WO2019209657A1 - Marquage de sondes moléculaires avec des isotopes non métalliques et métalloïdes pour une analyse multiplexée à haute résolution - Google Patents

Marquage de sondes moléculaires avec des isotopes non métalliques et métalloïdes pour une analyse multiplexée à haute résolution Download PDF

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WO2019209657A1
WO2019209657A1 PCT/US2019/028359 US2019028359W WO2019209657A1 WO 2019209657 A1 WO2019209657 A1 WO 2019209657A1 US 2019028359 W US2019028359 W US 2019028359W WO 2019209657 A1 WO2019209657 A1 WO 2019209657A1
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cells
cell
labeling
mass
labeling moiety
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Shih-Yu Chen
Felice A. BAVA
Xavier ROVIRA CLAVE
Garry P. Nolan
Sizun JIANG
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Leland Stanford Junior University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • 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
    • C12Q1/6804—Nucleic acid analysis using immunogens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/60—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances involving radioactive labelled substances

Definitions

  • Single cell mass cytometry and multiplexed ion beam imaging facilitate high dimensional, quantitative analysis of the effects of bioactive molecules on cell populations at single-cell resolution.
  • datasets are generated with antibody or oligonucleotide probes panels in which each antibody or oligonucleotide is conjugated to a polymer that chelates stable metal isotopes, usually in the lanthanide series of the periodic table, through metal-chelating chemical moieties, e.g., diethylene triamine pentaacetic acid (DTPA) and l,4,7,l0-tetraazacyclododecane-l,4,7,l0- tetraacetic acid (DOTA).
  • DTPA diethylene triamine pentaacetic acid
  • DOTA diethylene triamine pentaacetic acid
  • the current generation of isotope-chelating polymers can bear up to 120 lanthanide ions per antibody molecule with the detection limit of 300-1000 target protein copies and up to 50 simultaneously measureable parameters.
  • Current implementations of MIBI that use antibodies that have a metal chelate have a resolution that is similar to that of a light microscope, i.e., of 200-300 nm.
  • the labeling moiety may comprise multiple internal atoms of a non-biological stable nonmetal or metalloid isotope, wherein: i. the labeling moiety is or comprises a binding agent, e.g., an antibody, that specifically binds to a complementary site in or on a cell, or ii. the labeling moiety comprises a chemo selective group, e.g., may be a polymer such as an oligonucleotide that comprises a chemo selective group. In either case, the labeling moiety is not an anti-IgG antibody.
  • a binding agent e.g., an antibody
  • the labeling moiety comprises a chemo selective group
  • the labeling moiety is not an anti-IgG antibody.
  • the labeling moiety may be used for the multiplexed analysis of target biomolecules by elemental mass spectrometry, secondary ion mass spectrometry (SIMS), mass cytometry (CyTOF), laser-ablation inductively-coupled mass spectrometry (LA- ICP-MS), X-ray microscopy (XRM), X-ray fluorescence (XRF), laser-induced breakdown spectroscopy (LIBS), focused ion beam (FIB), energy-dispersive X-ray spectroscopy (EDX), electron microscopy, or nuclear magnetic resonance spectroscopy.
  • elemental mass spectrometry secondary ion mass spectrometry
  • CDT mass cytometry
  • LA- ICP-MS laser-ablation inductively-coupled mass spectrometry
  • XRM X-ray microscopy
  • XRF X-ray fluorescence
  • LIBS laser-induced breakdown spectroscopy
  • FIB focused ion beam
  • EDX energy-dispers
  • Labeling binding agents internally using e.g., using an non-biological isotope of carbon, nitrogen, chalcogen, or a halogen, etc. not only expands the number of available isotopes, thereby exponentially increasing the measureable parameters, the present labeling moiety may be superior to chelate-labeled binding agents for a variety of reasons.
  • the labeling moiety may contain a large number of atoms of the isotope (e.g., at least 5, at least 10, at least 100, at least 500, or at least 1,000), which should, in theory, make any assays that use the labeling moiety more sensitive.
  • monoclonal antibodies contain thousands of nonmetal atoms (for example Trastuzumab, is composed of C 6470 H 10012 N 1726 O 2013 S 42 ) and, in principle, synthesis of an antibody using amino acids that contain carbon 13 would greatly enhance the signal intensity by at least 50-fold compared to the current antibody reagents that are labeled by a metal chelate.
  • the labeling moiety may contain a defined number of isotopically labeled atoms.
  • the number of isotopically labeled atoms that are in a labeling moiety made by adding monomers that contain isotopically labeled atoms to a growing chain can be highly controlled by controlling the number of labeled monomers used. If each labeling moiety has the same or similar number of labeled atoms, then the signals from each of those labeling moieties should be very similar. The number of labeled atoms in a labeling moiety can therefore be increased or decreased, as desired.
  • labeling moieties that contain defined ratios of two or more labeled atoms can be made. This provides more options for multiplexing because different labeling moieties that are labeled with different ratios of the labeled atoms are readily distinguishable in some analysis method (e.g., SIMS).
  • SIMS some analysis method
  • some embodiments of the present labeling moiety provide higher resolution (e.g., sub-30nm resolution) results. At this resolution single binding events can be detected.
  • the present labeling moiety do not require a chelator that can potentially interfere with binding.
  • the absence of interference from the chelator allows for more accurate measurement of the biomolecules.
  • Fig ⁇ 1 shows data obtained from various embodiments of the present labeling moiety
  • (b) an Iodine-modified oligonucleotide was conjugated to an anti-H3K27Ac antibody provides a specific signal via secondary ion beam imaging.
  • Such signal can be amplified by varying the length or the structure of the oligonucleotide.
  • the oligonucleotide sequence is set forth in SEQ ID NO: 7.
  • oligonucleotides are assembled into a Cas9 protein, which retains activity and renders visualization of pericentromeres.
  • (d) shows a signal deriving from a 13 C/ 12 C modified anti-dsDNA antibody, which enables visualization of nuclear dsDNA by secondary ion beam imaging. This antibody was produced by culturing hybridoma cells in 13 C- rich media.
  • Fig. 2 (a) Schematic representation of ATAC-mass. Fluorine-labeled adaptors were loaded with Tn5 enzyme to specifically label open chromatin. Mass tags were detected directly by Ion Beam Imaging or through metal ( 156 Gd)-conjugated antibody by mass cytometry.
  • Fig. 3 (a) Representative image of DNA in Hela cells. IdU labelled DNA was detected by high-resolution secondary ion mass spectrometry (b) Serial scanning of IdU labeled,
  • Fig. 4 (a) ATAC-mass as measured by mass-cytometry. The indicated cell-cycle phases of Jurkat cells were measured along with ATAC-mass signal (b) Phosphorylation of retinoblastoma protein was measured along with ATAC-mass signal. Highlighted area defines cells in GO phase of cell-cycle (c) Heat map depicting each signaling protein and ATAC-mass in control, IL12 and IL18 priming conditions. The color scale indicates changes in median intensity(arcsinh ratio) compared to control (no priming) (d) Spearman correlation matrix from control, IL-12 and IL18 priming conditions.
  • Fig. 5 (a) Boolean gating analysis of polyfunctional NKL cells in response to PMA plus ionomycin after control, IL12 or IL18 priming. Color represents the indicated number of functional effectors coexpressed in a single cell (b) Heat map depicting each effector functions in control, IL12 and IL18 priming conditions. The color scale indicates changes in median intensity (arcsinh ratio) compared to control (no priming) (c) Representative biaxial plots showing the polyfunctional NKL cells after IL18 priming. Number indicates the percentage of cells in each quadrat.
  • Fig. 6 Workflow of High-Resolution 3D MIBI for drug visualization ⁇
  • Cells are treated with the drug of study, stained with antibodies conjugated to mass-oligos (SEQ ID NO: 11) and processed for MIBI.
  • the cells are rastered by a cesium primary ion beam, with a lateral resolution up to 50 nm and an axial resolution previously calculated to be 14.3 nm.
  • Serial scans of the sputtered negatively charged secondary ions and secondary electrons are recorded.
  • 3D rendering of the data summarizes sub-cellular location of the drug and markers of interest.
  • Fig. 7 Validation of High-Resolution MIBI.
  • (a-d) A schematic of the antibody conjugation strategy, immunofluorescence confocal microscopy image and high-resolution MIBI image of (a) a-Nucleolin- 19 F/FITC, (b) a-CENP-A- 81 Br/Cy3, (c) a-H3K27Ac- 127 FCy5 and (d) a-SC 35 -biotin:Streptavidin- 197 Au/FITC.
  • (e) Five-color high-resolution image of a HeLa cell nucleus. Scale bars: 4 pm.
  • the sequence of (a) is set forth in SEQ ID NO: 11.
  • the sequence of (b) is set forth in SEQ ID NO: 11.
  • the sequence of (c) is set forth in SEQ ID NO:
  • Fig. 8 Sub-cellular visualization of cisplatin distribution by High-Resolution MIBI.
  • e Two-color images of cell in (c). Summation of 10 (b) or 50 (c-e) planes. Scale bars: 4 pm.
  • Fig. 9 Three dimension rendering of high-resolution MIBI data (a) 3D rendering of a
  • TyKNU cell treated with 5 pM 194 Cisplatin for 24 hours (b) Single-color 3D rendered images of cell in (a).
  • Fig. 10 Validation of ATAC-mass.
  • ATAC DNA adaptors are modified to include fluorine-containing ATTO 514 at the 5’ end and 5-fluoro-2’-deoxyuridine and 5-fluoro-2’- deoxycytidine.
  • Tn5 transposase loaded in vitro with fluorine-labeled adaptors (Fluoro-Tn5) is used to specifically label open chromatin
  • the adaptor can be detected by five different modalities: (1) ATAC-seq with sequencing adaptors, (2) high-resolution ion beam imaging due to the presence of fluorine, (3) mass cytometry through metal ( 156 Gd)-conjugated antibody against ATTO 514, and (4) fluorescence microscopy and (5) flow cytometry via detection of the intrinsic fluorescence of ATTO 514.
  • X-axis genomic coordinates
  • Y-axis normalized ATAC-seq read counts
  • f Representative fluorescence microscopy image of ATAC-mass signal due to the presence of ATTO 514 in the modified adaptor (red) in HeFa cells. EDTA treatment served as a negative control. Nuclei were stained with DAPI (blue). The boxed areas are enlarged on the right. Scale bars, 5 pm.
  • g Histogram of ATAC-mass signals at indicated cell cycle phases in Jurkat cells analyzed by mass cytometry. The color scale represents the median intensity of ATAC-mass. Y- axis, cell cycle phases.
  • the color scale indicates changes in median intensity (arcsinh ratio) compared to control (b) tSNE plots of mass cytometry results depicted in Figure 12 a from NKL cells treated as indicated. Numbers indicate the percentage of cells in the gate (c) AT AC-mass signal intensity in the corresponding tSNE plot as in Figure 12 b. Color scale represents AT AC-mass intensity (d) Spearman correlation matrix of control and IL-12- and IL- l8-treated cells of mass cytometry results depicted in Figure 12 a. Yellow indicates a positive correlation; blue indicates no correlation.
  • Fig. 12 ATAC-seq reveals increased genome accessibility of quiescent enhancers in IF- l8-treated NK cells
  • the color scale indicates changes in median intensity (arcsinh ratio) compared to control (g) Representative biaxial plots showing the increased frequency of NKL cells with multiple cytokines produced after stimulation of IL- l8-treated cells compared to control and IL-l2-treated cells. Blue lines are the thresholds for multifunctional capacity. Numbers indicate the percentage of cells in each quadrant.
  • Fig. 14 Super-resolution visualization of nuclear structures using srIBI.
  • A Workflow of super-resolution ion beam imaging (srIBI). (1) Cells are treated with the drug of study, fixed, and stained with MoC-Abs. Endogenous elemental masses can also be detected. (2) Cells are rasterized by a cesium primary ion beam, and the secondary ions are collected by a magnetic sector mass spectrometer. (3) Spatial information is serially recorded in up to 8 channels simultaneously. (4) A composite image of the cell is reconstructed by combining and pseudo coloring the total number of channels at each depth.
  • srIBI yields a 3D rendering based on the depth profile that reveals the specific sub-cellular localization of endogenous and labeled targets.
  • Ion count extraction on a pixel-by-pixel basis and application of dimensional reduction methods identifies nuclear neighborhoods for feature extraction.
  • B Schematic of 19 F- based MoC-Ab.
  • the oligonucleotide includes a FITC fluorophore at the 3’ end and the modified base 2-F-Ac-C (green) in place of deoxycytidine.
  • the oligonucleotide sequence is set forth in SEQ ID NO: 11.
  • C Representative e and 19 F images of a HeLa cell stained with anti- nucleolin- 19 F/FITC.
  • srIBI image of a HeLa cell stained with anti-H3K27Ac- 19 F/FITC Overlay of ion images for 19 F (green) and 31 P (blue). The image is the sum of 10 consecutive planes.
  • E HeLa cells were labeled for 24 hours with IdU ( 127 I) and stained with anti-nucleolin- 19 F/FITC. Using srIBI, 40 depths of a single cell were acquired with an increase in the current every 10 scans. Images of newly synthesized DNA (top) and nucleolin (middle) and their overlay (bottom) show details of a region within the nucleus, at different currents.
  • Fig. 15 Multiparametric srIBI using MoC-Abs identifies unique subcellular features.
  • A-C (Left) Representative srIBI images of a HeLa cell stained with A) anti-CENP-A- 81 Br/Cy3, B) anti-H3K27Ac- 127 Ir/Cy5, and C) anti-SC35-biotin recognized by streptavidin- 197 AU/FITC. Overlay of ion images for MoC-Ab (red) and phosphorus ( 31 P; blue). (Right) A digital zoom of boxed area in the original image to show specificity of MoC-Ab signal. All images consist of the sums of 10 consecutive planes.
  • Cells were stained with anti-nucleolin- 19 F/FITC, anti-H3K9me3- 81 Br/Cy3, anti-H3K27Ac- 127 Ir/Cy5, and anti-SC35-biotin (recognized by streptavidin- 197 Au/FITC).
  • the image consists of the sum of 10 consecutive planes.
  • E-G Multiple enlarged images from boxed regions in panel D showing E) a nuclear speckle, F) a nucleolus, and G) heterochromatin.
  • the areas marked A-C indicate three distinct groups of nucleolin-positive pixels identified manually in the nucleolin map.
  • Grouped pixels from the unsupervised viSNE map are differentially distributed in space as shown in srIBI images of (1) total nucleolin ( 19 F) from the HeLa cell shown in panel D, (2) 19 F signals from pixels within gate A, (3) gate B, (4) gate C, and (5) overlay of 19 F signals from gates A (red), B (green), and C (blue).
