EP1929351A1 - Microscope et procede correspondant - Google Patents

Microscope et procede correspondant

Info

Publication number
EP1929351A1
EP1929351A1 EP06805709A EP06805709A EP1929351A1 EP 1929351 A1 EP1929351 A1 EP 1929351A1 EP 06805709 A EP06805709 A EP 06805709A EP 06805709 A EP06805709 A EP 06805709A EP 1929351 A1 EP1929351 A1 EP 1929351A1
Authority
EP
European Patent Office
Prior art keywords
sample
detection
detected
detection step
sample radiation
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.)
Withdrawn
Application number
EP06805709A
Other languages
German (de)
English (en)
Inventor
Ralf Wolleschensky
Michael Kempe
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 EP1929351A1 publication Critical patent/EP1929351A1/fr
Withdrawn legal-status Critical Current

Links

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

Definitions

  • the invention relates to a microscopy method for producing an image of an image field lying at a predetermined depth of a sample to be examined, comprising a plurality of illumination steps, in each of which a part of the image field is illuminated with a focused illumination beam that causes the generation of sample radiation due to an interaction with the sample causes detection steps in which the generated sample radiation is detected, and an evaluation step, in which the image is generated on the basis of the detected sample radiation.
  • the invention further relates to a microscope for generating an image of an image field lying at a predetermined depth of a sample to be examined, having an illumination module which illuminates the image field in a plurality of illumination steps, wherein in each illumination step a respective part of the image field is illuminated with a focused illumination beam, which causes the generation of sample radiation due to an interaction with the sample, a detection module that detects generated sample radiation, and an evaluation module that generates the image on the basis of the detected sample radiation.
  • a diaphragm is used, which shadows unwanted sample light.
  • structuring or intensity modulation of the illumination can be used to achieve confocal depth discrimination in the wide field or in the case of partial illumination of the image field (eg, a line illumination).
  • a phase shift of the structured illumination By means of a phase shift of the structured illumination, a deeply discriminated optical section can then be calculated and thus the desired image of the object can be generated.
  • MAA Neil et al. “Method of obtaining optical sectioning by using structured light in a conventional microscope "Optics Letters 22 (24) 1997, 1905-1907, this can be achieved with three phase images at 0 °, 120 ° and 240 °.
  • Structuring is required.
  • a different grating must be provided in the rule, the interference of the coherent sub-beams are changed or another diffractive optical element can be used.
  • the object is achieved in a microscopy / experience of the type mentioned in that during each illumination step, a first and a second detection step are performed, wherein in the first detection step in focus and out of focus generated sample radiation and in the second detection step, a lesser proportion of in Focus generated sample radiation is detected as generated in the first detection step and out of focus sample radiation, and that in the evaluation step, the sample radiation detected in the second detection step is used to reduce the non-focal portion in the detected in the first detection step sample radiation.
  • the spatial limitedness of the illumination within the image field due to the focusing is used to detect different proportions of the sample radiation generated in the focus in the first and second detection step with substantially the same sample radiation generated outside the focus. This is then advantageously used in the evaluation step to reduce the extra-focal portion of the sample radiation detected in the first detection step.
  • a low-discriminated cut can be generated with little effort, without having to provide a physical aperture for shading the extra-focal sample radiation.
  • the illumination beam z. B. focused point you can spectrally split the detected sample radiation and z. B. detect a line detector spectrally resolved.
  • the additional degree of freedom (or the additional spatial coordinate) can thus be used to spectrally detect in the detection steps and at the same time to realize the desired deeply discriminated generation of the image of the image field.
  • the two detection steps can be performed simultaneously during at least one illumination step.
  • the measurement time can be kept as low as possible.
  • both detection steps can be carried out chronologically successively during at least one illumination step.
  • the sample radiation emerging from a second section of the sample surface is detected from a first section of the sample surface and in the second detection step, wherein both sections adjoin one another or the second section only partially covers the first section.
  • the two sections may be immediately adjacent (touching) or spaced apart from each other.
