EP1678544A1 - Procede d'examen d'echantillon et microscope a eclairage evanescent de l'echantillon - Google Patents

Procede d'examen d'echantillon et microscope a eclairage evanescent de l'echantillon

Info

Publication number
EP1678544A1
EP1678544A1 EP04787190A EP04787190A EP1678544A1 EP 1678544 A1 EP1678544 A1 EP 1678544A1 EP 04787190 A EP04787190 A EP 04787190A EP 04787190 A EP04787190 A EP 04787190A EP 1678544 A1 EP1678544 A1 EP 1678544A1
Authority
EP
European Patent Office
Prior art keywords
sample
detection light
microscope
field
illuminating
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
EP04787190A
Other languages
German (de)
English (en)
Inventor
Andreas Hecker
Heinrich Ulrich
Werner Knebel
Kyra MÖLLMANN
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.)
Leica Microsystems CMS GmbH
Original Assignee
Leica Microsystems Heidelberg GmbH
Leica Microsystems CMS 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
Priority claimed from DE10344410A external-priority patent/DE10344410A1/de
Application filed by Leica Microsystems Heidelberg GmbH, Leica Microsystems CMS GmbH filed Critical Leica Microsystems Heidelberg GmbH
Publication of EP1678544A1 publication Critical patent/EP1678544A1/fr
Ceased legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/06—Means for illuminating specimens
    • G02B21/08—Condensers
    • G02B21/082—Condensers for incident illumination only
    • 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
    • G02B21/0032—Optical details of illumination, e.g. light-sources, pinholes, beam splitters, slits, fibers
    • 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
    • G02B21/008—Details of detection or image processing, including general computer control
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/02—Objectives
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/06—Means for illuminating specimens
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/06—Means for illuminating specimens
    • G02B21/08—Condensers
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/16—Microscopes adapted for ultraviolet illumination ; Fluorescence microscopes
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/32—Micromanipulators structurally combined with microscopes