  • Fig. 16 3D nanoscale imaging of the nucleus through iterative srIBI.
  • A Representative single-plane srIBI images at different depths of a HeLa cell nucleolus. HeLa cells were stained with anti-nucleolin- 19 F/FITC, and 785 individual planes were acquired to obtain srIBI images of a nucleolus from its appearance to its disappearance. Single planes every 100 depths show a distinctive molecular distribution of nucleolin in the 3D space. See Fig. 42 for images of each individual plane. Blue, red, and green arrows indicate x-axis, y-axis, and z-axis, respectively.
  • (B) (Left) 3D surface reconstruction of images of nucleolin staining of a nucleolus shown in panel A. (Right) Overviews of the same nucleolus along x-axis (blue arrow), y-axis (red arrow), and z-axis (green arrow) with the origin represented as a black dot.
  • the image consists of the 3D reconstruction of a stack of 40 consecutive planes.
  • D Representative 3D reconstruction of nucleolin (cyan), phosphorus (blue), H3K9me3 (magenta), H3K27Ac (green), and SC35 (red) in a HeLa cell stained with anti-nucleolin- 19 F/FITC, anti-H3K9me3- 81 Br/Cy3, anti-H3K27Ac- l 27 I/Cy5, and anti-SC35-biotin (recognized by streptavidin- 197 Au/FITC).
  • the image consists of the 3D reconstruction of a stack of 400 consecutive planes. Enlarged images from the boxed region show details of marker distribution.
  • HeLa cells were stained with anti-FBL- 81 Br/Cy3, anti nucleolin- 127 Ir/Cy5, and anti-NPMl -biotin (detected with streptavidin- 197 Au/FITC). Iterative srIBI was performed on a site with three nucleolin. The e image confirmed that the ROI was acquired. Nucleolin (cyan), phosphorus (blue), FBL (green), and NPM1 (red) were used for 3D reconstruction from 40 consecutive planes. These images identify the granular component (GC, NPM1 -positive), dense fibrillar component (FBL- and nucleolin-positive), and perinucleolar heterochromatin (PNC, phosphorus-high). Fig.
  • A Representative srIBI image of cisplatin ( 194 Pt) distribution within the cell.
  • B Overlay of ion images for cisplatin ( 194 Pt; red) and a second marker (green). All images consist of the sums of 10 consecutive planes.
  • C A schematic for the identification of nuclear neighborhoods.
  • SrIBI acquisition is performed on single TYK-nu cells treated with cisplatin and labeled with MoC-Abs as described in panel A.
  • a sliding window of 6 * 6 * 20 pixels is used for feature extraction for all channels imaged.
  • Unsupervised clustering is performed on extracted features to identify nuclear neighborhoods.
  • Identified clusters are recolored back onto the cell to visualize nuclear neighborhoods spatially.
  • D A heatmap of the 10 distinctive neighborhoods identified based on the five indicated nuclear markers. The intensity of each marker is denoted by the scale bar on the left.
  • the identified clusters of interactions, termed nuclear neighborhoods, are colored and numbered.
  • E Identified nuclear neighborhoods are used to recreate the cell, resulting in distinctive structures that resemble known features in the nucleus.
  • TYK-nu ovarian cancer cells were treated with 5 mM cisplatin for 24 hours and stained with anti-nucleolin- 19 F/FITC, anti-H3K9me3- 81 Br/Cy3, anti-H3K27Ac- l 27 I/Cy5, and anti-SC35- biotin (detected with streptavidin- 197 Au/FITC). Images of nucleolin ( 19 F), DNA ( 31 P), H3K9me3 ( 81 Br), H3K27Ac ( 127 I), cisplatin ( 194 Pt), and SC35 ( 197 Au) were simultaneously acquired. A l00-pm 2 ROI in the nucleus was acquired by iterative srIBI.
  • Each point represents a voxel. Voxels grouped in distinct regions based on the expression of each marker.
  • the 11 hierarchical clusters were identified by unsupervised hierarchically clustering, followed by manual annotation.
  • C An expression profile of the mean marker expression in each of the 11 distinctive nuclear neighborhoods. The scale intensity of each marker is denoted by the color bar on the top right (Z-score, normalized to each row).
  • Pairwise interaction frequency calculations A graphical schematic of the permutation test implemented to quantify the frequency of pairwise neighborhood interactions is shown. If two points (e.g., A and B) were within 5 pixels of each other, they were defined as being“attracted”. Point labels were shuffled 1000 times, and the mean interaction frequency noted. The final distribution of shuffled interaction frequencies was plotted, and the mean was taken as the expected interaction frequency. Real interaction frequencies below the expected were indicative of repulsion between the points, whereas those above were attraction. (Right) The neighborhood interaction frequency map was calculated using the log2 enrichment of the real over expected number of pairwise interaction frequencies. (G-H) Nuclear neighborhoods are colored as shown in the legend of panel D to show G) chromatin- specific neighborhoods, and H) cisplatin- enriched neighborhoods. Enlarged images from boxed regions exemplify different
  • Fig. 19 Sequences and validation of size and purify of mass adaptors
  • Fig. 20 Confirmation of purity of Tn5.
  • Pre-induction Pre-induction
  • Post post-induction
  • DTT-eluted products were separated on SDS-PAGE, and the gel was stained with Coomassie blue.
  • the expected sizes of the Tn5 fusion protein open arrowhead
  • cleaved Tn5 arrow
  • Marker molecular weights in kDa are listed to the left.
  • Fig. 21 Fluorescein/Oregon green antibody recognizes ATTO 514.
  • Fig. 22 Cell-cycle phase gating strategy. Strategy used to define each cell cycle phase of Jurkat cells (related to Figure 10 g and h).
  • Fig. 23 Correlation of ATAC-mass signal and phosphorylation of various signaling pathway markers
  • c Representative biaxial plots of ATAC-mass and pSLP-76 (Y128) signals in NKL cells treated as indicated. Numbers indicate the percentage of cells in each gate
  • Fig. 24 Principal component analysis of ATAC-seq open chromatin peaks from IL-12- treated, IL-l8-treated, and control NKL cells. Points indicate data from the two biological replicates per condition.
  • Fig. 25 Ion beam imaging enables high-resolution visualization of the nucleus
  • IL-18 treatment increases genome accessibility in uncompacted DNA in NKL cells
  • Fig. 28 Strategy for the synthesis and conjugation of mass-oligonucleotides to antibodies.
  • A Mass-oligonucleotides were synthesized with internal isotope-derivatized nucleotides and a maleimide cycloadduct at the 5’ position. Dashed box shows chemical structures of isotope-derivatized cytidines and thymidines that are commercially available or that can be synthesized with previously reported protocols (48, 49). In this study, we incorporated 11 fluorine, bromine, or iodine-derivatized cytidines (shown in red) into a 47- nucleotide long oligonucleotide.
  • Fig. 29 Protocol optimization for intracellular staining with MoC-Abs.
  • HeLa cells were fixed and permeabilized as indicated in the second column. Cells were then blocked for 30 minutes at room temperature as indicated in the third column, stained for 1 hour at room temperature with unconjugated, lanthanide-polymer conjugated ( 162 Dy) or mass- oligonucleotide-conjugated ( 127 I) primary anti-double- stranded DNA (anti-dsDNA), washed three times in PBS, and stained for 1 hour at room temperature with secondary anti-mouse- Alexa 647. Cells were then washed and mounted with fluoromount containing DAPI for confocal microscopy analysis.
  • HeLa cells stained with anti-dsDNA (sample 1), specific nuclear staining co-localizing with DAPI was observed as expected.
  • HeLa cells stained with anti-dsDNA- 162 Dy (sample 2) showed the same nuclear staining indicating that that the conjugation process did not alter antibody specificity.
  • the presence of high salt concentration and salmon sperm DNA in cells stained with anti-dsDNA- 162 Dy (sample 3) did not interfere with antibody staining.
  • HeLa cells fixed and permeabilized with methanol (sample 4), acetone (sample 6), or fixed with 1.6% paraformaldehyde and permeabilized with methanol (sample 8) and stained with anti-dsDNA- 127 I showed nonspecific cytoplasmic staining.
  • FIG. 30 Validation of 19 F/FITC MoC-Abs for srIBI.
  • A A schematic of the 19 F/FITC MoC-Ab. The antibody was conjugated to the 5’ end of an oligonucleotide in which all cytidines were replaced with 2-F-Ac-C. The oligonucleotide also had a 3’ FITC.
  • SEQ ID NO: 11 SEQ ID NO: 11
  • B Representative confocal microscopy image of a HeLa cell stained with unconjugated anti-nucleolin- 19 F/FITC and a secondary anti-mouse-Alexa488 (green) and DAPI (blue). Scale bar, 4 pm.
  • C Representative confocal microscopy images of control HeLa cells (no antibody) or HeLa cells stained with anti-nucleolin- 19 F/FITC (green) at different concentrations. 31 P is shown in blue. Cells in red boxes in the composite image are shown enlarged in the right column. Scale bars, 20 pm in standard images and 4 pm in enlarged images. A 1:100 dilution of this antibody was used in subsequent experiments.
  • D Representative srIBI images of a control HeLa cell (no antibody) and HeLa cells stained with anti-nucleolin- 19 F/FITC. Scale bar, 4 pm.
  • Fig. 31 High-phosphorus regions within the nucleus mainly originate from the DNA backbone.
  • A Representative srIBI image of a HeLa cell treated with 5-iodo-2'-deoxyuridine (IdU) for 24 hours to label DNA. Ion images for phosphorus ( 31 P; left panel) and newly synthesized DNA ( 127 I; middle panel) are overlaid in the right panel.
  • B Line scan along the dashed red line in the right image in panel A. Raw ion counts were scaled by max-min normalization. Newly replicated DNA ( 127 I; green line) and phosphorus ( 31 P; blue line) in the nucleus show a similar pattern, confirming that high-phosphorus regions mainly originate from the DNA backbone rather than from RNA or phosphorylated proteins.
  • Fig. 32 Regions of active transcription within the nucleus mainly originate from low- phosphorus regions.
  • A Representative srIBI image of a HeLa cell stained with anti- H3K27AC- 19 F/FITC. Ion images for H3K27Ac ( 19 F; left panel) and phosphorus ( 31 P; middle panel) are overlaid in the right panel. Scale bars, 4 pm. Cell in the composite image is also shown in Fig. 14 D.
  • B-C Line scan along the lines in the boxes outlined in B) red and C) yellow dashed lines in the image to the right in panel A. Raw ion counts were scaled by mix- man normalization.
  • Black arrows point to regions of high H3K27Ac counts (green line) that are anti-correlated with phosphorus counts (blue line). Red arrows point to low H3K27Ac counts (green line) that are anti-correlated with phosphorus counts (blue line). Images below the line scans are magnified from regions boxed in panel A. Scale bars, 800 nm.
  • Fig. 33 Beam current scales with ion counts per pixel at the expense of resolution.
  • HeLa cells were labeled with IdU for 24 hours and stained with anti-nucleolin 19 F/FITC. The images were acquired with a constant 25 * 25 pm field of view, 1 ms dwell time per pixel, and 512 * 512 pixels (xy). The beam current was increased every 10 planes by changing the Dl aperture width to obtain a beam diameter of -50 nm (Dl: 5), -75 nm (Dl: 4), -100 nm (Dl: 3) and -150 nm (Dl: 2).
  • Dl Representative srIBI images of nucleolin staining in the same HeLa cell using different aperture widths.
  • Fig. 34 Validation of 81 Br/Cy3 MoC-Abs for srIBI.
  • A A schematic of the 81 Br/Cy3 MoC-Ab. The antibody was conjugated to the 5’ end of an oligonucleotide in which all cytidines were replaced by 5-Br-dC. The oligonucleotide also had a 3’ Cy3.
  • SEQ ID NO:l l SEQ ID NO:l l
  • B Representative confocal microscopy image of a HeLa cell stained with unconjugated anti- CENP-A- 81 Br/Cy3 and a secondary anti-mouse-Alexa488 (green) and DAPI (blue). Scale bar, 4 pm.
  • (C) Representative confocal microscopy images of control HeLa cells (no antibody) and HeLa cells stained with anti-CENP-A- 81 Br/Cy3 (orange) at three different concentrations. 31 P is shown in blue. Cells in red boxes in the composite image are enlarged in the right column. Scale bars, 20 pm in standard images and 4 pm in enlarged images. A 1:500 dilution of this antibody was used in subsequent experiments.
  • FIG. 35 Validation of 1 27 I/C y 5 MoC-Abs for srIBI.
  • A A schematic of the 1 27 I/C y 5 MoC-Ab. The antibody was conjugated to the 5’ end of an oligonucleotide in which all cytidines were replaced by 5-I-dC. The oligonucleotide also had a 3’ Cy5. (SEQ ID NO: 11)
  • (C) Representative confocal microscopy images of control HeLa cells (no antibody) and HeLa cells stained with anti-H3K27Ac- 127 I/Cy5 (red) at three different concentrations. 31 P is shown in blue. Cells in red boxes in the composite image are enlarged in the right column. Scale bars, 20 pm in standard images and 4 pm in enlarged images. A 1:100 dilution of this antibody was used in subsequent experiments.
  • Fig. 36 Validation of Phalloidin-ATT05l4 for srIBI.
  • A Chemical structure of ATTO 514. Green arrows indicate the presence of the six 19 F atoms in ATTO 514.
  • B Representative confocal microscopy image of a HeLa cell stained with phalloidin-ATT0514. Scale bars, 4 pm.
  • C Representative srIBI images of a HeLa cell stained with phalloidin-ATT05l4. Scale bars, 4 pm.
  • Fig. 37 Validation of biotin-conjugated antibodies stained with 197 Au/FITC- conjugated streptavidin.
  • A A schematic of the antibody conjugation strategy. Primary antibodies were conjugated to biotin and subsequently stained with 197 Au/FITC-conjugated streptavidin. The 197 Au consists of 1.4 nm nanoparticles, a diameter below the theoretical srIBI resolution limit.
  • B Representative confocal microscopy image of a HeLa cell stained with unconjugated anti-SC35 and a secondary anti-mouse-Alexa488 (green). DAPI (blue) was used to stain the nucleus. Scale bars, 4 pm.
  • (C) Representative confocal microscopy images of HeLa cells stained with streptavidin- 197 Au/FITC and HeLa cells stained with anti-SC35-biotin followed by streptavidin- 197 Au/FITC at different concentrations of the primary antibody and 1:40 of streptavidin- 197 Au/FITC (green). DAPI (blue) was used to stain nuclei. Scale bars, 20 pm. Cells in red boxes in the composite image are magnified in the right column. Scale bars, 4 pm. A 1:100 dilution of this antibody was used on following experiments.
  • (D) Representative srIBI image of a control HeLa cell (no antibody) and a HeLa cell stained with anti-SC35-biotin followed by streptavidin- 197 Au/FITC. Scale bar, 4 pm.
