)^comprised^in^a^decoding^oligonucleotide,^and^a^signal^element^ facilitating^a^signal^which^is^specific^for^the^polymorphic^analyte; and wherein the^method^comprises^the monitoring^probe^according^to^any^of^the^claims^1^to^10;^and^ wherein^the^method comprises at^least^the^steps^of: 1.^contacting^the^binding^element with^the^analyte^nucleotide^sequence; 2.^contacting^the^binding^element with^the^decoding^element;^and 3.^contacting^the^signal^oligonucleotide with^the^decoding^element; and wherein^the^monitoring^probe^is^added before,^during^and/or^after^adding^the^decoding^element^ in^step^2. 13. The^method^according^to^any^of^claims 11 or^12,^wherein^the^method^comprises^a^set^of^ monitoring^ probes,^ wherein^ for^ each^ of^ the^ different^ identifier^ elements^ (t)^ of^ the^ decoding^ elements the^set^of^monitoring^probes^comprises^at^least^one^monitoring^probe with^the^same^ identifier^elements^(t). 14. The^method^according^to^any^of^claims^11 to^13,^wherein^the^nucleic^acid^of^the^monitoring^ probe is^attached^to^particle^selected^from^a^magnetic^bead^particle,^a^streptavidin^coated^particle,^ a^biotin^coated^particle,^and/or^any^combination^thereof. 15. The^method^according^to^any^of^claims^11 to^14,^wherein^monitoring^probe is^designed^to^ produce^a^fluorescent^signal^substantially^equivalent^to^the^signal^intensity^obtained^for^the^target^ biological^analytes. 16. A^ detection^mix comprising^ the^monitoring^ probe according^ to^ any^ of^ claims^ 1 to^ 10,^ further^ comprising^ at^ least^ one^ element^ selected^ from^ a^ decoy^ element consisting^ either^ of^ a^ magnetic^ bead^ particle,^ a^ streptavidin^ coated^ particle,^ a^ biotin^ coated^ particle,^ and/or^ any^ combination^ thereof;^ optionally^ bound^ to^ a^ nucleic^ acid^ oligonucleotide, which^ does^ not^ correspond^to^the^identifier^element^(t);^a^decoding^element comprising^an^identifier^element^(t)^ comprising^a^nucleotide^sequence^which^is^essentially^complementary^to^at^least^a^section^of^the^ unique^set^identifier^sequence^of^the^identifier^element^(T)^of^the^corresponding^binding^element,^ and^a^translator^element^(c)^comprising^a^nucleotide^sequence^allowing^a^specific^hybridization^ of^a^signal^oligonucleotide;^a^binding^element which^ is^essentially^complementary^ to^at^ least^a^ section^of^an^analyte^nucleotide^sequence^to^be^detected^and^an^identifier^element^(T)^comprising^ a^ nucleotide^ sequence^ which^ is^ unique^ to^ the^ analyte^ nucleotide^ sequence;^ and^ a^ signal^ RES-PA18-PCT^ oligonucleotide comprising^a^translator^connector^element^(C)^comprising^a^nucleotide^sequence^ which^is^essentially^complementary^to^at^least^a^section^of^the^nucleotide^sequence^of^a^translator^ element^(c)^comprised^ in^a^decoding^oligonucleotide,^and^a^signal^element^ facilitating^a^signal^ which^is^specific^for^the^polymorphic^analyte;^as^well^as^any^combination^thereof. 17. A^method^ to^monitor^ a^ in^ situ hybridization^multiplex^ reaction^with^ the^detection^mix^ according^to^claim^16,^ wherein^the^method^comprises^at^least^the^steps^of: 1.^contacting^the^analyte^nucleotide^sequence^with^at^least^one detecting nucleic^acid^sequence in^ order^to^conduct^an^in-situ^hybridization;^and 2.^adding^the^detection^mix before,^during^and/or^after^adding^the^detecting nucleic^acid^sequence in^step^1. 18. The^method^according^to^claim^17,^ wherein^the^in-situ hybridization^multiplex^reaction^is characterized^by^comprising^adding^a:^ a.) a^binding^element which^is^essentially^complementary^to^at^least^a^section^of^an^analyte^ nucleotide^ sequence^ to^ be^ detected^ and^ an^ identifier^ element^ (T)^ comprising^ a^ nucleotide^ sequence^which^is^unique^to^the^analyte^nucleotide^sequence;^and/or b.) a^decoding^element comprising^an^identifier^element^(t)^comprising^a^nucleotide^sequence^ which^is^essentially^complementary^to^at^least^a^section^of^the^unique^set^identifier^sequence^of^ the^ identifier^ element^ (T)^of^ the^ corresponding^binding^ element,^ and^a^ translator^ element^ (c)^ comprising^a^nucleotide^sequence^allowing^a^specific^hybridization^of^a^signal^oligonucleotide;^ and/or c.) a^ signal^ oligonucleotide comprising^ a^ translator^ connector^ element^ (C)^ comprising^ a^ nucleotide^sequence^which^ is^essentially^complementary^ to^at^ least^a^section^of^the^nucleotide^ sequence^of^a^translator^element^(c)^comprised^in^a^decoding^oligonucleotide,^and^a^signal^element^ facilitating^a^signal^which^is^specific^for^the^polymorphic^analyte;^and wherein^the^method^comprises^the^detection^mix according^to^claim^16;^and^ wherein^the^method^comprises^at^least^the^steps^of: 1.