Definitions

  • the invention relates to a method for microscopic examination of a sample.
  • the invention also relates to a microscope with evanescent sample illumination.
  • a microscope with evanescent illumination of a sample is known from US 2002/0097489 A1.
  • the microscope contains a white light source, the light of which is coupled via a slit diaphragm through the microscope objective into the specimen slide for evanescent illumination.
  • the illuminating light propagates in the slide by total internal reflection, the sample being illuminated only in the area of the evanescent field protruding from the slide.
  • Microscopes of this type are known under the term TIRFM (Total Internal Reflection Fluorescent Microscope).
  • the z-resolution of TIRF microscopes is extremely good due to the evanescent field that only projects into the sample by approx. 100 nm.
  • the lens consists of a first lens a positive refractive power, a second lens with a negative refractive power, the focal length ratio between the two lenses being in the range of - 0.4 and - 0.1 and the total refractive power being greater than zero. Furthermore, the lens contains two positive lenses, whose ratio diameter to the focal length is greater than 0.3 and less than 0.6.
  • the objective also includes a negative lens and a converging lens, the negative lens facing the front group and the focal length ratio of the negative lens and the converging lens being between ⁇ 0.5 and ⁇ 2.
  • the incident light illuminating arrangement contains an illuminating source that is polarized during operation
  • a microscope for TIRM Total Internal Reflection Microscopy
  • the microscope has a microscope housing and an objective.
  • the illuminating light emanating from an illuminating device can be coupled in via an adapter which can be inserted into the microscope housing.
  • an adapter which can be inserted into the microscope housing.
  • Illumination system includes a laser light source, the light of which is coupled into an optical fiber.
  • a coupling optic is also provided, which focuses the light emerging from the fiber into a rear focal point of the microscope objective.
  • the optical fiber can be displaced in a plane perpendicular to the optical axis of the microscope objective.
  • DE 102 29 935 A1 discloses a device for coupling light into a microscope. Laser light is directed onto the specimen in the light field diaphragm plane by an optical fiber coupling designed as a slide. The invention is particularly suitable for the TIRF process.
  • This task is solved by a method with the following steps: Illumination of the sample with a first evanescent field, the first evanscent field having a first depth of penetration into the sample, Detection of the first detection light which is illuminated by the one with the first evanscent field Part of the sample starts and generation of first detection light data, • illuminating the sample with a second evanescent field, the second evanscent field having a second depth of penetration into the sample which is greater than the first depth of penetration, • detecting a second detection light emitted by runs out of the part of the sample illuminated with the second evanscent field, and generate second detection light data, and • process the first and second detection light data. It is a further object of the present invention to provide
  • a microscope which is characterized in that a first evanscent field, which has a first depth of penetration into the sample, and a second evanscent field, which has a second depth of penetration into the sample, which is greater than the first depth of penetration , has, can be generated, and that at least one detector is provided, which detects the first detection light emanating from the part of the sample illuminated with the first evanscent field and generates first detection light data therefrom, and the second detection light that comes from that with the second evansescent Field illuminated part of the sample goes out, detected and second detection light data generated therefrom, and that a processing module is provided for processing the first and second detection light data.
  • the method according to the invention preferably comprises the further steps of illuminating the sample with one or more further evanescent fields of different penetration depth and detecting further detection light emanating from the part or parts of the sample illuminated with the further evansent field and generating of further detection light data.
  • the processing then preferably comprises the first, second and further detection light data.
  • a three-dimensional examination of the sample is consequently made possible by sequentially changing the penetration depth of the illuminating light. By increasing the penetration depth sequentially, additional sample layers are recorded.
  • the first and second detection light data contain data According to the invention, data from image objects and / or from parts of image objects.
  • the processing preferably includes assigning image objects to different layer depths (eg 20 nm - 40 nm, 40 nm - 60 nm, 60 nm - 80 nm ...) of the sample. This can be done with the help of a processing module.
  • the processing comprises the generation of a 3-D data stack, preferably with a processing module.
  • This data stack or the data record generated by the processing module can preferably be represented as a three-dimensional image of the sample or a sample area - preferably on a display.
  • the microscope according to the invention preferably has a lens with a lens pupil, the first and / or the second and / or the further evanescent field being generated by an illuminating light beam which has a focus in the area of the lens pupil of the lens.
  • the penetration depth of the first and / or the second and / or the further evanescent field can be set by adjusting the distance of the focus from the optical axis of the objective.
  • an adjustment means can be provided with which the spatial position of the focus can be changed within the plane of the objective pupil.
  • the setting means can comprise, for example, a beam deflection device with a plurality of rotating or tilting mirrors or with a gimbal-mounted mirror.
  • the setting means can also be designed as an acousto-optical element or contain micromirrors.
  • a displaceable optical fiber can also be used to adjust the spatial position of the focus of the illuminating light beam.
  • Illuminating light bundle leaves the lens depends on the spatial position of the focus in the lens pupil.
  • the penetration depth is set by adjusting the polarization of the illuminating light beam that generates the first and / or the second and / or the further evanescent field.
  • a rotatable ⁇ / 2 plate can be provided.