  • Fig. 38 Validation of antibody-based tools for srIBI by targeting other proteins.
  • A Representative srIBI image of a HeLa cell stained with anti-H3K9me3- 81 Br/Cy3 (red).
  • Phosphorus is shown in blue. Scale bar, 4 pm.
  • B Representative srIBI image of a HeLa cell stained with anti-nuclcolin- l 27 I/Cy5 (red). Phosphorus is shown in blue. Scale bar, 4 pm.
  • C Representative srIBI image of a HeLa cell stained with anti-NPMl -biotin followed by streptavidin- 197 Au/FITC (red). Phosphorus is shown in blue. Scale bars, 4 pm.
  • Fig. 39 Assessment of srIBI channel crosstalk. Representative srIBI images of a control HeLa cell control (no antibody) and HeLa cells stained with the indicated antibodies. Each row represents a staining condition and each column the ion channel extracted for the indicated isotope. Scale bars, 4 pm.
  • Fig. 40 Two-channel srIBI on HeLa cells using a beam diameter of -50 nm (Dl: 5). Representative confocal microscopy (left) and srIBI (right) images of HeLa cells stained with anti-nucleolin- 19 F/FITC and anti-CENP-A- 81 Br/Cy3 MoC-Ab. Scale bars, 4 pm (top 3 rows) and 1 pm (bottom row).
  • Fig. 41 Six-channel srIBI on HeLa cells using a beam diameter of -50 nm (Dl: 5).
  • Fig. 42 Sequential planes are similar in ion counts.
  • A Representative srIBI image of a HeLa cell stained with anti-nucleolin- 19 F/FITC. Scale bars, 4 pm; and 1 pm for enlarged images.
  • B srIBI images of the 10 individual planes summed in panel A in consecutive order. The 19 F signal is similar in all individual images. Scale bars, 4 pm
  • C Line scans on the nucleolus enlarged in panel A for each individual plane. Line scans were used to quantify ion counts per pixel in individual planes. Scale bars, 1 pm.
  • Fig. 43 3D surface reconstruction of CENP-A signal at 5 nm Z-depth shows centromeres with a spherical shape.
  • HeLa cells were stained with anti-CENP-A- 81 Br/Cy3, and 40 individual planes were acquired to obtain srIBI images of centromeres from its appearance to its disappearance. 3D surface reconstruction of images of CENP-A at 5 nm reveal centromeres with spherical shape.
  • Fig. 44 Individual planes of a whole nucleolus.
  • A (Left) Representative srIBI image of a HeLa cell stained with anti-nucleolin- 19 F/FITC. Scale bar, 4 pm.
  • (Right) Enlarged image of the nucleolus shown in Fig. 16 A-B. Scale bar, 400 mm.
  • B Individual planes of a whole nucleolus, in order from left to right and from top to bottom; these 783 images were used for the volumetric reconstruction of the nucleolus shown in Fig. 16 B. Images have the same scale for intensity, and sequential planes have similar 19 F ion counts.
  • Fig. 45 Iterative srIBI is able to resolve MoC-Ab signals at the nanoscale.
  • Left Representative enlarged image of a nucleolus of a HeLa cell stained with anti-nucleolin- 19 F/FITC. The same nucleolus is shown in Fig. 16 F. Scale bar, 300 nm.
  • Middle Higher magnification images of the area boxed in the image on the left.
  • Line scans of the lines in the middle images demonstrate that srIBI can resolve signals spaced a few tens of nm.
  • Fig. 46 Iterative super-resolution imaging reveals nucleolar substructures.
  • A Representative secondary electron image of a HeLa cell. Scale bar, 5 pm.
  • B (Left) High- resolution scan of the HeLa cell used to find a ROI. The HeLa cell shown in panel A was stained with anti-nucleolin- 19 F/FITC, anti-FIB-l- 81 Br/Cy3, and anti-NPMl -biotin (recognized by streptavidin- 197 Au/FITC). Scale bar, 5 pm.
  • Light Iterative super-resolution imaging for the visualization of the nucleolar structure.
  • NPM1 a marker of the granular component (GC) is distributed in regions distinct from those stained with FIB-l, a marker of the dense fibrillary component (DFC). Regions with high phosphorus surrounding the nucleolus indicate perinucleolar heterochromatin (PHC). Scale bar, 1 pm.
  • C Individual images of the super resolution image shown in panel B. Scale bars, 1 pm.
  • Fig. 47 Validation of cisplatin for srIBI.
  • A Chemical structure of cisplatin.
  • B Chemical structure of cisplatin.
  • CyTOF analysis of cisplatin distribution in TYK-nu ovarian cancer cells TYK-nu cells were treated with different concentrations of cisplatin for 24 hours, washed, and treated with 1 pM Rh-intercalator for 15 minutes (to discriminate dead from live cells) and analyzed by CyTOF. Maximal drug uptake with minimal cell death at 24 hours was observed with 5 pM.
  • C The five naturally occurring stable isotopes of cisplatin.
  • D Mean of ion count per pixel of all pixels in srIBI images from TYK-nu cells treated with cisplatin.
  • Cisplatin is observed in both the cytoplasm and nucleus. Representative srIBI images of a TYK-nu cell treated with DMSO (top) or 5 pM cisplatin for 24 hours without antibody staining (bottom). Scale bars, 4 pm.
  • F Assessment of 194 Pt channel crosstalk.
  • Fig. 48 Six-channel srIBI images on TYK-nu cells treated with cisplatin.
  • A-C Representative srIBI images of TYK-nu cells treated with 5 pM cisplatin for 24 hours and stained with anti-nucleolin- 19 F/FITC, anti-H3K9me3- 81 Br/Cy3, anti-H3K27Ac- l 27 I/Cy5, and anti-SC35-biotin (recognized by streptavidin- 197 Au).
  • nucleolin 19 F; cyan
  • DNA 31 P; blue
  • H3K9me3 81 Br; magenta
  • H3K27Ac 127 I; green
  • cisplatin 194 Pt; grey
  • SC35 197 AU; red
  • A A composite image of a TYK-nu cell nucleus. Scale bars, 2 pm (right image), 200 nm (middle image) and 67 nm (left images).
  • B Individual images of the cell shown in panel A. Scale bars, 2 pm.
  • C Composite images from three additional TYK-nu cell nuclei. Scale bars, 2 pm.
  • Fig. 49 Identification of nuclear neighborhoods by the iterative srIBI analysis framework.
  • B scaled intensity
  • Unsupervised hierarchical clustering was performed on the voxels to separate them into 29 distinct groups; the t-SNE map from panel A is shown recolored based on these groups.
  • C A heatmap representation of the 29 distinct clusters identified based on the average expression per cluster of SC35, H3K9me3, phosphorus, nucleolin, H3K27Ac, and cisplatin. The scale intensity of each marker is denoted by the color bar on the top right (Z-score, normalized to each row).
  • D Clusters were manually merged by tree distance, proximity on the t-SNE map and similar expression profiles. This resulted in a reduction of 29 clusters into 11 clusters, which were then annotated based on their final average expression profile.
  • FIG. 50 The spatial distribution of nuclear neighborhoods across cells reveals order and diversity in nuclear organization. Nuclear neighborhoods were recolored by each cluster in each of five cells to allow a visual understanding of nuclear neighborhood interactions and conserved and divergent features. Cells 1, 2, 4, and 5 are shown here. Cell 3 is shown in Fig. 18 F.
  • Fig. 51 Pairwise neighborhood interaction frequency at different cutoffs. Pairwise voxels were defined as neighbors using a cutoff of n pixels in 3D Euclidean space (see Materials and Methods for details). We tested two distances here, using (A) 5 pixels and (B) 25 pixels as the cutoff. The neighborhood interaction frequency map was calculated using the log2 enrichment of the real over expected number of pairwise interaction frequencies (left).
  • Fig. 52 Identification of a cisplatin gradient within SC35 nuclear speckles.
  • Neighborhoods 6 (Active SC35 and Cisplatin) and 7 (Inactive SC35 and Cisplatin) were plotted for all 5 cells, to represent the spatial distribution of these two neighborhoods relative to each other.
  • B Scaled cisplatin levels (Z-score) were plotted on neighborhoods 6 and 7. An increasing gradient of cisplatin concentration is observed across the inactive-active SC35 transition boundary.
  • Fig. 53 schematically illustrates a labeling system that can be used to labeled nucleic acid in a cell in situ.
  • Fig. 54 shows results obtained using the labeling system illustrated in Fig. 53.
  • nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.
  • nucleotide is intended to include those moieties that contain not only the natural purine and pyrimidine bases, but also other heterocyclic bases that have been modified. Such modifications include methylated purines or pyrimidines, acylated purines or
  • nucleotide includes those moieties that contain hapten or fluorescent labels and may contain not only conventional ribose and deoxyribose sugars, but other sugars as well.
  • Modified nucleosides or nucleotides also include modifications on the sugar moiety, e.g., wherein one or more of the hydroxyl groups are replaced with halogen atoms or aliphatic groups, or are functionalized as ethers, amines, or the like.
  • a nucleotide may be modified to contain a non-biological stable isotope. 2-F-Ac-C, 5-Br-dC, 5-I-dC are examples of such modified nucleotides.
  • nucleic acid and“polynucleotide” are used interchangeably herein to describe a polymer of any length, e.g., greater than about 2 bases, greater than about 10 bases, greater than about 100 bases, greater than about 500 bases, greater than 1000 bases, up to about 10,000 or more bases composed of nucleotides, e.g., deoxyribonucleotides or
  • ribonucleotides and may be produced enzymatically or synthetically (e.g., PNA as described in U.S. Patent No. 5,948,902 and the references cited therein) which can hybridize with naturally occurring nucleic acids in a sequence specific manner analogous to that of two naturally occurring nucleic acids, e.g., can participate in Watson-Crick base pairing
  • Naturally-occurring nucleotides include guanine, cytosine, adenine and thymine (G, C, A and T, respectively).
  • DNA and RNA have a deoxyribose and ribose sugar backbone, respectively, whereas PNA’s backbone is composed of repeating N-(2-aminoethyl)-glycine units linked by peptide bonds.
  • PNA PNA
  • a locked nucleic acid (LNA) often referred to as inaccessible RNA, is a modified RNA nucleotide.
  • LNA nucleotide The ribose moiety of an LNA nucleotide is modified with an extra bridge connecting the 2' oxygen and 4' carbon.
  • the bridge “locks” the ribose in the 3'-endo (North) conformation, which is often found in the A-form duplexes.
  • LNA nucleotides can be mixed with DNA or RNA residues in the oligonucleotide whenever desired.
  • the term“unstructured nucleic acid,” or“UNA,” is a nucleic acid containing non-natural nucleotides that bind to each other with reduced stability.
  • an unstructured nucleic acid may contain a G' residue and a C residue, where these residues correspond to non- naturally occurring forms, i.e., analogs, of G and C that base pair with each other with reduced stability, but retain an ability to base pair with naturally occurring C and G residues, respectively.
  • Unstructured nucleic acid is described in US20050233340, which is incorporated by reference herein for disclosure of UNA. Also included in this definition are ZNAs, i.e., zip nucleic acids.
  • biological feature of interest refers to any part of a cell that can be indicated by binding to a capture agent.
  • exemplary biological features of interest include cell walls, nuclei, cytoplasm, membrane, keratin, muscle fibers, collagen, bone, proteins, nucleic acid (e.g., mRNA or genomic DNA, etc) fat, etc.
  • a biological feature of interest can also be indicated by immunohistological methods, e.g., a capture agent that is linked to an oligonucleotide.
  • the capture agent binds to a site, e.g., a protein epitope, in the sample.
  • Exemplary epitopes include, but are not limited to carcinoembryonic antigen (for identification of adenocarcinomas, cytokeratins (for
  • CD15 and CD30 for Hodgkin's disease
  • alpha fetoprotein for yolk sac tumors and hepatocellular carcinoma
  • CD117 for gastrointestinal stromal tumors
  • CD10 for renal cell carcinoma and acute lymphoblastic leukemia
  • prostate specific antigen for prostate cancer
  • estrogens and progesterone for tumour identification
  • CD20 for identification of B-cell lymphomas
  • CD3 for identification of T-cell lymphomas.
  • Complementary nucleic acid molecules e.g., DNA and/or RNA
  • in the sample provide binding complementary sites for oligonucleotide probes.
  • multiplexing refers to using more than one distinguishably labeled reagent (e.g., at least 5, at least 10, at least 50, at least 100, up to 200 or 500 or more distinguishably labeled reagents), for the simultaneous or sequential detection and
  • antibody and“immunoglobulin” are used interchangeably herein and are well understood by those in the field. Those terms refer to a protein consisting of one or more polypeptides that specifically binds an antigen.
  • One form of antibody constitutes the basic structural unit of an antibody. This form is a tetramer and consists of two identical pairs of antibody chains, each pair having one light and one heavy chain. In each pair, the light and heavy chain variable regions are together responsible for binding to an antigen, and the constant regions are responsible for the antibody effector functions.
  • the recognized immunoglobulin polypeptides include the kappa and lambda light chains and the alpha, gamma (IgGi, IgG 2 , IgG 3 , IgG 4 ), delta, epsilon and mu heavy chains or equivalents in other species.
  • Full-length immunoglobulin "light chains" (of about 25 kDa or about 214 amino acids) comprise a variable region of about 110 amino acids at the NH 2 - terminus and a kappa or lambda constant region at the COOH-terminus.
  • immunoglobulin "heavy chains” (of about 50 kDa or about 446 amino acids), similarly comprise a variable region (of about 116 amino acids) and one of the aforementioned heavy chain constant regions, e.g., gamma (of about 330 amino acids).
  • antibodies and“immunoglobulin” include antibodies or immunoglobulins of any isotype, fragments of antibodies which retain specific binding to antigen, including, but not limited to, Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, minibodies, single-chain antibodies, and fusion proteins comprising an antigen-binding portion of an antibody and a non-antibody protein. Also encompassed by the term are Fab’, Fv, F(ab’) 2 , and or other antibody fragments that retain specific binding to antigen, and
  • Antibodies may exist in a variety of other forms including, for example, Fv, Fab, and (Fab') 2 , as well as bi-functional (i.e. bi-specific) hybrid antibodies (e.g., Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)) and in single chains (e. g., Huston et al., Proc. Natl. Acad. Sci. U.S.A., 85, 5879-5883 (1988) and Bird et al., Science, 242, 423-426 (1988), which are incorporated herein by reference).