  • the focus area in the image field can be imaged onto a detector, while in the second detection step, an adjacent area in the image field is detected.
  • the signal detected in the second detection step can be subtracted from the signal detected in the first detection step.
  • a relative weighting of the two signals to each other can be taken into account.
  • the illumination steps, the detection steps and the evaluation step for a plurality of image fields can be carried out at different predetermined depths of the sample, in order thus to be able to produce a plurality of deeply discriminated sectional images of the sample. From this, three-dimensional sample images can then be generated by known methods.
  • the illumination beam (preferably diffraction-limited) can be focused point or line.
  • the object is further achieved in a microscope of the type mentioned in that during each illumination step, the detection module performs a first and a second detection step, wherein in the first detection step in focus and out of focus generated sample radiation and in the second detection step, a lesser proportion of the focus generated sample radiation is detected as generated in the first detection step and out of focus sample radiation, and that the evaluation module uses the detected in the second detection step sample radiation to reduce in the detected in the first detection step sample radiation the non-focal portion.
  • the sample radiation can be detected spectrally resolved in the two detection steps. It is exploited that no physical aperture for shading the extra-focal portion of the sample radiation is necessary and that there is a further degree of freedom on the detection side due to the spatial limitedness of the focused illumination beam.
  • the sample radiation can be spectrally split and spectrally detected by means of a line detector. The spectral splitting takes place in the direction of extension of the line detector.
  • a line detector z. B. only one row or one column of a spatially resolving surface detector can be used.
  • the spectral splitting preferably takes place transversely to the direction of extent of the linear focus.
  • the spectral splitting can be carried out with any suitable optical element (with suitable dispersion), for. B. by means of a prism or a diffraction grating.
  • the detection module can thus optics for spectral splitting Have the sample radiation and at least one spectrally split sample radiation spectrally resolved detecting detector.
  • the detection module may comprise two detectors, whereby a simultaneous execution of the two detection steps over both detectors is possible.
  • the detection module may also have a single detector, so that both Detechnischs intimide are performed sequentially in time.
  • the sample radiation emerging from a second portion of the sample surface can be detected from a first portion of the sample surface and in the second detection step, the two portions of the sample surface adjacent (directly or spaced apart) or the second portion only partially the first portion covered.
  • the detection module can be designed such that the focus area in the image field is imaged on a detector in the first detection step and in the second detection step an area of the image field adjacent to the focus area is imaged on a detector of the detection module.
  • the evaluation module can subtract the signal detected in the second detection step from the signal detected in the first detection step, wherein a weighting of the two signals relative to one another is possible.
  • a weighting of the two signals relative to one another is possible.
  • the illumination module may include a scanner module that deflects the illumination beam to illuminate the entire field of view.
  • the illumination module can direct the illumination beam to the field of view as a focused spot or line-focused illumination beam.
  • FIG. 1 is a schematic view of the microscope according to the invention
  • FIG. 2 is an enlarged view of the detection module of FIG. 1
  • 3 is a cross-sectional view of the sample to be examined
  • Fig. 4 is a plan view of the detectors of Fig. 2;
  • Fig. 5 is a modification of the detectors of Fig. 4; Fig. 6 shows an alternative embodiment of the Delementsmoduls of Figure 1, and
  • FIGS. 7 and 8 show a further alternative embodiment of the detection module of FIG. 1.
  • the microscope is designed as a laser scanning microscope comprising a light source module 1, a scanning module 2, a lens 3, a recording module 4 and an evaluation module 5.
  • the light source module 1 which has a laser 8 and a beam shaping optics 9, generates a laser beam LS1 which is directed to the scanning module 2 via a beam splitter 6 connected between the light source module 1 and the scan module 2, thus deflecting the beam LS1 over the sample 7, an image field within the sample is completely illuminated.
  • the laser beam LS1 is focused by means of the lens 3 in the sample 7, in the depth of the image field from which an image is to be generated (optical section).
  • the laser beam LS1 is focused in a punk shape (preferably diffraction-limited) so that the scanning module 2 deflects the laser beam LS1 in two independent directions so as to be able to cover the entire image field within the sample 7 with the focused laser beam LS1.