  • the microscope is advantageously calibrated - regardless of the method with which the penetration depth into the sample is set - so that reproducible examinations are made possible and quantitative statements about the nature of the sample - in particular about the arrangement of the individual sample components within the sample - are made can.
  • Detection is preferably carried out with at least one detector which comprises a camera and / or a CCD element and / or an EMCCD element and / or a multiband detector.
  • Bandpass filters and / or edge filters are preferably arranged in front of the detector and are matched to the respective emission focal length of the fluorescence signal.
  • a dispersive element can be provided for color selection, which generates a spectral splitting from which the wavelength components to be detected are masked out.
  • the detector can also be designed as a color detector, for example as a color camera. It is also possible for a dispersive element to split the detection light over several detectors in order to achieve spectral detection.
  • the detector is designed as a point detector and the detection comprises a point-by-point scanning of the respectively illuminated part of the sample.
  • a further adjustable beam deflection device can preferably be provided in the beam path of the detection light.
  • the microscope comprises a scanning microscope; especially a confocal scanning microscope.
  • the microscope according to the invention preferably has at least one multi-line light source and / or at least one broadband light source. The wavelengths or the wavelength of the first illuminating light beam and / or of the second illuminating light beam are preferably adjustable.
  • the first illuminating light beam and also the second illuminating light beam to contain light of one or more wavelengths, the wavelengths of the first illuminating light beam and the second illuminating light beam being able to differ from one another.
  • the diameter of the illuminating light beam is adjustable.
  • the opening angle of the illuminating light beam bundle converging to form a focus lying in the objective pupil can be adjusted. By changing the opening angle when focusing in the objective pupil, the size of the surface that is illuminated evanescently changes.
  • Fig. 2 shows another microscope according to the invention
  • Fig. 3 is an illustration of the method according to the invention.
  • the illuminating light beam 9 emanating from the light source 5 serves for the evanescent illumination of a sample 11 which is attached to a specimen slide 13.
  • the illuminating light beam 9 has a focus 19 represented by a point in the plane 15 of the objective pupil 17.
  • several optical elements for beam guidance and beam shaping are arranged. For example, there are first optics 21, second optics 23 and optics 25 that generate a first intermediate image plane 27 and a second intermediate image plane 29.
  • the spatial position of the focus 19 within the plane 15 of the objective pupil 17 can be changed with the aid of an adjusting means 31, which comprises an adjustable beam deflection device 33.
  • the adjustable beam deflection device 33 includes a gimbaled rotating mirror, not shown.
  • the detection light 37 emanating from the sample 11 passes through the lens 3 and through the beam splitter 39, which directs the illuminating light beam 9 to the lens 3, to a detector 41 which comprises a CCD camera 43 and which generates detection light data.
  • the beam splitter 39 is designed as a dichroic beam splitter and is designed such that light of the wavelength of the illuminating light beam is reflected while light of the wavelength of the detection light 37 can pass through.
  • the first detection light data are forwarded to a processing module 45. Then the distance between the focus of the illuminating light beam 9 and the optical axis 35 is increased and a second evanescent field is generated, the second
  • Depth of penetration into the sample is greater than the first depth of penetration.
  • the second detection light is emitted, which emanates from the part of the sample 11 illuminated with the second evanscent field, and that
  • the second detection light data are also forwarded to the processing module 45.
  • the Processing module 45 uses the first and second detection light data to assign image objects to different layer depths of the sample and to generate a 3-D data stack which is shown as a three-dimensional image of the sample or the illuminated sample area on the display 47 of a PC 49.
  • FIG. 2 shows a microscope according to the invention in which the penetration depth of the first and / or the second and / or the further evanescent field is set by adjusting the polarization of the illuminating light beam.
  • a ⁇ / 2 plate 51 is rotatably arranged in the beam path of the illuminating light beam 9 with which the direction of polarization of the illuminating light beam 9 can be rotated.
  • a beam splitter 53 is arranged in the further beam path of the illuminating light beam 9, which splits off a small part of the illuminating light beam 9 for polarization control as a measuring beam 55.
  • the measuring beam 55 is split by a polarization beam splitter 57 into an s-polarized partial beam 63, which is detected by a first detector 59, and into a p-polarized partial beam 65, which is detected by a second detector 61.
  • the polarization of the illuminating light beam 9 can be inferred from the ratio of the light powers measured with the first detector 59 and with the second detector 61.
  • the rotary position ⁇ / 2 plate 51 is set in accordance with the user specifications by means of a control circuit (not shown).
  • the sample is illuminated with a first evanescent field with a penetration depth of 20 nm, for example, and the first detection light for generating first detection light data 67 is detected.
  • the first detection light data comprise 3 image objects.
  • the sample is then illuminated with a second evanescent field with a penetration depth of 40 nm, for example, and the second detection light is detected to generate second detection light data 69.
  • the second detection light data comprise 5 image objects, 3 of which are already from the first Detection light data are known.
  • the sample is then illuminated with a further evanescent field with a penetration depth of 60 nm, for example, and the further detection light is detected to generate further detection light data 71.
  • the further detection light data comprise 8 image objects, 5 of which are already known from the first detection light data and the second detection light data.
  • the first, second and further detection light data are then processed 73.
  • the processing includes an assignment of which image objects belong to first sample layer data 75 (0-20 nm), which second sample layer data 77 (20-40 nm) and which to third sample layer data 79 (40-60 nm).
  • the data is stored as a 3-D data stack and can be displayed to the user, for example on a monitor.