  • bi-functional hybrid antibodies e.g., Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)
  • single chains e.g., Huston et al., Proc. Natl. Acad. Sci. U.S.A., 85, 5879-5883 (1988) and Bird et al., Science
  • telomere binding agent refers to the ability of a binding agent to preferentially bind to a particular analyte that is present in a homogeneous mixture of different analytes. In certain embodiments, a specific binding interaction will discriminate between desirable and undesirable analytes in a sample, in some embodiments more than about 10 to lOO-fold or more (e.g., more than about 1000- or 10, 000-fold).
  • the affinity between a binding agent and analyte when they are specifically bound in a binding agent/analyte complex is characterized by a K D (dissociation constant) of less than lO 6 M, less than 10 7 M, less than 10 8 M, less than 10 9 M, less than 10 9 M, less than 10 11 M, or less than about 10 12 M or less.
  • A“plurality” contains at least 2 members. In certain cases, a plurality may have at least
  • labeling refers to attaching a label to specific sites in a sample (e.g., sites containing an epitope for the antibody being used, for example) such that the presence and/or abundance of the sites can be determined by evaluating the presence and/or abundance of the label.
  • biological sample refers to a sample, which may be substantially planar, i.e., two dimensional, that contains cells.
  • a biological sample can be made by, e.g., growing cells on a planar surface, depositing cells on a planar surface, e.g., by centrifugation, by cutting a three dimensional object that contains cells into sections and mounting the sections onto a planar surface, i.e., producing a tissue section, or any other means.
  • the cells may be fixed using any number of reagents including formalin, methanol, paraformaldehyde, methanol: acetic acid etc.
  • a planar sample has a thickness.
  • tissue section refers to a piece of tissue that has been obtained from a subject, and optionally fixed, sectioned, and mounted on a planar surface, e.g., a microscope slide.
  • FFPE tissue section refers to a piece of tissue, e.g., a biopsy that has been obtained from a subject, fixed in formaldehyde (e.g., 3%-5% formaldehyde in phosphate buffered saline) or Bouin solution, embedded in wax, and optionally cut into thin sections, and then mounted on a microscope slide.
  • formaldehyde e.g., 3%-5% formaldehyde in phosphate buffered saline
  • Bouin solution embedded in wax
  • spatialally-addressable measurements refers to a set of values that are each associated with a specific position on a surface. Spatially-addressable
  • measurements can be mapped to a position in a sample and can be used to reconstruct a 2d or 3d image of the sample.
  • A“diagnostic marker” is a specific biochemical in the body which has a particular molecular feature that makes it useful for detecting a disease, measuring the progress of disease or the effects of treatment, or for measuring a process of interest.
  • A“pathoindicative” cell is a cell which, when present in a tissue, indicates that the animal in which the tissue is located (or from which the tissue was obtained) is afflicted with a disease or disorder.
  • the presence of one or more breast cells in a lung tissue of an animal is an indication that the animal is afflicted with metastatic breast cancer.
  • complementary site is used to refer to an epitope for an antibody or aptamer, or a nucleic acid molecule if the capture agent is an oligonucleotide probe.
  • a binding agent is an antibody
  • the complementary site for the binding agent is the epitope in the sample to which the antibody binds.
  • the capture agent is an oligonucleotide probe
  • the complementary site for the capture agent is a complementary sequence in a DNA or RNA molecule in the sample.
  • epitope is defined as small chemical groups on the antigen molecule that is bound to by an antibody.
  • An antigen can have one or more epitopes. In many cases, an epitope is roughly five amino acids or sugars in size.
  • an epitope is roughly five amino acids or sugars in size.
  • One skilled in the art understands that generally the overall three-dimensional structure or the specific linear sequence of the molecule can be the main criterion of antigenic specificity.
  • A“subject” of diagnosis or treatment is a plant or animal, including a human.
  • Non- human animals subject to diagnosis or treatment include, for example, livestock and pets.
  • the term“incubating” refers to maintaining a biological sample and binding agent under conditions (which conditions include a period of time, a temperature, an appropriate binding buffer and a wash) that are suitable for specific binding of the binding agent to molecules (e.g., epitopes or complementary nucleic acid) in the biological sample.
  • binding agent refers to an agent that can specifically bind to complementary sites in a biological sample.
  • exemplary capture agents include, e.g., an antibody, an ap tamer, and an oligonucleotide probe (which may be DNA or RNA) that hybridizes to a binding site.
  • binding agent that is linked to a polymer refers to a binding agent, e.g., an antibody or an oligonucleotide probe covalently linked to a polymer, e.g., an oligonucleotide.
  • the polymer and the capture agent may be linked via a number of different methods, including those that use maleimide or halogen-containing group, which are cysteine- reactive.
  • oligonucleotide refers to a multimer of at least 10, e.g., at least 15 or at least 30 nucleotides. In some embodiments, an oligonucleotide may be in the range of 15-200 nucleotides in length.
  • reading in the context of reading a signal, refers to obtaining an image by scanning or by microscopy, where the image shows the pattern of fluorescence as well as the intensity of fluorescence in a field of view.
  • the term“isotopically tagged” refers to a molecule that is tagged with either a single kind of stable isotope that is identifiable by its unique mass or mass profile or a combination of the same, where the combination of stable isotopes provides an identifier. Combinations of stable isotopes permit channel compression and/or barcoding. Examples of elements that are identifiable by their mass are listed below, although other elements may be employed.
  • the terms“mass tagged” and“elementally tagged” may be used interchangeably herein.
  • isotopically tag means any isotope of any element that is identifiable by its mass, distinguishable from other mass tags, and used to tag a biologically active material or analyte.
  • a mass tag has an atomic mass that is distinguishable from the atomic masses present in the analytical sample and in the particle of interest.
  • monoisotopic means that a tag contains a single type of metal isotope (although any one tag may contain multiple metal atoms of the same type).
  • the term“elemental analysis” refers to a method by which the presence and/or abundance of elements of a sample are evaluated.
  • sample comprising cells is a sample of biological origin that contains intact, e.g., fixed, cells.
  • the sample may be substantially planar. Examples of such samples include tissue sections, samples that are made by depositing disassociated cells onto a planar surface, and samples that are made by growing a sheet of cells on a planar surface
  • the term“scanning” refers to a method by which a source of radiation
  • a laser e.g., a laser
  • zig-zagged or rastered over a surface until a substantial two dimensional area has been irradiated by the source of energy.
  • the term“across an area”, in the context of spatially-addressable measurements of the abundance of a isotope tag across an area of a sample, refers to measurements of mass tags that are at or under (e.g., on or within cells that are proximal to) the surface of the sample.
  • the depth of the area analyzed can vary depending on the energy of the ion beam.
  • mass cytometry refers to a method in which cells are separated from one another by use of a flow cell and then subjected to elemental analysis.
  • the term“substantially pure” indicates that a reagent has been isolated from other components by purification and is relatively pure form, e.g., at least 50% free, at least 70% free or at least 90% free of other components that were present prior to purification. Reagents that are made synthetically are substantially pure.
  • the term“internal atoms” refers to atoms that are part of the molecule, not non-covalently attached the molecule. Atoms that are chelated to a molecule are not internal atoms.
  • non-biological refers to isotopes that have low abundance in biological samples, e.g., human cells.
  • the labeling moiety may be a binding agent that specifically binds to a complementary site in or on a cell (in which case the isotopically- labeled atoms may be part of the binding agent), or the labeling moiety may comprise a binding agent that specifically binds to a complementary site in or on a cell (in which case the isotopically-labeled atoms may be part of an adjunct such as a polymer that is covalently linked to the binding agent).
  • the labeling moiety may comprise a chemo selective group, such that the labeling moiety can be readily covalently linked with a binding agent via any suitable reaction.
  • the labeling moiety is not antibody that binds to other antibodies.
  • the labeling moiety is not or does not comprise an anti-IgG antibody (an antibody that binds to IgG antibodies).
  • the isotope used should fulfill two requirements: it should be efficiently ionized upon impact of a positively charged ion and it should have low abundance in cells. As such, in many cases, any nonmetal or metalloid isotope (except for 3 ⁇ 4 12 C, 14 N, 16 0 and 32 S, which are commonly found in cells) can be used.
  • the labeling moiety comprises a non-biological stable nonmetal isotope of carbon (C), nitrogen (N), oxygen (O), fluorine (F), phosphorus (P), sulfur (S), chlorine (Cl), selenium (Se), bromine (Br), or iodine (I), with the exception of 3 ⁇ 4 12 C, 14 N,
  • the labeling moiety may comprise a stable isotope of a halogen (e.g., fluorine, chlorine, bromine, or iodine). 19 F, 81 Br and 127 I are examples of halogens that can be used herein.
  • the labeling moiety may comprise a stable metalloid isotope of boron (B), silicon (Si), germanium (Ge), arsenic (As), antimony (Sb) or tellurium (Te).
  • B stable metalloid isotope of boron
  • Si silicon
  • Ge germanium
  • As arsenic
  • Sb antimony
  • Te tellurium
  • the isotope used should be“non-biological” in that it is not found in abundance in biological samples, i.e., samples that contain cells.
  • a nonmetal or metalloid isotope may be selected from the following table:
  • Fig. 1 Exemplary types of labeling moiety are shown in Fig. 1.
  • the labeling reagent may be (Fig. 1, d) or may comprise (Fig. 1, b) a binding agent (e.g., an antibody).
  • a binding agent e.g., an antibody
  • the labeling reagent itself may comprise internal atoms of the non-biological stable nonmetal or metalloid isotope.
  • the labeling reagent may be a protein, e.g., an antibody.
  • the labeling reagent may be made by growing cells comprising a nucleic acid encoding the labeling reagent on a medium that comprises isotopically labeled amino acids, then purifying the labeling reagent.
  • the labeling reagent may contain isotopically labeled C, N or O (but not 12 C, 14 N, 16 0), because these atoms are abundant in proteins.
  • the isotopically-labeled atoms may be internal to an adjunct that is covalently linked to a binding agent, e.g., an antibody.
  • the labeling moiety may be composed of: i. of a polymer and ii. a binding agent that specifically binds to a complementary site in or on a cell, wherein only the polymer comprises the atoms of the stable nonmetal or metalloid isotope.
  • the polymer may be an oligonucleotide, although any polymer that can be built to a defined size, monomer-by-monomer, can be used.
  • the isotope may be part of the nucleotides that make up the oligonucleotide.
  • the monomers may be joined to one another using, e.g., phosphor amidite chemistry, which is well known.
  • the oligonucleotide may contain non-abundant isotopes of C, N, O or P but not 12 C, 14 N, 16 0 or 15 P, since these atoms are abundant in oligonucleotides) since these atoms are abundant in oligonucleotides, or one or more of the atoms of a nucleotide may be substitute with the stable isotope.
  • an oligonucleotide may be composed of nucleotides that have a group attached thereto, where the group has the labeled isotope.
  • the group may have a fluorophore or another label that contains a halogen atom.
  • These labeling moieties may be made by: i. making the binding agent part of the moiety (e.g., an antibody) in a cell, ii. making the isotopically-labeled polymer synthetically, and iii.
  • oligonucleotides to proteins, e.g., antibodies, are well known (see, e.g., Gong et ah, Bioconjugate Chem. 2016 27: 217-225 and Kazane et al., Proc Natl Acad Sci 2012 109: 3731-3736). A variety of linkage methods are available. For example, an oligonucleotide may be linked to an antibody directly using any suitable chemical moiety on the antibody (e.g., a cysteine residue or via an engineered site).
  • One advantage of using polymers to label a binding agent is that the number of types of atoms of a non-biological stable nonmetal or metalloid isotope that are incorporated into the polymer can be highly controlled.
  • the number, position and identity of the isotope can be accurately defined in the oligonucleotide and, as such, binding moieties that can be identified by a specific combination of isotopes (e.g., two different halogens, for example) or by a specific ratios of isotopes (e.g., a 1:4, 1:2, 1:1, 2:1 or 4:1 ratio of different isotopes, for example) can be readily made.
  • a polymer of up to, e.g., 500 monomers, such as a polymer of 10-400 or 20-300 monomers, may be used in some embodiments.
  • the binding moiety may be a non-covalently associated complex comprising at least two components, where one of the components is labeled with one or more isotopes, and the other component specifically binds to sites in or on a cell.
  • the binding moiety may be a transposase complex that comprises i. a transposase and ii. a pair of double stranded nucleic acids comprising transposon end sequences, where the nucleic acids are isotopically labeled, as described above.
  • the transposase complex can be contacted with chromatin in the presence of a divalent cation, and the transposase inserts the labeled nucleic acids into the open chromatin, thereby allowing the open chromatin to be analyzed.
  • the isotopically-labeled atoms may be part of a nucleic acid that specifically binds to protein.
  • the nucleic acid is a guide nucleic acid and the protein may be a nucleic acid guided endonuclease, e.g., Cas9 or the like.
  • the labeling moiety may be a polymer (e.g., an
  • the oligonucleotide that has a chemoselective group.
  • the chemo selective group may be amine-reactive, thiol reactive or hydroxyl reactive.
  • the chemoselective group may be capable of participating in a 1,3 cycloaddition (or“click”) reaction.
  • groups include azido and alkynyl (e.g., cyclooctyne) groups, but others are known.
  • the polymer may comprises a biotin moiety, i.e., a tag that includes biotin or a biotin analogue such as desthiobiotin, oxybiotin, 2-iminobiotin, diaminobiotin, biotin sulfoxide, biocytin, etc.
  • Biotin moieties bind to streptavidin with an affinity of at least 10 8 M.
  • labeled polymers e.g., oligonucleotides
  • a labeling moiety may comprise two or more (e.g., two, three, four, five or more) different stable nonmetal and/or metalloid isotopes.
  • about 95% of the isotopically labeled atoms in the moiety may be of one isotope, and about 5% of the isotopically labeled atoms in the moiety may be of the other isotope, although the percentage of the different isotopes may be different, e.g., 90%/l0%, 80%:20%, 50%:50%, 20%: 80%, 10% 190%, or 5%:95% in other embodiments.
  • a set may comprise at least 5, at least 10, at least 20, at least 30, at least 50, or at least 100, at least 150, at least 200, up to 300 or 500 or more labeling moieties that binds to different complementary sites (e.g., epitopes) in or on a cell.
  • Each labeling moiety may be labeled with a single isotope or multiple isotopes, and the different labeling moieties may be distinguishable by the identity of the isotope in a labeling moiety, the combination of isotopes in a labeling moiety, and/or the ratio of isotopes in a labeling moiety.
  • the moieties may comprise multiple internal atoms of at least two stable nonmetal or metalloid isotopes, wherein the moieties are distinguishable by the identity and/or ratio of the different isotopes.
  • a panel of at least 5, at least 10, at least 20, at least 30, at least 50, or at least 100, at least 150, at least 200, up to 300 or 500 or more antibodies, each conjugated to a distinguishably labeled polymers, e.g., oligonucleotide is provided.