  • sample radiation LS2 is generated, which impinges on the scan module 2 via the objective 3, which scans the sample radiation LS2 coming from the sample 7, so that the sample radiation LS2 is present behind the scan module 2 as stationary radiation beam LS2 ,
  • the beam splitter 6 is designed so that it transmits the sample radiation LS2 so that it strikes the detection module 4.
  • the generated sample radiation can, for. B. fluorescent light, luminescent light, reflected, transmitted and / or scattered light.
  • the detection module 4 comprises a detection optics 11 and a first and second detector D1, D2 whose signals are supplied to the evaluation module 5.
  • the focus area of the image field ie the area in which the laser beam LS1 focuses
  • the focus area of the image field is imaged onto the detector D1, while an area adjacent thereto is imaged onto the detector D2.
  • FIG. 3 shows a cross section through the sample to be examined 7, wherein the beam waist 12 of the illumination beam LS1 is shown. Between the dashed lines horizontal lines L1 and L2 is the depth range (field of view) of the sample 7 to be displayed. In this area is the laser beam LS1 focused. This can be seen from the fact that the beam waist 12 has the smallest diameter in this area.
  • the sample radiation generated in the obliquely hatched area B1 reaches the detector. It can be seen that in addition to the desired confocal sample radiation from the section of the area B1 between the dashed lines L1 and L2 nor the extra-focal sample radiation passes, which is generated above and below the line L1 and L2 in the area B1.
  • the sample radiation generated in the horizontally hatched region B2 passes to the detector D2. Since only a region adjacent to the focus region is detected with the detector D2, only the sample radiation which is generated outside the focal region (between L1 and L2) by the beam LS1 within the region B2 strikes the detector D2. In the example described here, the detector D2 thus sees only sample radiation LS2 generated outside the focus.
  • Fig. 4 the detector-side illumination distribution is shown in focus for clarity.
  • the focused laser beam LS1 is imaged only on the detector D1 (circle F1), so that the detector D2 does not receive any confocal signals.
  • the confocal signal Sc and the non-confocal signal Snc may be given as follows:
  • n can be empirically z. B. from the minimization of the background signal during the measurement. Typical values for n are 1 to 1, 3.
  • the detectors It is generally convenient to arrange the detectors so that the boundary between both detectors D1 and D2 is about 1 to 2 Airy Unit radii away from the center of the spot distribution on the detector D1.
  • a spacing of the two areas B1 and B2, as shown in Fig. 3 for simplified graphical representation, for example, less 1AU between two detectors D1 and D2 (for example, by a web) is not a problem.
  • the detectors D1 and D2 are shown for the case that there is not a punctiform focusing, but a linear focusing.
  • the linear focus (ellipse F2) is then imaged onto the detector D1, as shown in FIG.
  • the scan module 2 is adapted accordingly, so that under certain circumstances (if the line is so long that it covers the entire image field in the line direction) only a deflection transverse to the extension direction of the linear focus is necessary.
  • the microscope has a control unit 10.
  • the focusing has a local boundary in at least one direction in the image field, wherein this limit is preferably as sharp as possible.
  • the steepness of the boundary should preferably correspond to at least one spatial frequency at half the cutoff frequency of the objective 3.
  • FIG. 6 shows an alternative embodiment of the detection module 4.
  • the detection module 4 has a beam splitter 13, which, for example, reflects and transmits half of the incident sample light LS2 and thus splits it into two detection arms.
  • the focal component LS2c and the non-focal component LS2nc are shown, with only the confocal component LS2c shown in the detection arm pointing upwards for the sake of simplicity. Due to the local arrangement of the detectors D1, D2 in the detection arms, only the non-focal component LS2nc strikes the detector D2, while the focal component LS2c strikes the detector D1.
  • FIG. 7 and 8 an embodiment of the detection module 4 is shown in which only a single
  • Detector D1 is provided. Between the detection optics 10 and the detector D1, a rotatable glass plate 14 is arranged, which, depending on the rotational position, ensures that either the focal component LS2c or the extra-focal component LS2nc strikes the active surface of the detector 1.
  • the line detectors can of Fig. 5 are used for spectrally resolved detection.
  • the detected sample radiation must be split only spectrally in the direction of extension of the line detector before it hits the line detectors D1, D2.
  • the laser beam LS1 is focused linearly. Then at least one spatially resolving planar detector is necessary, wherein z. B. in the column direction spatially resolved line focus and in the line direction of the spectrally resolved in this direction line focus is detected spectrally resolved.

