EP1636624A1 - Procede de microscopie par fluorescence - Google Patents

Procede de microscopie par fluorescence

Info

Publication number
EP1636624A1
EP1636624A1 EP04739720A EP04739720A EP1636624A1 EP 1636624 A1 EP1636624 A1 EP 1636624A1 EP 04739720 A EP04739720 A EP 04739720A EP 04739720 A EP04739720 A EP 04739720A EP 1636624 A1 EP1636624 A1 EP 1636624A1
Authority
EP
European Patent Office
Prior art keywords
dyes
reference spectra
detection
spectra
inclusion
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
EP04739720A
Other languages
German (de)
English (en)
Inventor
Ralf Wolleschensky
Bernhard Zimmermann
Richard Ankerhold
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.)
Jenoptik AG
Original Assignee
Carl Zeiss Jena 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 Jena GmbH filed Critical Carl Zeiss Jena GmbH
Publication of EP1636624A1 publication Critical patent/EP1636624A1/fr
Ceased legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • G01N21/6458—Fluorescence microscopy
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6402—Atomic fluorescence; Laser induced fluorescence
    • 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
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/16—Microscopes adapted for ultraviolet illumination ; Fluorescence microscopes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N2021/6417—Spectrofluorimetric devices

Definitions

  • the invention relates to a method in fluorescence microscopy, in particular laser scanning microscopy.
  • the irradiated photons of a certain energy excite the dye molecules through the absorption of a photon from the ground state into an excited state.
  • This excitation is usually referred to as single-photon absorption (Fig. 1a).
  • the dye molecules excited in this way can return to the ground state in various ways.
  • fluorescence microscopy the transition with the emission of a fluorescence photon is most important.
  • the wavelength of the emitted photon is generally red-shifted due to the Stokes shift compared to the excitation radiation, so it has a longer wavelength. The Stokes shift enables the fluorescence radiation to be separated from the excitation radiation.
  • Fig. 1 b shows a multi-photon excitation
  • the fluorescent light is split off from the excitation radiation with suitable dichroic beam splitters in combination with block filters and observed separately. This makes it possible to display individual cell parts stained with different dyes. In principle, however, several parts of a preparation can also be colored simultaneously with different specific dyes (multiple fluorescence). Special dichroic beam splitters are used to differentiate the fluorescence signals emitted by the individual dyes.
  • LSM confocal laser scanning microscope
  • An LSM is essentially divided into 4 modules: light source, scan module, detection unit and microscope. These modules are described in more detail below. Reference is also made to DE19702753A1.
  • lasers with different wavelengths are used in an LSM. The choice of the excitation wavelength depends on the absorption properties of the dyes to be examined.
  • the excitation radiation is generated in the light source module.
  • Various lasers are used here (argon, argon krypton, TiSa laser).
  • the wavelengths are also selected in the light source module and the intensity of the required excitation wavelength is set, for example by using an acousto-optical crystal.
  • the laser radiation then arrives in the scan module via a fiber or a suitable mirror arrangement.
  • the laser radiation generated in the light source is focused into the specimen with the aid of the objective (2) and diffraction-limited via the scanner, the scanning optics and the tube lens.
  • the focus raster scans the sample in the x-y direction.
  • the pixel dwell times when scanning over the sample are usually in the range of less than a microsecond to a few seconds.
  • confocal detection descanned detection
  • MDB dichroic beam splitter
  • the fluorescent light is then focused on a diaphragm (confocal diaphragm / pinhole) which is located exactly in a plane conjugate to the focal plane. This suppresses fluorescent light components outside of the focus.
  • the optical resolution of the microscope can be adjusted by varying the aperture size.
  • Another dirchroic block filter (EF) is located behind the diaphragm, which again suppresses the excitation radiation.
  • the fluorescent light is measured using a point detector (PMT).