Landscapes

  • Physics & Mathematics (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Optics & Photonics (AREA)
  • Engineering & Computer Science (AREA)
  • Computer Vision & Pattern Recognition (AREA)
  • General Engineering & Computer Science (AREA)
  • Microscoopes, Condenser (AREA)

Abstract

L'invention concerne un microscope (1) à éclairage évanescent (9) de l'échantillon, et un procédé d'examen d'échantillon. Selon l'invention, on produit un premier champ évanescent présentant une première profondeur de pénétration dans l'échantillon (11), et un deuxième champ évanescent présentant une deuxième profondeur de pénétration dans l'échantillon, supérieure à la première. Ledit microscope comporte un détecteur (43) détectant une première lumière de détection émise par la partie de l'échantillon (11) éclairée au moyen du premier champ évanescent, produisant à partir de ceci des premières données de lumière de détection, et détectant une deuxième lumière de détection émise par la partie de l'échantillon (11) éclairée au moyen du deuxième champ évanescent, et produisant à partir de ceci des deuxièmes données de lumière de détection. Ledit microscope comporte par ailleurs un module de traitement (45) destiné au traitement des premières et deuxièmes données de lumière de détection (37).
EP04787190A 2003-09-25 2004-09-22 Procede d'examen d'echantillon et microscope a eclairage evanescent de l'echantillon Ceased EP1678544A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE10344410A DE10344410A1 (de) 2003-09-25 2003-09-25 Rastermikroskop mit evaneszenter Beleuchtung
DE102004044311 2004-09-10
PCT/EP2004/052273 WO2005031427A1 (fr) 2003-09-25 2004-09-22 Procede d'examen d'echantillon et microscope a eclairage evanescent de l'echantillon

Publications (1)

Publication Number Publication Date
EP1678544A1 true EP1678544A1 (fr) 2006-07-12

Family

ID=34395049

Family Applications (1)

Application Number Title Priority Date Filing Date
EP04787190A Ceased EP1678544A1 (fr) 2003-09-25 2004-09-22 Procede d'examen d'echantillon et microscope a eclairage evanescent de l'echantillon

Country Status (3)

Country Link
US (1) US7633622B2 (fr)
EP (1) EP1678544A1 (fr)
WO (1) WO2005031427A1 (fr)

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EP1690122B8 (fr) * 2003-09-25 2009-02-18 Leica Microsystems CMS GmbH Module d'eclairage pour eclairage evanescent, et microscope correspondant
DE102005023768B4 (de) * 2005-05-19 2017-06-29 Leica Microsystems Cms Gmbh Verfahren zur Ermittlung der Orientierung von Molekülen in biologischen Proben
DE102006033306A1 (de) * 2006-07-17 2008-01-31 Leica Microsystems Cms Gmbh Tirf Mikroskop
DE102007004346B4 (de) * 2007-01-29 2021-02-11 Syntegon Technology Gmbh Vorrichtung zur optischen Charakterisierung
US9335533B2 (en) * 2010-10-13 2016-05-10 The University Of Vermont And State Agricultural College Adjustable total internal reflectance microscopy (TIRFM) illuminator apparatus
WO2013007726A1 (fr) 2011-07-11 2013-01-17 Facultes Universitaires Notre-Dame De La Paix Procédé de génération d'une fréquence somme à haute résolution et microscopie infrarouge
US9897536B2 (en) 2013-01-04 2018-02-20 University Of Limerick Differential infra red nanoscopy system and method
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CN111624756A (zh) * 2020-06-28 2020-09-04 中国海洋大学 一种景深扩展显微成像三维重建装置与方法
CN112816410A (zh) * 2020-12-31 2021-05-18 中科院长春应化所黄埔先进材料研究院 一种tirf照明的深度成像方法和系统

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Also Published As

Publication number Publication date
WO2005031427A1 (fr) 2005-04-07
US7633622B2 (en) 2009-12-15
US20080151226A1 (en) 2008-06-26

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