Landscapes

  • Physics & Mathematics (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Optics & Photonics (AREA)
  • Microscoopes, Condenser (AREA)
  • Spectrometry And Color Measurement (AREA)

Abstract

L'invention concerne un procédé de microscopie destiné à produire une image d'un champ d'image situé à une profondeur définie dans un échantillon à étudier. Ledit procédé comporte plusieurs étapes d'éclairage au cours desquelles respectivement une partie du champ d'image est éclairée avec un faisceau de rayons d'éclairage focalisé, produisant un rayonnement d'échantillon par interaction avec l'échantillon ; des étapes de détection au cours desquelles le rayonnement d'échantillon produit est détecté ; et une étape d'évaluation au cours de laquelle l'image est produite sur la base du rayonnement d'échantillon détecté. Au cours de chaque étape d'éclairage, une première et une deuxième étape de détection sont réalisées. Au cours de la première étape de détection, le rayonnement d'échantillon produit dans le foyer et en-dehors du foyer est détecté, et au cours de la deuxième étape de détection, une fraction réduite du rayonnement d'échantillon produit dans le foyer et en-dehors du foyer est détectée. Au cours de l'étape d'évaluation, le rayonnement d'échantillon détecté au cours de la deuxième étape de détection est employé afin de réduire la fraction extra-focale du rayonnement d'échantillon détecté dans la première étape de détection.
EP06805709A 2005-09-29 2006-09-14 Microscope et procede correspondant Withdrawn EP1929351A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102005046753A DE102005046753A1 (de) 2005-09-29 2005-09-29 Mikroskopierverfahren und Mikroskop
PCT/EP2006/008943 WO2007036303A1 (fr) 2005-09-29 2006-09-14 Microscope et procede correspondant

Publications (1)

Publication Number Publication Date
EP1929351A1 true EP1929351A1 (fr) 2008-06-11

Family

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Family Applications (1)

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EP06805709A Withdrawn EP1929351A1 (fr) 2005-09-29 2006-09-14 Microscope et procede correspondant

Country Status (5)

Country Link
US (1) US7728270B2 (fr)
EP (1) EP1929351A1 (fr)
JP (1) JP5214448B2 (fr)
DE (1) DE102005046753A1 (fr)
WO (1) WO2007036303A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102012017920B4 (de) * 2012-09-11 2023-11-30 Carl Zeiss Microscopy Gmbh Optikanordnung und Lichtmikroskop
WO2015163261A1 (fr) * 2014-04-24 2015-10-29 オリンパス株式会社 Microscope et procédé d'observation microscopique
WO2017046863A1 (fr) 2015-09-15 2017-03-23 オリンパス株式会社 Microscope et procédé d'observation au microscope
JPWO2017221356A1 (ja) * 2016-06-22 2019-04-18 オリンパス株式会社 顕微鏡
JP2020502558A (ja) 2016-11-10 2020-01-23 ザ トラスティーズ オブ コロンビア ユニバーシティ イン ザ シティ オブ ニューヨーク 大型試料のための高速・高解像度イメージング方法

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EP0485803B1 (fr) * 1990-11-10 1996-05-01 Grosskopf, Rudolf, Dr.-Ing. Dispositif de balayage optique avec trajet du faisceau confocal utilisant un réseau de sources de lumière et de détecteurs
EP0542962B2 (fr) 1991-06-08 2002-03-13 RENISHAW plc Spectroscopie a foyer commun
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DE10029680B4 (de) * 2000-06-23 2016-06-16 Leica Microsystems Cms Gmbh Mikroskop-Aufbau
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GB0106342D0 (en) * 2001-03-15 2001-05-02 Renishaw Plc Spectroscopy apparatus and method
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
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JP4894161B2 (ja) * 2005-05-10 2012-03-14 株式会社ニコン 共焦点顕微鏡

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Also Published As

Publication number Publication date
US20090128898A1 (en) 2009-05-21
JP5214448B2 (ja) 2013-06-19
US7728270B2 (en) 2010-06-01
JP2009510498A (ja) 2009-03-12
DE102005046753A1 (de) 2007-04-12
WO2007036303A1 (fr) 2007-04-05

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