  • a panel of distinguishably labeled polymers e.g., a panel of distinguishably labeled polymers, e.g.,
  • oligonucleotides each containing the same chemoselective groups is provided.
  • labeling moieties may be in separate tubes (particularly if they contain a chemoselective group) or they may be mixed together.
  • the labeling reagents described above may be employed in a variety of analysis methods, e.g., elemental mass spectrometry, secondary ion mass spectrometry (SIMS), mass cytometry (CyTOF), laser- ablation inductively-coupled mass spectrometry (LA-ICP-MS), X- ray microscopy (XRM), X-ray fluorescence (XRF), laser-induced breakdown spectroscopy (LIBS), focused ion beam (FIB), energy-dispersive X-ray spectroscopy (EDX), electron microscopy, or nuclear magnetic resonance spectroscopy, for example.
  • the method may comprise labeling biological sample with a plurality of distinguishably labeled binding moieties, where each moiety recognizes a different complementary site in the sample, and analyzing the sample.
  • the labeling moieties may be used to generate a high resolution image by secondary ion mass spectrometry (SIMS).
  • This method may comprise incubating a sample with one or more labeling moieties, thereby producing a labeled sample in which a biological feature of interest is bound to the one or more labeling moieties, scanning the sample by secondary ion mass spectrometry (SIMS) using a positively charged ion beam (e.g., a Cs ion beam) to generate a data set that comprises spatially-addres sable measurements of the abundance of said at least one stable nonmetal or metalloid isotope across said sample; and outputting the data set.
  • SIMS secondary ion mass spectrometry
  • MIBI Magnetic Infrared spectroscopy
  • methods by which samples may be made methods by which cells can be labeled using, e.g., mass-tagged antibodies, methods for ionizing the tags, and methods for analyzing the data, as well as hardware that can be employed in MIBI, including, mass spectrometers and computer control systems are known and are reviewed in a variety of publications including, but not limited to Angelo et al. Nature Medicine 2014 20:436, Rost et al Lab. Invest. 2017 97: 992-1003, US patent 9,766,224, US patent 9,312,111 and US20150080233, among many others, which publications are
  • the labeling moieties may be used to analyze cells by mass cytometry.
  • This method may comprise incubating the cells with one or more labeling moieties, thereby producing a labeled sample in which the labeling moieties are bound to the cells; analyzing the cells by mass cytometry, using a positively charged plasma beam to atomize the cells and generate a data set that comprises spatially-addres sable measurements of the abundance of the stable nonmetal or metalloid isotopes in or on the cells and outputting the data set.
  • mass cytometry including methods by which single cell suspensions can be made, methods by which cells can be labeled using, e.g., mass-tagged antibodies, methods for atomizing particles and methods for performing elemental analysis on particles, as well as hardware that can be employed in mass cytometry, including flow cells, ionization chambers, reagents, mass spectrometers and computer control systems are known and are reviewed in a variety of publications including, but not limited to Bandura et al Analytical Chemistry 2009 81 6813-6822), Tanner et al (Pure Appl. Chem 2008 80: 2627- 2641), U.S. Patent Nos.
  • the method described above may be employed in a multiplex assay in which a heterogeneous population of cells is labeled with a plurality of distinguishably mass tagged binding agents (e.g., a number of different antibodies).
  • the population of cells may be labeled using at least 5, at least 10, at least 20, at least 30, at least 50, or at least 100, up to 150 or more different binding agents (that bind to, for example different cell surface markers) that are each tagged with a different isotope.
  • the cells are introduced into the flow cell, individually analyzed using the method described above, and the cells are separated based on the mass tags associated with each of the cells.
  • a cell having a particular profile of mass tags is desired, and the machine performing the method may be programmed to sort cells having the profile away from other cells that do not have the profile.
  • the nucleic acid of a cell may be stained in situ using: (a) a primary oligonucleotide and (b) a labeled oligonucleotide that contains one or more contains non-metal/metalloid isotopes, where primary oligonucleotide has (i) a first, target- specific, sequence, i.e., a sequence that is complementary to a sequence in a cell (e.g., to a sequence in the genome of a mammalian cell) and (b) a second sequence that is complementary to the labeled oligonucleotide.
  • a first, target-specific, sequence i.e., a sequence that is complementary to a sequence in a cell (e.g., to a sequence in the genome of a mammalian cell)
  • a second sequence that is complementary to the labeled oligonucleotide.
  • multiple (at least two, at least 3 at least 5, etc.) sets of primary oligonucleotides may be used wherein, within each set, the oligonucleotides can have: i.
  • the different sets may have different second sequences, thereby allowing different nucleic acid sequences targeted by the oligonucleotides (e.g. different chromosomes or chromosomal regions) to be labeled with different labels.
  • this method may be used to stain different nucleic acid sequences targeted by the oligonucleotides (e.g. different chromosomes or chromosomal regions) to be labeled with different labels.
  • this method may be used to stain different nucleic acid sequences targeted by the oligonucleotides (e.g. different chromosomes or chromosomal regions) to be labeled with different labels.
  • this method may be used to stain different
  • chromosomes different chromosomal regions, or different structures (A/B compartments, etc.) with different labels, thereby allowing those regions to be analyzed at high resolution.
  • the above-described method can be used to analyze a cell from a subject to determine, for example, whether the cell is normal or not or to determine whether the cells are responding to a treatment.
  • the method may be employed to determine the degree of dysplasia in cancer cells.
  • the cells may be from a sample of from a multicellular organism or a microbe.
  • a biological sample may be isolated from an individual, e.g., from a soft tissue or from a bodily fluid, or from a cell culture that is grown in vitro.
  • a biological sample may be made from a soft tissue such as brain, adrenal gland, skin, lung, spleen, kidney, liver, spleen, lymph node, bone marrow, bladder stomach, small intestine, large intestine or muscle, etc.
  • Bodily fluids include blood, plasma, saliva, mucous, phlegm, cerebral spinal fluid, pleural fluid, tears, lactal duct fluid, lymph, sputum, cerebrospinal fluid, synovial fluid, urine, amniotic fluid, and semen, etc.
  • Biological samples also include cells grown in culture in vitro.
  • a cell may be a cell of a tissue biopsy, scrape or lavage or cells.
  • the cell may of a cell in a formalin fixed paraffin embedded (FFPE), or frozen sample.
  • the method may be used to distinguish different types of cancer cells in FFPE samples.
  • the method may be used to produce a signature that may be diagnostic (e.g., may provide a diagnosis of a disease or condition or the type or stage of a disease or condition, etc.), prognostic (e.g., indicating a clinical outcome, e.g., survival or death within a time frame) or theranostic (e.g., indicating which treatment would be the most effective).
  • diagnostic e.g., may provide a diagnosis of a disease or condition or the type or stage of a disease or condition, etc.
  • prognostic e.g., indicating a clinical outcome, e.g., survival or death within a time frame
  • theranostic e.g., indicating which treatment would be the most effective.
  • the binding moieties specifically bind to biomarkers, including cancer biomarkers, that may be proteinaceous or a nucleic acid.
  • cancer biomarkers include, but are not limited to carcinoembryonic antigen (for identification of adenocarcinomas), cytokeratins (for identification of carcinomas but may also be expressed in some sarcomas), CD 15 and CD30 (for Hodgkin's disease), alpha fetoprotein (for yolk sac tumors and hepatocellular carcinoma), CD 117 (for gastrointestinal stromal tumors), CD 10 (for renal cell carcinoma and acute lymphoblastic leukemia), prostate specific antigen (for prostate cancer), estrogens and progesterone (for tumour identification), CD20 (for identification of B- cell lymphomas) and CD3 (for identification of T-cell lymphomas).
  • carcinoembryonic antigen for identification of adenocarcinomas
  • cytokeratins for identification of carcinomas but may also be expressed in some sarcomas
  • the binding moieties may bind to a drug, and the method may be used to examine the distribution of the drug in a cell after it has been administered to by a subject, e.g., a human.
  • the drug itself may be isotopically labeled, and the binding moieties may be used to identify cellular proteins.
  • the above-described method can be used to analyze cells from a subject to determine, for example, whether the cell is normal or not or to determine whether the cells are responding to a treatment.
  • the method may be employed to determine the degree of dysplasia in cancer cells.
  • the cells may be a sample from a multicellular organism.
  • a biological sample may be isolated from an individual, e.g., from a soft tissue.
  • the method may be used to distinguish different types of cancer cells in FFPE samples.
  • the analysis may be to determine an appropriate therapy.
  • the method described above finds particular utility in examining planar samples using a plurality of antibodies, each antibodies recognizing a different marker. Examples of cancers, and biomarkers that can be used to identify those cancers, are shown below. In these embodiments, one does not need to examine all of the markers listed below in order to make a diagnosis.
  • the method may involve obtaining an image as described above (an electronic form of which may have been forwarded from a remote location) and may be analyzed by a doctor or other medical professional to determine whether a patient has abnormal cells (e.g., cancerous cells) or which type of abnormal cells are present.
  • the image may be used as a diagnostic to determine whether the subject has a disease or condition, e.g., a cancer.
  • the method may be used to determine the stage of a cancer, to identify metastasized cells, or to monitor a patient’s response to a treatment, for example.
  • data can be forwarded to a“remote location”, where“remote location,” means a location other than the location at which the image is examined.
  • a remote location could be another location (e.g., office, lab, etc.) in the same city, another location in a different city, another location in a different state, another location in a different country, etc.
  • office, lab, etc. another location in the same city
  • another location in a different city e.g., another location in a different city
  • another location in a different state e.g., another location in a different state
  • another location in a different country etc.
  • the two items can be in the same room but separated, or at least in different rooms or different buildings, and can be at least one mile, ten miles, or at least one hundred miles apart.
  • Communication references transmitting the data representing that information as electrical signals over a suitable communication channel (e.g., a private or public network).
  • a suitable communication channel e.g., a private or public network.
  • "Forwarding" an item refers to any means of getting that item from one location to the next, whether by physically transporting that item or otherwise (where that is possible) and includes, at least in the case of data, physically transporting a medium carrying the data or communicating the data. Examples of communicating media include radio or infra-red transmission channels as well as a network connection to another computer or networked device, and the internet or including email transmissions and information recorded on websites and the like.
  • the image may be analyzed by an MD or other qualified medical professional, and a report based on the results of the analysis of the image may be forwarded to the patient from which the sample was obtained.
  • the method may be employed in a variety of diagnostic, drug discovery, and research applications that include, but are not limited to, diagnosis or monitoring of a disease or condition (where the image identifies a marker for the disease or condition), discovery of drug targets (where the a marker in the image may be targeted for drug therapy), drug screening (where the effects of a drug are monitored by a marker shown in the image), determining drug susceptibility (where drug susceptibility is associated with a marker) and basic research (where is it desirable to measure the differences between cells in a sample).
  • diagnosis or monitoring of a disease or condition where the image identifies a marker for the disease or condition
  • discovery of drug targets where the a marker in the image may be targeted for drug therapy
  • drug screening where the effects of a drug are monitored by a marker shown in the image
  • determining drug susceptibility where drug susceptibility is associated with a marker
  • basic research where is it desirable to measure the differences between cells in a sample).
  • two different samples may be compared using the above methods.
  • the different samples may be composed of an“experimental” sample, i.e., a sample of interest, and a“control” sample to which the experimental sample may be compared.
  • the different samples are pairs of cell types or fractions thereof, one cell type being a cell type of interest, e.g., an abnormal cell, and the other a control, e.g., normal, cell. If two fractions of cells are compared, the fractions are usually the same fraction from each of the two cells. In certain embodiments, however, two fractions of the same cell may be compared.
  • Exemplary cell type pairs include, for example, cells isolated from a tissue biopsy (e.g., from a tissue having a disease such as colon, breast, prostate, lung, skin cancer, or infected with a pathogen etc.) and normal cells from the same tissue, usually from the same patient; cells grown in tissue culture that are immortal (e.g., cells with a proliferative mutation or an immortalizing transgene), infected with a pathogen, or treated (e.g., with environmental or chemical agents such as peptides, hormones, altered temperature, growth condition, physical stress, cellular transformation, etc.), and a normal cell (e.g., a cell that is otherwise identical to the experimental cell except that it is not immortal, infected, or treated, etc.); a cell isolated from a mammal with a cancer, a disease, a geriatric mammal, or a mammal exposed to a condition, and a cell from a mammal of the same species, preferably from the same family, that is healthy
  • cells of different types e.g., neuronal and non-neuronal cells, or cells of different status (e.g., before and after a stimulus on the cells) may be employed.
  • the experimental material is cells susceptible to infection by a pathogen such as a virus, e.g., human immunodeficiency virus (HIV), etc.
  • the control material is cells resistant to infection by the pathogen.
  • the sample pair is represented by undifferentiated cells, e.g., stem cells, and differentiated cells.
  • the images produced by the method may be viewed side-by-side or, in some embodiments, the images may be superimposed, combined or staked to produce a 3-d image. In some cases, the images may be in color, where the colors used in the images may correspond to the labels used.
  • Cells any organism, e.g., from bacteria, yeast, plants and animals, such as fish, birds, reptiles, amphibians and mammals may be used in the subject methods.
  • bacteria e.g., from bacteria, yeast, plants and animals, such as fish, birds, reptiles, amphibians and mammals
  • animals such as fish, birds, reptiles, amphibians and mammals
  • mammalian cells i.e., cells from mice, rabbits, primates, or humans, or cultured derivatives thereof, may be used.
  • GM12878 were grown in RPMI 1640 (Life Technologies), 15% fetal bovine serum (FBS; Omega Scientific) and 2mM L-glutamine (Life Technologies).
  • Jurkat and K562 cells were grown in RPMI 1640, 10% FBS and 2mM L-glutamine.
  • HeLa cells were grown in DMEM (Life Technologies), 10% FBS and passaged with TrypLE Express (Gibco). Cells were maintained in 100 U/mL penicillin (Life Technologies) and 100 mg/mL
  • ESI-MS Electrospray Ionization Mass Spectrometry
  • LC-MS was run on a Thermo linear ion trap.
  • a short 8 minute gradient using HFIP ion pairing buffer for A and methanol for B was used at a flow rate of 0.4 ml/min on a phenomenex clarity 2.1 mm column (00B-4746-AN) at 60°C.
  • Spray voltage was set at -1.5 kV.
  • LC-MS was run in Negative Ion mode set at full scan.
  • the fluorine-labeled oligonucleotides sequences are as follows (5’ to 3’):
  • Tn5 transposome assembly The assembly of Tn5 transposome was performed as described 2 . Briefly, fluorine-labeled oligonucleotides were each resuspended in Nuclease-Free water (Ambion) to 100 mM. The Tn5MERev-5FdU-5FdC/Tn5ME-A-ATT0514-5FdU and Tn5MERev-5FdU-5FdC/Tn5ME-B-ATT0514-5FdU duplexes were created by mixing oligonucleotides at molar ratio of 1:1 (final concentration of 50 pM) in sterile microcentrifuge tubes.
