WO2008043459A2 - Dispositif de division de lumière de détection - Google Patents

Dispositif de division de lumière de détection Download PDF

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Publication number
WO2008043459A2
WO2008043459A2 PCT/EP2007/008555 EP2007008555W WO2008043459A2 WO 2008043459 A2 WO2008043459 A2 WO 2008043459A2 EP 2007008555 W EP2007008555 W EP 2007008555W WO 2008043459 A2 WO2008043459 A2 WO 2008043459A2
Authority
WO
WIPO (PCT)
Prior art keywords
filter
arrangement according
optical axis
degrees
detection light
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/EP2007/008555
Other languages
German (de)
English (en)
Other versions
WO2008043459A3 (fr
Inventor
Ralf Wolleschensky
Wolfgang Bathe
Jörg STEINERT
Dieter Huhse
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Carl Zeiss Microscopy GmbH
Original Assignee
Carl Zeiss MicroImaging GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Carl Zeiss MicroImaging GmbH filed Critical Carl Zeiss MicroImaging GmbH
Publication of WO2008043459A2 publication Critical patent/WO2008043459A2/fr
Publication of WO2008043459A3 publication Critical patent/WO2008043459A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/002—Scanning microscopes
    • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/0052—Optical details of the image generation
    • G02B21/0064—Optical details of the image generation multi-spectral or wavelength-selective arrangements, e.g. wavelength fan-out, chromatic profiling
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B27/00—Optical systems or apparatus not provided for by any of the groups G02B1/00 - G02B26/00, G02B30/00
    • G02B27/10—Beam splitting or combining systems
    • G02B27/1006—Beam splitting or combining systems for splitting or combining different wavelengths
    • G02B27/1013—Beam splitting or combining systems for splitting or combining different wavelengths for colour or multispectral image sensors, e.g. splitting an image into monochromatic image components on respective sensors
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B27/00—Optical systems or apparatus not provided for by any of the groups G02B1/00 - G02B26/00, G02B30/00
    • G02B27/10—Beam splitting or combining systems
    • G02B27/14—Beam splitting or combining systems operating by reflection only
    • G02B27/145—Beam splitting or combining systems operating by reflection only having sequential partially reflecting surfaces

Definitions

  • a confocal scanning microscope includes a light source module, which preferably consists of a plurality of laser beam sources that generate illumination light of different wavelengths.
  • a scanning device in which the illumination light is coupled as an illumination beam, has a main color splitter, an x-y scanner and a scanning objective to guide the illumination beam by beam deflection over a sample which is located on a microscope stage of a microscope unit.
  • a measurement light beam generated thereby from the sample is directed via a main color splitter and imaging optics to at least one confocal detection aperture (detection pinhole) of a detection channel.
  • FIG. 1 a beam path of a laser scanning microscope is shown schematically.
  • An LSM is essentially divided into 4 modules as shown in FIG. 1: light source, scanning module, detection unit and microscope. These modules are described in more detail below. Reference is additionally made to DE19702753A1.
  • Excitation wavelength depends on the absorption properties of the dyes to be investigated.
  • the excitation radiation is generated in the light source module.
  • Various lasers are used here (argon, argon krypton, TiSa laser).
  • the selection of the wavelengths and the adjustment of the intensity of the required excitation wavelength e.g. through the use of an acousto - optic crystal.
  • the laser radiation passes through a fiber or a suitable mirror arrangement in the scan module.
  • the laser radiation generated in the light source is focused by means of the diffraction-limited diffraction lens via the scanner, the scanning optics and the tube lens into the specimen.
  • the focus scans the sample punctiformly in the x-y direction.
  • the pixel dwell times when scanning over the sample are usually in the range of less than one microsecond to several 100 microseconds.
  • a confocal detection (descanned detection) of the fluorescent light the light passes from the focal plane (Specimen) and from the above and below Layers is emitted via the scanner to a dichroic beam splitter (MD). This separates the fluorescent light from the excitation light. Subsequently, the fluorescent light is focused on a diaphragm (confocal aperture / pinhole), which is located exactly in a plane conjugate to the focal plane. As a result, fluorescent light portions outside the focus are suppressed. By varying the aperture size, the optical depth resolution of the microscope can be adjusted. Behind the aperture is another dichroic block filter (EF) which again suppresses the excitation radiation.
  • EF dichroic block filter
  • the fluorescent light is measured by means of a point detector (PMT).
  • PMT point detector
  • the excitation of dye fluorescence occurs in a small volume where the excitation intensity is particularly high. This area is only marginally larger than the detected area using a confocal array. The use of a confocal aperture can thus be dispensed with and the detection can take place directly after the objective (non-descanned detection).
  • a descanned detection also takes place, but this time the pupil of the objective is imaged into the detection unit (nonconfocally descanned detection).
  • the LSM is therefore suitable for the examination of thick specimens.
  • the excitation wavelengths are determined by the dye used with its specific absorption properties. Dichroic filters tuned to the emission characteristics of the dye ensure that only the fluorescent light emitted by the respective dye is measured by the point detector.
  • connection of the light source modules with the scan module is usually about
  • Fig. 2 the area after the main color splitter in the direction of detection is shown.
  • the secondary color splitters designated as DBS in FIG. 1 are here NFT1 and NFT2, which are the
  • Wavelength-dependent detection radiation in the direction of detectorsDI-
  • Main selection of the wavelength are arranged upstream.
  • NFT1 and 2 and EF 1-3 can be designed as a motorized filter wheels to the flexibility in the setting of detected
  • the pinholes PH1-4 shown in FIG. 1 are not present for reasons of clarity in FIG. 2-5 n, since a pinhole for all beam paths .these vorby executable would be.
  • the emission filters suppress the light outside the selected spectral range
  • Dichroic beam splitters (with significantly lower suppression properties outside their transmissive range of action) for preselection of wavelength ranges, also, also changeable, non-perpendicular, typically less than 45 ° to the optical axis
  • 5 filter wheels are required to detect three spectral ranges, and 5 drives for changing the filters are required for a maximum of variable detection.
  • advantageously coated emission filters have virtually no absorption. These filters reflect all light with wavelengths outside the selected spectral range. It therefore seems possible to use this reflected light for other spectral channels. In order for the reflected light to be able to be separated from the incident light without further filters, the emission filters are easily tilted out of the vertical incidence. A small angle ( «20 °) was found to be particularly advantageous because the spectral properties of typical emission filters (high suppression, steep band edges) can be implemented particularly well for vertical incidence and small angles deviating from the normal incidence.
  • Emission filters NEF 1-3 arranged according to the invention, detectors D1-D3, jet trap S
  • the emission filters are designed such that they transmit a defined part of the light radiation and reflect the rest almost completely.
  • they are arranged at an angle other than 90 degrees in the beam path, which realizes an angle of incidence on the non-zero-degree filter
  • a downstream, preferably switchable, emission filter follows in front of the detector with the following advantages:
  • the reflected light from the downstream filter can also be used.
  • the unused portion of the spectrum may also be directed into a beam trap S. Detection of the light with another detector e.g. for control purposes is also conceivable.
  • Fig. 4 shows a simple structure without downstream filters:
  • the filters can also be switched interchangeable:
  • Fig. 5 shows fixed filters for main selection, additional switchable filters, preferably long and short passes, for narrowing the spectra of the fixed filters:
  • Fig. 6 shows an arrangement with arranged in the beam path "conventional" emission filter wheels NEF 1-3.
  • arrangements with 3 channels have always been shown here, but it is also possible for only two or any number of channels.