  • PMT point detector
  • the dye fluorescence is excited in a small volume at which the excitation intensity is particularly high. This area is only slightly larger than the detected area when using a confocal arrangement. The use of a confocal Aperture can thus be omitted and detection can take place directly after the lens (non-descanned detection).
  • a descanned detection still takes place, but this time the pupil of the objective is imaged in the detection unit (non-confocal descanned detection).
  • the plane (optical section) that is in the focal plane of the objective is reproduced by both detection arrangements in conjunction with the corresponding single-photon or multi-photon absorption.
  • a three-dimensional image of the sample can then be generated with the aid of a computer.
  • the LSM is therefore suitable for examining thick specimens.
  • the excitation wavelengths are determined by the dye used with its specific absorption properties. Dichroic filters matched to the emission properties of the dye ensure that only the fluorescent light emitted by the respective dye is measured by the point detector.
  • the spectral detection range must be restricted. The area in which the two dyes overlap is simply cut out and not detected. The efficiency of the detection unit thus deteriorates.
  • the same signal-to-noise ratio can only be achieved by increasing the excitation power, which can damage the specimen. For this reason, up to 6 different dye probes are used simultaneously at the same time, since otherwise the dyes cannot be separated due to the strongly overlapping emission bands.
  • fluorescent dyes are used for the specific labeling of the preparations
  • the fluorescence is spectrally split in the ZEISS META Laser Scanning Microscope.
  • the emission light is split off from the excitation light in the scan module or in the microscope (in the case of multi-photon absorption) using the main color splitter (MDB).
  • MDB main color splitter
  • a block diagram of the following detector unit is shown in Fig. 5.
  • the light of the sample is now focused by means of an imaging optics PO with confocal detection through an aperture (pinhole) PH, whereby fluorescence that has arisen out of focus is suppressed.
  • the aperture is omitted for undescanned detection.
  • the light is now broken down into its spectral components using an angle-dispersive element DI.
  • Prisms, gratings and acousto-optical elements come into question as angle-dispersive elements.
  • the light split by the dispersive element into its spectral components is subsequently imaged on a line detector DE.
  • This line detector DE therefore measures the emission signal as a function of the wavelength and converts it into electrical signals.
  • a line filter for suppressing the excitation wavelengths can be connected upstream of the detection unit.
  • the structure shown essentially describes a Cerny Turner structure.
  • the light L of the sample is focused with the pinhole optics PO through the confocal aperture PH.
  • this aperture can be omitted.
  • the first imaging mirror S1 collimates the fluorescent light.
  • the light hits a line grating G, for example a grating with a number of lines of 651 lines per mm.
  • the grating bends the light in different directions according to its wavelength.
  • the second imaging mirror S2 focuses the individual spectrally split wavelength components on the corresponding channels of the line detector DE.
  • the use of a line secondary electron multiplier from Hamamatsu H7260 is particularly advantageous.
  • the detector has 32 channels and a high sensitivity.
  • the free spectral range of the embodiment described above is approximately 350 nm. In this arrangement, the free spectral range is evenly distributed over the 32 channels of the line detector, resulting in an optical resolution of approximately 10 nm. This arrangement is therefore only conditionally suitable for spectroscopy. However, their use in an imaging system is advantageous since the signal per detection channel is still relatively large due to the relatively broad spectral band detected. A shift the free spectral range can also be achieved by rotating the grating, for example.
  • Another possible embodiment could involve the use of a matrix detector (e.g. a CCD).
  • the dispersive element splits into different wavelength components in a coordinate.
  • a complete line (or column) of the scanned image is imaged in the remaining direction on the matrix detector.