^contacting^the^binding^element with^the^analyte^nucleotide^sequence; 2.^contacting^the^binding^element with^the^decoding^element;^and 3.^contacting^the^signal^oligonucleotide with^the^decoding^element;^and wherein^the^detection^mix is^added before,^during^and/or^after^adding^the^decoding^element^in^ step^2. RES-PA18-PCT^ 19. A^ method,^ wherein^ the monitoring^ probe according^ to^ any^ of^ claims^ 1 to^ 10,^ or^ the^ detection^mix according^ to^ claim^ 16,^ can^ be^ detected^ by^ a^ signal^ element^with^ a^ fluorophore^ different^ from^ the^ fluorophore^ used^ in^ the^ detection^ of^ the^ target^ nucleic^ acid^ sequence^ that^ permits^normalizing^the^signal^of^the^immobilized^monitoring^probe. 20. The^method^ according^ to^ claim^ 19,^ wherein^ a^ first^ signal^ element^ with^ a^ fluorophore allows^ for^measurement^of^hybridization^efficiency^of^ the^decoder^ and^readout^probe^ labelled with^a^second^signal^element^with^a^fluorophore by^measuring^the^ratio^between^the^first^and^the^ second^fluorophore. 21. The^method^according^to^any^of^claims^claim^19 or^20,^wherein^the^first^signal^element with^a^fluorophore^enables^the^detection^of^general^nonspecific^hybridization^by^identifying^cycles^ in^ which^ the^ particle^ is^ labelled with^ a^ second^ signal^ element with^ a^ fluorophore^ that^ is^ inconsistent^with^the^decoder^element. 22. A^method^for^generating^a^detection^mix according^to^claim^16. 23. A^method^for^controlling^the^density^of^the^monitoring^probes according^to^any^of^claims^ 1 to^10 on^a slide^surface by^attaching^azide^or^alkyne^partner^to^a poly-lysine^before^coating^it. 24. The method^ for^ controlling^ the^ density^ of^ monitoring^ probes^ according^ to^ claim^ 23, wherein^unreactive^azide^or^alkyne is^removed, followed^by^a^reaction step with^detection^mix according^to^claim^16. 25. A^method, wherein^ the^monitoring^ probes^ according^ to^ any^ of^ claims^ 1 to^ 10,^ or^ the^ detection^mix according^to^claim^16, are^placed^underneath^a tissue in^order^to^assess^the^efficacy^ of^ a tissue^ sample^ preparation^ process^ comprising in^ situ oligonucleotides^ which penetrate^ through^the^tissue^and^hybridize^to^their^intended^targets. 26. A method^to^monitor^temperature^ranges^of^an in-situ^environment^in^which^monitoring^ probes^according^to^any^of^claims^1 to^10 or^the^detection^mix according^to^claim^16, have^different^ delta^G^values^corresponding^to^different^melting^points^of^the^secondary^structure^in^order^to^ monitor^temperature^fluctuations^that^occur^during^the^hybridization process. RES-PA18-PCT^ 27. The method^according^to^claim^26,^wherein^the^delta^G^value^of^the^monitoring^probes is adjusted^by the^sequence^length^or^sequence^composition.^ 28. The^ method^ according^ to^ any^ of^ claims^ 26 or^ 27,^ wherein^ the^ delta^ G^ value^ of^ the^ monitoring^probes is^adjusted^by^using^modified^bases^which^have^higher^stability^than^standard^ bases. 29. The^method^according^to^any^of^claims^26 to^28,^wherein^temperature^fluctuation^can^be^ read-out^by^specific^change^in^the^fluorescence^and/or^colour. 30. A^ set^ of^ monitoring^ probes according^ to^ any^ of^ claims^ 1 to^ 10,^ or^ the^ detection^ mix according^to^claim^15,^adapted^to^any^of^the^methods^according^to^any^of^claims^25^to^29. 31. A^ kit^ to^ enable^ optimizing^ in^ situ^ hybridization^methods^ comprising slides^ containing^ immobilized^monitoring^probes according^to^any^of^claims^1^to^10,^or^the^detection^mix according^ to^claim^16,^and/or^binding^sites^for monitoring^probes according^to^any^of^claims^1^to^10 32. The^kit^according^to^claim^31 for^monitoring^a^tissue^sample^preparation.^ 33. A^ control^ slide^ for^ quality^ control comprising a^ set^ of^ immobilized^monitoring^ probes according^to^any^of^claims^1^to^9,^or^the^detection^mix according^to^claim^16^and/or^binding^sites^ for monitoring^probes according^to^any^of^claims^1^to^10. 34. The^ control^ slide^ according^ to^ claim^ 33,^ for^ assessing^ in-situ hybridization^ instrument installations.
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