Landscapes

  • Physics & Mathematics (AREA)
  • General Physics & Mathematics (AREA)
  • Optics & Photonics (AREA)
  • Spectroscopy & Molecular Physics (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Microscoopes, Condenser (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)

Abstract

La présente invention concerne un dispositif conçu pour diviser une lumière de détection dans un microscope à balayage laser. Les différentes composantes spectrales font l'objet d'une séparation spectrale en composantes transmises et diaphragmées. La division est effectuée à travers au moins un filtre à revêtement qui est incliné selon un angle différent de 90° ou d'un angle nul par rapport à l'axe optique. L'angle par rapport à l'axe optique est inférieur à 20°, de préférence inférieur à 10°.
PCT/EP2007/008555 2006-10-06 2007-10-02 Dispositif de division de lumière de détection Ceased WO2008043459A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102006047911.4 2006-10-06
DE200610047911 DE102006047911A1 (de) 2006-10-06 2006-10-06 Anordnung zur Aufteilung von Detektionslicht

Publications (2)

Publication Number Publication Date
WO2008043459A2 true WO2008043459A2 (fr) 2008-04-17
WO2008043459A3 WO2008043459A3 (fr) 2008-07-10

Family

ID=39154696

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2007/008555 Ceased WO2008043459A2 (fr) 2006-10-06 2007-10-02 Dispositif de division de lumière de détection

Country Status (2)

Country Link
DE (1) DE102006047911A1 (fr)
WO (1) WO2008043459A2 (fr)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102008034008B4 (de) 2008-07-21 2010-07-01 Carl Zeiss Surgical Gmbh Filtersatz zur Beobachtung von Fluoreszenzstrahlung in biologischem Gewebe
DE102011083847A1 (de) 2011-09-30 2013-04-04 Carl Zeiss Microscopy Gmbh Mikroskop für die Weitfeldmikroskopie
DE102018126232B3 (de) * 2018-10-22 2020-03-26 Abberior Instruments Gmbh Scanning-Lichtmikroskop mit verschiedenen Eingängen für Licht unterschiedlicher Wellenlängen zum Abtasten einer Probe
DE102019101773B9 (de) * 2019-01-24 2021-11-25 Carl Zeiss Meditec Ag Mikroskopiesystem und Verfahren zum Betreiben eines Mikroskopiesystems

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19758746C2 (de) * 1997-01-27 2003-07-31 Zeiss Carl Jena Gmbh Laser-Scanning-Mikroskop
DE19859314A1 (de) * 1998-12-22 2000-06-29 Zeiss Carl Jena Gmbh Anordnung zur Separierung von Anregungs- und Emissionslicht in einem Mikroskop
US7508507B2 (en) * 2004-10-12 2009-03-24 Leica Microsystems Cms Gmbh Device for selecting and detecting at least two spectral regions of a light beam

Also Published As

Publication number Publication date
WO2008043459A3 (fr) 2008-07-10
DE102006047911A1 (de) 2008-04-10

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