  • This embodiment is particularly advantageous when building a line scanner (Lit .: Code, Kino; "Confocal Scanning Optical Microscopy and Related Imaging Systems”; Academic Press 1996).
  • the basic structure corresponds essentially to that of an LSM according to Fig. 2. However, instead A line is mapped into the focus of a point focus and the sample to be examined is only scanned in one direction.
  • a slit diaphragm is used as the confocal diaphragm instead of a pin diaphragm. Detection using a multiphoton absorption can also be carried out with this arrangement. Again, the confocal aperture can be omitted.
  • the spectral splitting of the fluorescent light enables the spectral components to be recorded separately after the fluorescence spectra of fluorescence markers have been recorded in pure form and the spectra with fluorescence components of several markers have been recorded by a “unmixing” method (DE 19915137 A1).
  • the number of fluorescent markers used for fluorescent labeling can be reduced or combinatorics can be used if not only pure fluorescence spectra are used as reference spectra but also reference spectra of mixed forms are recorded.
  • These mixed forms can, for example, be characterized by the time-dependent color state of a biological material when a fluorescent marker slowly leads to staining.
  • Such mixed states can furthermore be characterized by a mixed color when a fluorescent marker changes its color or its excitation properties.
  • Mixing ratios of this type can be generated in various ways: they can be present in the sample, they can be generated by irradiating the sample or they can be the result of a biological process that is stimulated by irradiation.
  • Mixed spectra can characterize a biological process, for example a change in concentration, a first spectrum corresponding to a lower concentration state and at least one further spectrum corresponding to a higher concentration state.
  • Image channels are defined and evaluated accordingly using the different reference spectra.
  • Such references can be generated over the entire image or advantageously via marked "regions of interest” (ROI).
  • ROI can also be used for targeted manipulation by defined irradiation.
  • a reference can be determined in a first region and in at least one In a further region, targeted irradiation and measurement can be carried out by extracting mixed spectra. The spectra can be separated and displayed after the image has been taken or during the image red colored (Lit: Ando, R., Hama, H., Yamamoto-Hino, M., Mizuno, H., and Miyawaki, A. (2002), An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein.
  • PNAS 99/20, 12651-12656 in the detection of organic processes, for example inter- and intracellular processes and to the marrow ation of individual cells or cell populations, can be used and spectrally detected.
  • Photoconvertible dyes that dynamically change their spectra due to intracellular processes or dyes that are used for FRET but also other indicator dyes can be used advantageously by the method according to the invention.
  • different cells or cell groups can have different lengths with UV or violet
  • Color mixing ratios which are recorded as reference spectra. Different cell populations can then be recorded individually over time.
  • An area can be selected using the ROI.
  • An analysis of transport processes at cellular and subcellular level can be carried out.
  • only one dye can be used, for example
  • Irradiation or other effects are placed in different states that are clearly identifiable via reference formation.
  • ROls can be defined interactively directly in the image.
  • the selected laser is switched on and off pixel-precisely at the border of these regions.
  • the radiation parameters for changing the radiation can also be automatically integrated here
  • Dye properties through photoactivation or photoconversion lead for example, repetition rate, wavelength, intensity, position.
  • the evaluation can be done after the experiment or online during the simulation
  • the META detector makes it possible to record the entire spectrum of the emission, for example from Kaede, to spectrally separate the respective mixed forms during the measurement and to display the segregated channels.
  • 4 shows schematically how different image channels CH1-CH3 are formed, wherein, as shown, different spectral mixed distributions CH1-3 are used as references and are used for image evaluation.