  • Tn5 transposome 0.1 vol SF-Tn5 (50 pM) was mixed with 0.125 vol Tn5MERev-5FdU-5FdC/Tn5ME-A-ATT0514-5FdU and 0.125 vol Tn5MERev-5FdU- 5FdC/Tn5ME-B-ATT0514-5FdU in 0.12 vol 2x dialysis buffer (100 mM HEPES-KOH, pH
  • Preliminary 133 Cs + implantation was done by scanning the region of interest for 1 to 5 minutes without limiting the angular aperture of the primary beam (Dl aperture 0).
  • High spatial resolution negative-ion maps were acquired with a ⁇ 2-4 pA 133 Cs + primary beam focused to a -100-200 nm diameter spot. Images over areas of 15 pm 2 containing 256 x 256 pixels were acquired with entrance slit 0 and aperture slit 0 and a pixel dwell time of 1 ms/pixel. Ion maps were obtained simultaneously for 19 F and 127 T ion species in multicollection mode. Up to 70 sequential scans over a single area were collected for 3D image reconstruction.
  • the resulting ATTO 514 Dynabeads were stained with either isotype control or anti-Fluorescein/Oregon green antibody (cat# A- 889, Life Technologies) for 30 min at room temperature in cell staining media (CSM; PBS containing 0.5% BSA), followed by two washes with CSM, and staining with Alexa 647 conjugated anti-rabbit secondary antibody (cat# 4414S, Cell Signaling Technology). After two washes with CSM, beads were analyzed using a BD LSRII. For mass cytometry analysis of ATAC-mass, Jurkat cells were washed with PBS and incubated with 10 mM IdU in complete RPMI 1640 media for 10 min at 37°C to label S-phase cells.
  • Viability staining was performed by incubating the cells with 50 pM cisplatin (cat# P4394, Sigma- Aldrich) in 1 ml of serum- free RPMI 1640 for 1 min at room temperature. An equal volume of complete RPMI 1640 medium was added and cells were centrifuged, followed by fixation with 1%
  • FCS files were analyzed by first gating out doublets, debris, and dead cells based on cell length, DNA content, cisplatin staining and cleaved caspase-3. Data analysis was done in Cytobank.
  • ATAC-mass ATAC-seq in GM12878 cells using with Illumina Tn5 transposase (Nextera kit; cat# FC-121-1030, Illumina) and Fluoro-Tn5 was performed as described 1 . Briefly, 50,000 cells were centrifuged at 500 g for 5 min at 4°C and washed once in cold PBS. The cell pellet was gently resuspended in 50 pl pre-chilled lysis buffer (10 mM Tris-Cl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% Igepal CA-630) and centrifuged immediately at 500 g for 10 min at 4 °C.
  • lysis buffer 10 mM Tris-Cl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% Igepal CA-630
  • the cell pellet was resuspended in 50 pl transposase mixture (25 m ⁇ 2x TD buffer (Nextera kit), 2.5 m ⁇ Illumina Tn5 transposase or final concentration of 100 nM Fluoro-Tn5, and Nuclease-Free water up to 40 m ⁇ ) and incubated at 37 °C 30 min. After transposition, the mixture was purified with Qiagen Min-Elute PCR purification kit (cat# 28004, Qiagen) and eluted in 10 m ⁇ Qiagen buffer EB. Sequencing libraries were prepared following the original ATAC-seq protocol 1 . The sequencing was performed on Illumina NextSeq at the Stanford Functional Genomics Facility.
  • ATAC-seq library data preprocessing and analysis ATAC-seq paired-end reads were trimmed for Illumina adaptor sequences and transposase sequences using an in-house script, and they were mapped to hgl9 using Bowtie2 v2.l.O with parameters - very-sensitive. Over 50 million mapped reads were generated in each sequencing library and used for downstream data mining. Duplicate reads were removed with Picard vl.79. Peak calling was performed by MACS2 narrow peak mode with parameters -q 0.01 -nomodel -shift 0.
  • Overlapping peaks from all samples were merged together to a consensus peak list, and number of unique-mapped and properly paired reads mapped to each peak for each individual samples was quantified to calculate the Pearson correlation.
  • For ATAC-seq signal intensity around TSS a 2 kb window centered on TSS was divided into 40 equally sized bins of 50 bp. The number of unique-mapped and properly paired ATAC-seq tags overlapping each bin was counted.
  • modified AT AC DNA- adaptors were syntheized. 5-fluoro-2’-deoxyuridine and 5-fluoro-2’-deoxycytidine nucleotides were incorporated into the adaptors sequence, while ATTO 514 was conjugated at the 5'-end of each of them (Fig. 2, a). Through these modifications, a fluorescent molecule (ATTO 514) and multiple atoms of fluorine per adapter were incorporated (please note that ATTO 514 also contains 6 fluorines per fluorophore; Fig. 2, a). The size and purity of these adaptors were validated by electrospray ionization mass spectrometry.
  • Tn5 transposase was purified as previously reported, loaded it with fluorine-labeled adaptors (Fluoro-Tn5; Fig. 2, a) and tested whether Fluoro-Tn5 retained the activity and selectivity for open chromatin sites.
  • ATAC-seq of human B cells(GM 12878) performed using Fluoro-Tn5 showed similar genomic distribution of ATAC-seq peaks and transcriptional start site enrichment when compared to results using Nextera-Tn5 (the commercially available adaptors and enzyme)
  • modified adaptors allow genome accessibility to be analyzed by sequencing and fluorescence-microscopy, demonstrating retention of Tn5 enzymatic activity and specificity.
  • ATAC-mass reveals increased chromatin accessibility in interleukin- 18 primed NK cells'
  • Synergistic interaction between cytokines is a common feature that immune cells may react in a non-linear way to challenges from the environment. For example, it is well established that during acute infection, interleukin- l2(IL- 12) and IL-18 secreted by the first line defenders, such as dendritic cells and macrophages, could synergistically activate natural killer cells(NK cells) and promote the secretion of IFNg from NK cells. However, it is still not clear how this synergistic effect is achieved and what the role of individual cytokines is. The ATAC-mass was applied together with multiple intracellular signaling pathways staining to interrogate the effect of IL-12 or IL-18 on epigenetic states as well as signaling networks.
  • ATAC-mass signals were observed in IL-18 primed NK cell line, NKL cells, in comparison to either without priming or IL-12 primed cells as shown in Fig. 4, c indicating that IL-18 affect NKL cells at epigenetic level by opening up genome accessibility.
  • ATAC-mass is highly correlated with NFkB, c-Jun and CD3zeta- SLP76 pathway (Fig. 4, d).
  • phospho-Stat4 which are downstream of IL-12 did increase after IL-12 priming but did not correlated with ATAC-mass signals (Fig. 4, c,d).
  • ATAC-seq was performed on cells of these three conditions and also saw significant increase of ATAC-peak in IL-18 group but not IL-12 or without priming group (Fig. 4, e). Consistently, the upregulated promoter regions are enriched with NFkB and AP-l binding motif, which brings up the possibility that IL-18 promotes the binding of NFkB and c-Jun to enhancers and subsequently facilitate the recruitment of chromatin remodeling factors to open the promoter region similar to the mechanism of transcription factor synergism in the induction of human interferon-b (IFNB) gene.
  • IFNB human interferon-b
  • IL-18 priming opens the genome accessibility, it was hypothesized that IL-18 could be a mediator of more potent effector functions in polyfunctional immune cells, which are commonly observed in the setting of effectively controlled viral infections. Indeed, when NKL are primed with IL-18 first and then stimulated with PMA plus ionomycin, percentage of polyfunctional NK cells are significantly increased (Fig. 5, a,c). In contrast, IL-12 only enhances the intensity of IFNg but has no effect on the number of functional effectors (Fig. 5, a,b,c).
  • NanoSIMs For secondary ion beam imaging, it has been previous demonstrated how NanoSIMs instruments enable multiplexed imaging through an oxygen beam. In the present case the same instrument (NanoSIMs) is used, but with a different ion beam (i.e. Cesium), which enables high-resolution secondary ion beam imaging.
  • Cesium a different ion beam
  • Detection 19 F secondary ions empowers imaging of accessible genome with higher resolution than previously reported (Fig. 3). Through co-detection of metabolic DNA labeling , the nuclear ultrastructure can be examined in a multiplexed fashion.
  • NanoSIMs instruments are limited by the number of detectors installed in each specific machine. However, given the very high Z-resolution, higher order multiplexing can be achieved by rendering a different set of masses at each scan, thus de facto bringing the theoretical multiplexing capability up to the number of isotopes potentially detectable after bombardment with each specific
  • GM12878 cells were grown in RPMI 1640 (Life Technologies), 15% fetal bovine serum (FBS; Omega Scientific), and 2 mM L-glutamine (Life Technologies).
  • Jurkat and K562 cells were grown in RPMI 1640, 10% FBS, and 2 mM L-glutamine.
  • NKL cells were grown in RPMI 1640, 10% FBS, 2 mM L-glutamine, and 200 U/mL rhIL-2 (National Cancer Institute).
  • HeLa cells were grown in DMEM (Life Technologies), 10% FBS and passaged with TrypLE Express (Gibco).
  • NKL cells were maintained in 100 U/mL penicillin (Life Technologies) and 100 mg/mL streptomycin (Life Technologies) at 37 °C in 5% C0 2 conditions.
  • cytokine treatment of NKL cells cells were incubated with 200 U/mL rhIL-2 plus 20 ng/ml human IL- 18 (R&D Systems) or 10 ng/ml human IL-12 (R&D Systems) for 24 h.
  • NKL cells were stimulated with IX PMA plus ionomycin (eBioscience) in the presence of IX brefeldin A plus monensin (eBioscience) and 1:250 anti-CDl07a 151 Eu (Fluidigm) antibody for 4 h.
  • the fluorine-labeled oligonucleotides for the Tn5 transposase adaptor were synthesized at Biosynthesis Inc., and a Thermo linear ion trap was used for LC-MS analyses of adaptors.
  • a short 8-min gradient using HFIP ion pairing buffer for A and methanol for B was used at a flow rate of 0.4 ml/min on a Phenomenex clarity 2.
  • l-mm column (00B-4746-AN) run at 60 °C. Spray voltage was set at -1.5 kV.
  • LC-MS was run in negative ion mode set at full scan.
  • Tn5 transposome was performed as described 2 . Briefly, fluorine- labeled oligonucleotides were each resuspended in nuclease-free water (Ambion) to 100 mM. The Tn5MERev-5FdU-5FdC/Tn5ME-A-ATT0514-5FdU and Tn5MERev-5FdU- 5FdC/Tn5ME-B-ATT0514-5FdU duplexes were created by mixing oligonucleotides at molar ratio of 1:1 (final concentration of 50 pM) in sterile microcentrifuge tubes. The mixtures were heated at 95 °C for 5 min and cooled in the thermocycler by turning it off.
  • Tn5 transposome 0.1 volume SL-Tn5 (50 mM) was mixed with 0.125 volume Tn5MERev- 5FdU-5FdC/Tn5ME-A-ATT05l4-5FdU and 0.125 volume Tn5MERev-5FdU-5FdC/Tn5ME- B-ATT05 l4-5FdU in 0.12 volume 2x dialysis buffer (100 mM HEPES-KOH, pH 7.2, 0.2 M NaCl, 0.2 mM EDTA, 2 mM DTT, 0.2% Triton X-100, 20% glycerol), 0.4 volume glycerol (100% solution), and 0.13 volume nuclease-free water. After gentle pipetting, the mixture was incubated at room temperature for 1 h protected from light to assemble the complex and then was stored at -20 °C.
  • 0.12 volume 2x dialysis buffer 100 mM HEPES-KOH, pH 7.2, 0.2 M NaCl, 0.2
  • Cells were grown in l2-mm glass coverslips until 80-90% confluent, fixed with 1% paraformaldehyde for 10 min at room temperature, and washed three times with PBS. Cells were permeabilized in lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl 2 , 0.1% Igepal CA-630) at room temperature for 10 min.
  • lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl 2 , 0.1% Igepal CA-630
  • Silicon wafers (Silicon Valley Microelectronics) were rinsed twice with methanol and cleaned with a cotton-tipped applicator. Substrates were then immersed in acetone for 5 min, placed in Vectabond reagent solution (Vector Laboratories) for 30 min at room temperature, washed once in acetone, air-dried using compressed air, and baked at 70 °C for 1 h. K562 cells were incubated with 10 mM 5-iodo-2’-deoxyuridine (Sigma-Aldrich) in complete RPMI 1640 for 24 h to label DNA.
  • Samples were imaged with a NanoSIMS 50L mass spectrometer (Cameca) using a cesium primary ion beam supplied by a microbeam cesium source.
  • the primary optics, secondary optics, mass spectrometer, and detector trolleys were tuned before each experiment.
  • Preliminary cesium implantation was done by scanning the region of interest for 1 to 5 min without limiting the angular aperture of the primary beam (Dl aperture 0).
  • High spatial resolution negative-ion maps were acquired with a 2-4 pA cesium primary beam focused to a 100-200 nm diameter spot.
  • Dynabeads M-280 Streptavidin (Life Technologies) were incubated with ATTO 5l4-biotin (ATTO-TEC) in PBS for 30 min at room temperature and washed twice with PBS.
  • the resulting ATTO 514 Dynabeads were stained with either isotype control or anti-fluorescein/Oregon green antibody (cat# A-889, Life Technologies) for 30 min at room temperature in cell staining media (CSM; PBS containing 0.5% BSA), followed by two washes with CSM, and staining with Alexa 647-conjugated anti-rabbit secondary antibody (cat# 4414S, Cell Signaling Technology).
  • Fluoro-Tn5 tagmentation was performed as described above, and then cells were permeabilized with ice-cold methanol for 10 min on ice and washed twice with CSM.