Landscapes

  • Physics & Mathematics (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Health & Medical Sciences (AREA)
  • Optics & Photonics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
  • Microscoopes, Condenser (AREA)

Abstract

L'invention concerne un procédé de microscopie par fluorescence, en particulier au moyen d'un microscope à laser à balayage, avec détection, au moins partiellement dispersée spectralement, du spectre de fluorescence, et utilisation des spectres de référence en vue d'une séparation spectrale, procédé caractérisé en ce qu'on effectue l'enregistrement des spectres de référence des colorants et/ou des combinaisons de colorants, modifiables en fonction du temps et/ou spectralement, ledit enregistrement servant à l'évaluation de l'image.
EP04739720A 2003-06-16 2004-06-09 Procede de microscopie par fluorescence Ceased EP1636624A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE10327382A DE10327382A1 (de) 2003-06-16 2003-06-16 Verfahren zur Fluoreszenzmikroskopie
PCT/EP2004/006202 WO2004113987A1 (fr) 2003-06-16 2004-06-09 Procede de microscopie par fluorescence

Publications (1)

Publication Number Publication Date
EP1636624A1 true EP1636624A1 (fr) 2006-03-22

Family

ID=33495111

Family Applications (1)

Application Number Title Priority Date Filing Date
EP04739720A Ceased EP1636624A1 (fr) 2003-06-16 2004-06-09 Procede de microscopie par fluorescence

Country Status (5)

Country Link
US (1) US7688442B2 (fr)
EP (1) EP1636624A1 (fr)
JP (1) JP2006527858A (fr)
DE (1) DE10327382A1 (fr)
WO (1) WO2004113987A1 (fr)

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102005022880B4 (de) * 2005-05-18 2010-12-30 Olympus Soft Imaging Solutions Gmbh Trennung spektral oder farblich überlagerter Bildbeiträge in einem Mehrfarbbild, insbesondere in transmissionsmikroskopischen Mehrfarbbildern
WO2010022330A2 (fr) * 2008-08-21 2010-02-25 University Of Florida Research Foundation, Inc. Perturbation différentielle induite par laser (dlip) pour bioimagerie et détection chimique
EP2359745A1 (fr) 2010-02-12 2011-08-24 Helmholtz Zentrum München Deutsches Forschungszentrum für Gesundheit und Umwelt (GmbH) Procédé et dispositif pour imagerie photonique multi-spectrale
DE102012219136A1 (de) 2012-10-19 2014-05-28 Leica Microsystems Cms Gmbh Mikroskop und ein Verfahren zur Untersuchung einer Probe mit einem Mikroskop
CA2899158C (fr) * 2013-03-15 2021-05-04 Ventana Medical Systems, Inc. Discrimination spectrale

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JPH04127039A (ja) * 1990-09-19 1992-04-28 Hitachi Ltd 蛍光スペクトルによる物質の同定方法
JP3649823B2 (ja) * 1996-09-17 2005-05-18 株式会社トプコン 有機物の分析装置
US6167173A (en) * 1997-01-27 2000-12-26 Carl Zeiss Jena Gmbh Laser scanning microscope
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DE19829657A1 (de) * 1997-08-01 1999-02-04 Ingo Klimant Verfahren und Vorrichtung zur Referenzierung von Fluoreszenzintensitätssignalen
EP1000345B1 (fr) * 1997-08-01 2003-05-07 PreSens Precision Sensing GmbH Procede et dispositif pour referencer des signaux d'intensite de fluorescence
DE19900135A1 (de) * 1998-01-07 1999-08-05 Univ Rockefeller Verfahren zur Bestimmung der Temperatur einer einzelnen Zelle in einer Zellprobe oder Gewebebiopsie sowie Verfahren zu dessen Verwendung
DE19915137C2 (de) * 1999-03-26 2001-10-18 Michael Schaefer Verfahren zur Quantifizierung mehrerer Fluorochrome in einer mehrfach gefärbten Probe bei der Fluoreszenzmikroskopie und Verwendungen des Verfahrens
DE10033180B4 (de) * 2000-06-29 2006-08-31 Carl Zeiss Jena Gmbh Verfahren zur Detektion von Farbstoffen in der Fluoreszenzmikroskopie
DE10038526B4 (de) * 2000-08-08 2004-09-02 Carl Zeiss Jena Gmbh Verfahren und Anordnung zur Erfassung des wellenlängenabhängigen Verhaltens einer beleuchteten Probe
JP4827335B2 (ja) * 2001-08-13 2011-11-30 オリンパス株式会社 走査型レーザ顕微鏡
DE10151217B4 (de) * 2001-10-16 2012-05-16 Carl Zeiss Microlmaging Gmbh Verfahren zum Betrieb eines Laser-Scanning-Mikroskops

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Also Published As

Publication number Publication date
US20070178602A1 (en) 2007-08-02
JP2006527858A (ja) 2006-12-07
US7688442B2 (en) 2010-03-30
DE10327382A1 (de) 2005-01-05
WO2004113987A1 (fr) 2004-12-29

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