  • Jurkat cells were stained with the following antibodies: cyclin Bl 164 Dy (cat# 3164010A, Fluidigm, 1:50), p-Rb (S807/811) 165 Ho (clone Jl 12-906, BD Biosciences, 2 pg/mL), p-Histone H3 (S28) 143 Nd (clone HTA28, Biolegend, 1 pg/mL), Fluorescein/Oregon green 156 Gd (cat# A-
  • FCS files were analyzed by first gating out doublets, debris, and dead cells based on cell length, DNA content, and cisplatin and cleaved caspase-3 staining. Data analysis was done in Cytobank (svwsv.c vtobank.org). For intracellular cytokine staining, cells were fixed with 1.5%
  • ATAC-seq in GM12878 and NKL cells was performed using Illumina Tn5 transposase (Nextera kit; cat# FC-121-1030, Illumina), and ATAC-seq was performed in GM12878 cells with Fluoro-Tn5 as described previously 1 . Briefly, 50,000 cells were centrifuged at 500 g for 5 min at 4 °C and washed once in cold PBS. The cell pellet was gently resuspended in 50 pl pre chilled lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl 2 , 0.1% Igepal CA- 630) and centrifuged immediately at 500 g for 10 min at 4 °C. The cell pellet was resuspended in 50 m ⁇ transposase mixture (25 m ⁇ 2x TD buffer (Nextera kit), 2.5 m ⁇ Illumina Tn5
  • Sequencing libraries were assessed using the 2100 Bioanalyzer (Agilent) for nucleosome profiles, and the library concentration was estimated by analysis of library fragments between 50 and 450 base pairs (bps). The sequencing was performed on Illumina NextSeq or HiSeq at the Stanford University
  • ATAC-seq paired-end reads were trimmed for Illumina adaptor sequences and transposase sequences using an in-house script and were mapped to hgl9 using Bowtie2 v2.l.O with the parameter -very-sensitive. Over 50 million mapped reads were generated in each sequencing library and used for downstream data mining. Duplicate reads were removed with Picard vl.79. Peak calling was performed using MACS2 in narrow peak mode with parameters -q 0.01, -nomodel, -shift 0. Overlapping peaks from all samples were merged into a consensus peak list, and the number of uniquely mapped and properly paired reads mapped to each peak for each individual sample was quantified to calculate the Pearson correlation.
  • a 2-kbp window centered on a TSS was divided into 40 equally sized bins of 50 bp.
  • the number of uniquely mapped and properly paired ATAC- seq tags overlapping each bin was counted.
  • the replicate samples were merged, and differential peak analysis was performed with DESeq2 65 .
  • the peaks of significantly different intensity between different conditions were selected based on (log2 (fold change) > 1 or ⁇ - 1 and BH-adjusted p-value ⁇ 0.01 65 .
  • the motif enrichment analysis was performed with Homer using the whole genome as background 66 .
  • Raw ion beam images were converted to TIFF files by OpenMIMS ImageJ, and the signal to noise was computationally enhanced in each channel. Briefly, multiple depth ion images (e.g., 3-5 slices) were combined to generate ion images with lower noise, followed by a digital sharpening of the images using a custom deconvolution method 47 . These improved ion images were then imported to the 3D rendering software (Imaris, Bitplane). The surfaces function in the rendering module was used to define ATAC-mass signal and DNA signal in the 3D visualization images ( Figure 13 a) and 3D videos. Line-scan analyses of 2D images were performed to plot pixel intensities for each isotope along straight lines crossing features of interest.
  • Isotope-modified ATAC adaptors are compatible with multiple technology platforms
  • SIMS is a powerful tool for studying biological samples due to its high spatial resolution and ability to detect multiple ion species simultaneously 33 35 .
  • the primary cesium ion beam in the NanoSIMS instrument currently achieves one of the highest lateral resolutions in the SIMS arena 31, 36, 37 .
  • NanoSIMS operates by rastering the finely focused primary ion beam over the sample surface and detecting the secondary ions released using a magnetic sector mass spectrometer. The elemental information of the secondary ions is registered to the position of the primary ion beam, and the ion map image is reconstructed pixel by pixel.
  • Serial plane acquisitions result in a stack of composite images that can be used to reconstruct cellular features of interest.
  • transposon insertion tags are used to label DNA.
  • ATAC DNA adaptors were modified to contain 5-fluoro-2’-deoxyuridine and 5-fluoro-2’-deoxycytidine nucleotides and a 5’ ATTO 514 ( Figure 10 a and Figure 19 a).
  • This design facilitates five applications ( Figure 10 b): ATAC-seq analysis, high-resolution ion beam imaging (enabled by the fluorine atoms), fluorescence microscopy and flow cytometry (by detection of the ATTO 514 tag), and mass cytometry (using a metal-conjugated antibody to the ATTO 514 tag).
  • Transcriptional start site (TSS) enrichment ( Figure 10 d) and ATAC-seq profiles (Figure 10 e) were similar, although there were minor differences in peak height in certain nuclear domains.
  • TSS Transcriptional start site
  • Figure 10 e ATAC-seq profiles
  • ATAC-mass enables multi-parametric single-cell mass cytometric accessible genome analysis
  • the CyTQF mass cytometer is a multi-parameter tool that exploits isotopic masses as tags.
  • the inductively coupled plasma mass spectrometer deployed in the CyTOF is not able to evaluate masses below 75 atomic mass units due to a mass cutoff that filters out abundant low mass ions; thus, it is unable to directly detect the fluorine atoms in the ATAC-mass adaptors.
  • ATAC-mass reveals increased genome accessibility in NK cells treated with interleukin-18
  • ATAC-mass was used to study the regulation of chromatin states in natural killer (NK) cells treated with the interleukins IL-12 and IL-18.
  • IL-12 and IL-18 function cooperatively to enhance NK cell activity both in vitro and in vivo 38, 39 .
  • NK cells from the NKL line were treated or not with IL-12 or IL-18, stained with a panel of previously validated metal-conjugated antibodies against key signaling components 40, 41 , and subjected to ATAC-mass.
  • IL-l8-treated NKL cells showed increased genome accessibility (i.e., higher ATAC-mass signal) and increased levels of phosphorylation of TBK1, p65/RELA (NFkB), CD3zeta, SLP76, Stat3, Stat4, Stat5, c-Jun, and ribosomal protein S6 ( Figure 11 a).
  • IL-12 treatment of NKL cells did not cause an increase in ATAC-mass signal as compared to untreated cells, although it did induce the expected patterns of signaling with phosphorylation of Stat3, Stat4, and S6 (Figure 11 a) 42, 43 .
  • a t-SNE visualization revealed that a subpopulation of cells with high ATAC-mass intensity was induced by treatment with IL-18 but not with IL-12 ( Figure 11 b and c).
  • Interleukin-18 treatment increases accessibility of quiescent genomic regions in NK cells
  • NanoSIMS Fluorescence-based approaches such as super-resolution microscopy could be used to investigate DNA architecture, but these methods are time consuming and only a few parameters can be evaluated simultaneously.
  • cellular features can be visualized at a resolution below the diffraction limit of light 46, 47 .
  • IdU 5-iodo-2'-deoxyuridine
  • K562 cells metabolically labeled with IdU were tagmented using Fluoro-Tn5 to visualize the spatial organization of the accessible genome in relation to DNA compactness.
  • Serial scanning at different depth planes showed non-uniform distribution of fluorine ions ( 19 F) ( Figure 26 a).
  • the lateral resolution of the ATAC-mass signals was at about 100 nm, calculated using the 16-84% criterion 36 ( Figure 26 b), enabling higher resolution visualization of the accessible genome than previously reported methods 7 .
  • a 3D reconstruction of ATAC- mass signals overlaid with that of the 127 I signal ( Figure 13 a) revealed that some foci with high ATAC-mass signals are clearly separated from compact DNA regions ( Figure 13 b, closed arrows).
  • Control and IL-l8-treated NKL cells had similar proportions of the analyzed nuclear territories, whereas IL-l2-treated cells had significantly more areas with ATAC-mass low regions associated with uncompact DNA as compared to control and IL-18 cells (77.6%, 54.2%, and 59.2%, respectively, Figure 13 c).
  • Interleukin-18 induces NK polyfunctionality
  • the next step was to determine the functional effects of the observed nuclear remodeling.
  • the frequency of polyfunctional immune cells defined as cells that produce multiple cytokines, is known to be regulated at the epigenetic level 50 52 . Therefore, it was asked whether the observed effects of IL-18 on the genome accessibility of NK cells was a result of an increase of cytokine production.
  • ATAC-mass for in situ visualization of genome accessibility in tissues should facilitate application of single-cell epigenomics and proteomics to clinically relevant specimens.
  • Stable isotopes such as 13 C and 15 N can be used as labels for metabolite visualization because of the excellent mass resolution of high-resolution ion beam imaging 31, 32, 53, 54 .
  • Chromatin remodeling is a highly energy-consuming process 55 , thus, it will be of interest to visualize genome accessibility in the presence of different metabolic labels to identify the metabolic determinants of chromatin remodeling at the nanoscale.
  • AT AC-mass coupled with DNA-FISH will enable high-throughput quantification of accessibility at the single-locus level, which will be especially relevant for drug screens targeting chromatin remodeling using barcoding techniques already developed for mass cytometry 56 .
  • ATAC-mass provides the opportunity to integrate epigenomics, proteomics, and high- resolution imaging at the single-cell level.
  • the effect of IL-12 and IL-18 on NK cells was focused on. Synergistic interactions between cytokines allow immune cells to respond in a non-linear way to challenges from the environment 57 .
  • IL- 12 and IL-18 secreted by first-line defenders such as dendritic cells and macrophages, synergistically activate and promote the secretion of IFNy from NK cells 58 60 .
  • IL-18 may function as the initiator of stepwise transcription activation, recruiting chromatin remodeling factors to the quiescent genomic regions, with subsequent synergistic interaction with promoter regions targeted by IL-12.
  • this mechanism of transcription factor synergism might be an efficient regulatory principle for innate immune system as similarly ordered epigenetic regulation has been reported for human interferon-b expression in response to viral infection 61, 62 .
  • IL-18 is an epigenetic modulator and, in combination with other ATAC methods, pinpointed mechanisms of gene regulation that could be exploited for vaccine or infection-control therapy development. Elevated IL-18 levels have been detected in the serum of patients suffering from several autoimmune disorders 63, 64 . It will be useful to investigate the epigenetic states of different immune subsets from such patients by
  • ATAC-mass especially in patients with diseases associated with NK cell dysfunction.
  • CD45 as a regulator of IL-2 synergy in the NKG2D-mediated activation of immature human NK cells. Science Signaling 10 (2017).
  • Multifunctional TH1 cells define a correlate of vaccine-mediated protection against Leishmania major. Nature Medicine 13, 843 (2007).
  • super-resolution MIBI using a positively charged cesium primary ion beam that allows visualization of subcellular structures with lateral (XY) and axial (Z) resolutions down to approximately 30 nm and 5 nm, respectively.
  • This method which is called super-resolution ion beam imaging (srIBI)
  • srIBI is capable of determining the precise subcellular locations of multiple small molecules, proteins, and/or nucleic acids at single-molecule resolution.
  • the capabilities of srIBI are demonstrated by simultaneously imaging five distinct subnuclear structures and the chemotherapeutic drug cisplatin.
  • Cisplatin is enriched in nuclear speckles, suggesting that this drug influences pre- mRNA processing.
  • a framework incorporating dimensional reduction and clustering methods to analyze srIBI data microenvironments within the nucleus, termed nuclear neighborhoods, are identified. Studying the interaction of these nuclear neighborhoods, the results suggest a directionality of cisplatin action within nuclear speckles.
  • srIBI joins the growing suite of techniques that image molecular component in situ. Use of these approaches will allow study of the roles of small molecules in biological processes and the functions of distinct biomolecules in multi-component molecular pathways.
  • HeLa cells were grown in DMEM (Gibco, Invitrogen). Media were supplemented with 10% heat-inactivated fetal bovine serum, 100 U/mL penicillin (Gibco, Invitrogen), and 100 mg/mL streptomycin (Gibco, Invitrogen). Cells were cultured in a humidified cell incubator at 37 °C with 5% C0 2 conditions and split with TrypLE Express (Gibco, Invitrogen) every 2-3 days.
  • Oligonucleotides (Table 3) were synthesized at the Stanford Protein and Nucleic Acid Facility with internal isotope-derivatized nucleotides, a fluorophore at the 3’ position, and a maleimide cycloadduct at the 5’ position.
  • the maleimide was deprotected by a retro Diels- Alder reaction.
  • the lyophilized oligonucleotide was suspended in 1 mL anhydrous toluene (MTX07327, Millipore) for 4 hours at 90 °C, washed four times with anhydrous ethanol, and solubilized in buffer C (2 mM Tris, 150 mM NaCl, 1 mM EDTA, pH 7.2).
  • oligonucleotide concentration was determined using a Nanodrop spectrophotometer. Aliquots of 8.5 nmol were prepared, lyophilized overnight, and stored in a desiccator at -20 °C.
  • Antibodies (Table 4) in carrier-free PBS were conjugated to the deprotected oligonucleotides. Briefly, 50 pg of antibody was loaded into a 50-KDa 0.5-mL centrifugal filter column with 400 pL PBS and reduced with 400 pL reduction buffer (PBS with 2.5 mM TCEP and 2.5 mM EDTA) for 30 minutes at room temperature.
  • Antibodies were then washed with 400 pL of C buffer into a 50- KDa 0.5-mL centrifugal filter column and conjugated to 8.5 nmol of oligonucleotide in 400 pL conjugation buffer (buffer C with 0.5 M NaCl) for 2 hours at room temperature.
  • Antibodies were washed five times with 400 pL of high- salt PBS (PBS with 1 M NaCl), diluted into storage buffer (Candor PBS Antibody Stabilization Solution with 0.5 M NaCl and 5 mM EDTA) and stored at 4 °C.
  • Each MoC-Ab was titrated by immunofluorescence using HeLa cells, as exemplified in Fig. 30, and the staining pattern was compared to the staining pattern of the unconjugated antibody.
  • Antibodies (Table 4) in carrier-free PBS were conjugated to metal-chelated polymers (MaxPAR Antibody Conjugation Kit, Fluidigm) or Sulfo-NHS-SS-Biotin (A39258,
  • Antibodies were diluted to 0.2 mg/mL in Candor PBS Antibody Stabilization Solution and stored at 4 °C.
  • TYK-nu cells were cultured in 0.5, 5, or 50 mM of cisplatin (P4394, Sigma- Aldrich) for 24 hours, washed, and treated with 1 mM Rh-intercalator (201103B, Fluidigm) for 15 minutes to discriminate dead from live cells. Cells were analyzed in a CyTOF2 instrument (Fluidigm) as previously described (63, 64).
  • compositions of buffers used for staining are given in Table 5.
  • Cells were fixed and permeabilized during 30 minutes at 4 °C in Fixation/Permeabilization buffer. Cells were then gently washed three times with Wash Buffer and blocked in Block Buffer 1 during 30 minutes at room temperature, washed three times with Wash Buffer, blocked in Block Buffer 2 during 30 minutes at room temperature, and washed three times with Wash Buffer.
  • the cells were stained with a mixture of MoC-Abs in Reaction Buffer for 3 hours at room temperature. Following staining with MoC-Abs, the Reaction Buffer was removed by gently touching the sample with a precision wipe (05511, Kimtech Science).
  • confocal microscopy analysis cells were grown in l2-mm diameter glass coverslips (72226-01, Electron Microscopy Sciences) until 80-90% confluent and stained as described. After staining, cells were washed three times with PBS, rinsed once with water, and mounted using Vectashield with DAPI (H-1200, Vector Laboratories). Anti-mouse- Alexa488 (1:2000 titer, 4408S, Molecular Probes) or anti-mouse- Alexa647 (1:2000 titer, 4410S, Molecular Probes) were the secondary antibodies used to detect unconjugated antibodies. Cells were analyzed in a LSM 880 (Zeiss) with a 63x oil-immersion objective (Zeiss Plan- Apochromat 63x/l.4 Oil).
  • srIBI analysis cells were grown on silicon wafers (7 nm * 7 nm or 18 nm * 18 mm, Silicon Valley Microelectronics). Wafers were rinsed twice in methanol, air-dried with compressed air, washed with ethanol for 10 minutes, and rinsed three times with sterile PBS in a cell culture hood prior to cell seeding. When cells reached 80-90% confluence, they were stained as described above. After staining, cells were washed twice in PBS, fixed for 5 minutes in Post-Fixation buffer, and rinsed five times with water. Cells were dehydrated using a graded ethanol series, air dried in a desiccator chamber, and stored at room temperature in a vacuum desiccator until analysis. srIBI images were acquired with the NanoSIMS 50L mass spectrometer (Cameca) at Stanford University using the CAMECA Microbeam Cesium
  • Counts were log2 transformed after adding a“0.0001” value to avoid zeros.
  • Unsupervised hierarchical clustering was performed on all voxels using the markers SC35, H3K9me3, phosphorus, nucleolin, H3K27Ac, and cisplatin.
  • a t-Distributed Stochastic Neighbor Embedding (t-SNE) using a Bames-Hut implementation was performed, and each identified cluster was differentially colored. The means of each cluster was also plotted and subsequently manually merged by similarity into 11 final clusters. The t-SNE plot was also colored by the cell of origin of each voxel to ensure minimal batch effects.
  • Equation 1 Next, we defined interacting voxels as those less than or equal to 5 pixels away from each other (between the center of each voxel). After 1000 permutations, where the cluster annotation was shuffled, and the p- value calculated on either tail (Equation 2).
  • srIBI uses a positively charged cesium primary ion beam with a small spot size to obtain 3D super-resolution multiparametric visualization of cellular features (Fig. 14 A), including the distribution of small molecules.
  • Cells are cultured on a conductive substrate and treated with a small molecule that carries a stable isotope that can be efficiently ionized by the cesium beam.
  • Cells are then simultaneously stained with multiple isotope-tagged antibodies using a protocol optimized for intracellular staining, dried under vacuum, and loaded into the instrument.
  • a cell of interest is identified with an optical camera and then iteratively rasterized with the cesium primary ion beam. This process releases a cloud of negative secondary ions at the point of contact on the cell surface.
  • the liberated secondary ions are collected and recorded, pixel-by-pixel, using a magnetic sector mass spectrometer. This results in a two- dimensional (2D) image for each analyzed isotope.
  • Serial acquisition of hundreds of planes yields a stack of 2D images for each isotope that can be merged to obtain a multiparameter visualization of the cellular features of interest and volumetrically reconstructed to provide a 3D composite image of the analyzed cell at super-resolution.
  • MoC-Abs can be used for intracellular protein staining in an optimized protocol that involves fixation with 1.6% paraformaldehyde, permeabilization, and blocking with a high salt concentration in the presence of sheared salmon sperm DNA to avoid non-specific binding (Fig. 29).
  • srIBI secondary electron (e) and elemental ion information are simultaneously recorded for each plane.
  • the e image akin to that from a scanning electron microscope, enables the identification of certain subnuclear structures such as the nucleolus (Fig. 14 C, left image).
  • the 19 F and e signals were merged and a specific enrichment of the 19 F signal in nucleolar structures identified through the e image was observed (Fig. 14 C, overlay and right image).
  • transcriptionally active chromatin was labeled using a 19 F/FITC MoC-Ab that binds to H3K27Ac (Fig. 14 D).
  • the beam size dictates the lateral resolution and ion yield of the acquired image (30).
  • the size of the beam is proportional to its current.
  • the effect of beam current on the ion count per pixel in the samples was quantified by acquiring sequential images of the nucleolus of a HeLa cell. Raster size, pixel dwell time, and number of pixels acquired were maintained at constant levels, and the beam current was varied (Fig. 33 A and B). It was observed that the increase in current scaled with ion counts per pixel (Fig. 33 C and D) at the expense of resolution (Fig. 33 B).
  • srIBI srIBI
  • HeLa cells were stained with phalloidin conjugated to ATT0514, a fluorophore that contains six 19 F atoms (Fig. 36A).
  • the distribution of actin filaments observed in HeLa cells labeled with this reagent was comparable in confocal microscopy and srIBI images (Fig. 36 B and C).
  • Nuclear speckles are nuclear domains containing inter-chromatin material enriched in pre-mRNA splicing components (33). Detection of SC35, a nuclear speckle protein, was validated using anti-SC35-biotin with streptavidin-labeled 1.4 nm gold
  • nanoparticles streptavidin- 197 Au/FITC by srIBI (Fig. 15 C and Fig. 37).
  • transcriptionally silent chromatin anti-H3K9me3- 81 Br/Cy3
  • nucleoli anti-nucleolin- 127 I/Cy5 and anti-NPMl- biotin recognized by streptavidin- 197 Au/FITC were labeled in HeLa cells, obtaining the expected patterns for all (Fig. 38).
  • Fig. 15 H and viSNE was used to identify subnuclear structures (Fig. 15 I) as previously described (34). Pixels with high expression of nucleolin were grouped in three distinct regions that had variable levels of H3K9me3 and low levels of SC35, H3K27Ac, and phosphorus (Fig. 15 I; gates A to C).
  • srIBI can be used to visualize multiple isotopes simultaneously, and the application of dimensionality reduction techniques to the data enables identification of distinct subnuclear structures.
  • ion beam strength and residence time at the spot can yield secondary ion information for each plane that results from the ablation of a just a few nanometers of the sample surface, and consecutive planes have similar ion counts (Fig. 42). It was reasoned that whole 3D nuclear reconstruction could be achieved with srIBI if data were acquired on many consecutive planes. Previous axial resolution quantifications in biological samples on the nanoSIMS was determined to be around 5 nm (35). In agreement with this measurement, reconstructions of centromeres from srIBI (Fig. 43) resembled those identified by recent super-resolution microscopy experiments (36).
  • srIBI is capable of resolving juxtaposed subnuclear structures as a 50- plane acquisition was sufficient to distinguish between the centromere, nucleolus, and nuclear speckles in a HeLa cell (Fig. 16 C).
  • a lateral resolution down to 30 nm in biological samples has been previously reported using the cesium primary ion beam by focusing it to an area of interest measuring a few microns (17, 24). During each pass of the primary ion beam only the top material contacted is ablated. The majority of the sample is preserved due to the high axial resolution and thus is available for further imaging. It was reasoned that an initial acquisition of a target cell to define a region of interest (ROI) could be performed and that sub-cellular feature could be focused on for higher resolution profiling (Fig. 16 E, workflow). Indeed, re -probing a region of interest in an IdU-treated cell enabled iterative imaging at higher resolution (Fig. 16 E, IdU images).
  • ROI region of interest
  • SIMS enables subcellular visualization of exogenously incorporated small molecules and has been previously used to study the cellular distribution of the metallo-drug cisplatin (38), which is used to treat various types of cancer (39).
  • the platinum atom of cisplatin (Fig. 47 A) can be readily ionized using the cesium beam.
  • TYK-nu cells which are immortalized ovarian cancer cells, were incubated for 24 hours with a range of concentrations of cisplatin to identify a concentration at which drug was internalized but caused minimal cell death (Fig. 47 B).
  • Naturally occurring platinum is composed of five stable isotopes (Fig. 47 C); the study took advantage of isotopically pure cisplatin ( 194 Pt) to maximize Pt counts.
  • Biomolecules in the nucleus are organized spatially to form pockets of interaction (40, 41), akin to tissue microenvironments (3, 14, 42). In such a nuclear neighborhood, the function of various biomolecules are dependent on their spatial localization and the context of other nearby constituents.
  • the multiplexed data generated from srIBI allowed the testing of the hypothesis that biomolecules in the nucleus have context-dependent functions.
  • Whole cell srIBI acquisition was performed on cisplatin-treated cells (Fig. 17 C, top left).
  • a sliding window consisting of 6 * 6 * 20 pixels, was applied to extract the average isotope counts per window for five channel (H3K9me3, H3K27Ac, SC35, nucleolin and phosphorus; Fig. 17 C, top right).
  • Unsupervised clustering was then performed on these extracted features to group windows based on similarity (Fig. 17 C, bottom right). Individual voxels were then pseudo colored based on these groups for visualization purposes (Fig. 17 C, bottom left). In total, 20,000 randomly sampled 3D voxels from the middle 40 Z-planes were extracted from each of the 2 cells, for a sum of 40,000 voxels.
  • Fig. 17 E Each nucleus was then colored based on these groups and the 3D voxels were projected onto a 2D plane to better visualize the spatial distribution of the nuclear neighborhoods (Fig. 17 E).
  • These images reveal distinctive nuclear structures, such as nucleolin-like structures (Neighborhood 1), nuclear speckles (Neighborhood 4), and chromatin that resembles lamin-associated domains
  • characterization of atomic components from small molecules offers the ability to create subcellular maps for multiple types of biomolecules.
  • the study set out to quantify cisplatin levels in each nuclear neighborhood. It was observed that nuclear speckles contained high levels of cisplatin (Fig. 17 F, Neighborhood 4). Cisplatin was depleted from heterochromatin regions and lamin-associated domains (Fig. 17 F, Neighborhoods 5 and 8). To obtain a super-resolution image of cisplatin localization, iterative srIBI was used, to reveal at an unprecedented level, the spatial enrichment of cisplatin along the nuclear speckles and their depletion from closed chromatin (Fig. 18 A and Fig. 48). These results demonstrate a differential distribution of cisplatin in distinct nuclear
  • Probes for detecting centromeres were designed in accordance with the illustration in Fig. 53 and hybridized in situ. The stained cells were analyzed. The results of this assay, as shown in Fig. 54, demonstrate that the probes can be used to label centromeres in situ. Discussion for Example 6
  • srIBI is an MSI method enabled by a new set of reagents, MoC-Abs, that allow for the specific detection of proteins using MIBI coupled to a cesium primary ion beam. This method enables targeted super-resolution, 3D, multiplexed imaging of different biomolecule types in cells. srIBI detection of MoC-Abs yielded results akin to those obtained using standard confocal light microscopy but use of iterative srIBI imaging resulted in lateral resolutions of approximately 30 nm. Application of unsupervised dimensional reduction methods enabled the identification of subcellular structures and localization of the small-molecule drug cisplatin.
  • srIBI will allow for study of localization of a variety of agents. Drug metabolism in and out of cells might also be determined by such imaging. Understanding the subcellular localization of drugs may maximize the success rates of drug candidates in clinical trials, holding promise for a more efficient and cost-effective drug discovery process (44).
  • the MoC-Abs used in this work each contain 44 to 66 isotopic labels.
  • An increase in labels per antibody can be readily achieved by increasing the number of substituted nucleotides, increasing the length of labeling oligonucleotide, or through nucleotide-based amplification methods (28, 45-47).
  • MIBI and IMC are orthogonal approaches to use of fluorescence for imaging the tissue microenvironment (13-15).
  • these methods are focused on large fields of view and fast acquisition times, due to their focus on large-scale tissue cellular contexts.
  • IMC is based on laser-ablation inductively coupled plasma mass spectrometry with a lOOO-nm laser beam.
  • the cutoff at 75 atomic mass units in the Helios system does not allow imaging of naturally occurring organic isotopes.
  • MIBI has a higher lateral resolution than IMC due to a smaller beam size (260 to 500 nm), but the negative charge of the oxygen duoplasmatron limits detection of intrinsic biomolecules.
  • srIBI acquires data on multiple biomolecules simultaneously, it is an alternative to current super-resolution light microscopy techniques.
  • srIBI samples can be stored for extended periods as the isotopes detected are stable; this is not the case for fluorophores used in light imaging.
  • srIBI enables specific protein staining compatible with direct imaging of exogenously added small molecules.
  • Extension of fluorescent-based methods for specific nucleic acid staining, like CRISPR/Cas-FISH (55), Oligopaint (27, 56), and ATAC- see (57), to srIBI will provide a unique opportunity for an integrative understanding of cellular processes at the nanoscale beyond current means.
  • cisplatin has been shown to reduce pre-mRNA splicing in a dose-dependent manner which might be additive to its well-known role inducing adducts, single strand gaps post repair and other lesions in DNA (39, 62). These results suggest potential vulnerabilities of mRNA processing that can be targeted for combinatorial therapies to increase the effectiveness of cisplatin.
  • SrIBI is an imaging method that can be applied in single-cell studies of the diverse molecular interactions in subcellular microenvironments. Combination with other unbiased or population-based methods, such as small molecule screens and multiplexed proteomics, promises to reveal new understanding into metabolic pathways or mechanisms of drug resistance. This has the potential to drive new therapeutic discoveries and inform decision making processes of small molecules.
  • RNAscope A Novel in situ RNA Analysis Platform for Formalin- Fixed, Paraffin-Embedded Tissues. The Journal of Molecular Diagnostics 14, 22-29 (2012).

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Abstract

L'invention concerne, entre autres, une fraction de marquage pour l'analyse d'échantillons biologiques. Selon certains modes de réalisation, la fraction de marquage comprend de multiples atomes internes d'un isotope non métallique ou métalloïde stable non biologique, dans laquelle : i. la fraction de marquage est ou comprend un agent de liaison qui se lie spécifiquement à un site complémentaire dans ou sur une cellule, ou ii. la fraction de marquage comprend un groupe chimiosélectif. Dans les deux cas, la fraction de marquage n'est pas un anticorps anti-IgG. L'invention concerne également des procédés d'utilisation de la fraction de marquage, et un mélange multiplexé de cette dernière.
PCT/US2019/028359 2018-04-23 2019-04-19 Marquage de sondes moléculaires avec des isotopes non métalliques et métalloïdes pour une analyse multiplexée à haute résolution Ceased WO2019209657A1 (fr)

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CN110853040A (zh) * 2019-11-12 2020-02-28 北京深境智能科技有限公司 一种基于超分辨率重建的图像协同分割方法
CN112233060A (zh) * 2020-09-04 2021-01-15 广州金域医学检验中心有限公司 数字病理图像异常样本的筛选方法、装置、计算机设备及存储介质
WO2023081243A1 (fr) * 2021-11-03 2023-05-11 Toreador Therapeutics, Inc. Procédés et systèmes pour l'étude à super-résolution de thérapies

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