WO2008012056A1 - Microscope à balayage laser - Google Patents

Microscope à balayage laser Download PDF

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Publication number
WO2008012056A1
WO2008012056A1 PCT/EP2007/006547 EP2007006547W WO2008012056A1 WO 2008012056 A1 WO2008012056 A1 WO 2008012056A1 EP 2007006547 W EP2007006547 W EP 2007006547W WO 2008012056 A1 WO2008012056 A1 WO 2008012056A1
Authority
WO
WIPO (PCT)
Prior art keywords
sample light
laser scanning
scanning microscope
beam path
housing
Prior art date
Application number
PCT/EP2007/006547
Other languages
German (de)
English (en)
Inventor
Jörg PACHOLIK
Dieter Huhse
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 WO2008012056A1 publication Critical patent/WO2008012056A1/fr

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
    • 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
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
    • G01J3/02—Details
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
    • G01J3/02—Details
    • G01J3/0205—Optical elements not provided otherwise, e.g. optical manifolds, diffusers, windows
    • G01J3/021—Optical elements not provided otherwise, e.g. optical manifolds, diffusers, windows using plane or convex mirrors, parallel phase plates, or particular reflectors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
    • G01J3/02—Details
    • G01J3/0205—Optical elements not provided otherwise, e.g. optical manifolds, diffusers, windows
    • G01J3/0237—Adjustable, e.g. focussing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
    • G01J3/12—Generating the spectrum; Monochromators
    • G01J3/14—Generating the spectrum; Monochromators using refracting elements, e.g. prisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
    • G01J3/28—Investigating the spectrum
    • G01J3/30—Measuring the intensity of spectral lines directly on the spectrum itself
    • G01J3/36—Investigating two or more bands of a spectrum by separate detectors
    • 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
    • G02B21/0076—Optical details of the image generation arrangements using fluorescence or luminescence

Definitions

  • a laser scanning system In a laser scanning system lasers of different power classes are used. Furthermore, a laser scanning system is characterized by a large number of variable modules that serve as a detector or for illumination. In 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.
  • lasers with different wavelengths are used in one LSM. The choice of the 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. Subsequently, 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 which is emitted from the focal plane (specimen) and from the planes above and below passes through the scanners 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 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 plane (optical section) which is located in the focal plane of the objective is reproduced by both detection arrangements in conjunction with the corresponding one-photon absorption or multiphoton absorption.
  • a three-dimensional image of the sample can then be generated computer-aided.
  • 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.
  • a collimator KO1 for collimating light from an optical fiber LF1 in the direction of a prism P and lenses L1, L2 for imaging the light spectrally split by the prism onto inputs of optical fibers LF2, LF3 are provided in a housing, preferably fixedly aligned with one another. At least in part of the spectrally split light coming from P, a displaceable mirror SP is arranged, which protrudes more or less and displaceably into the spectral distribution and reflects a selectable part of the detection light in the direction LF2.
  • LF2 and LF3 may be directed towards external detectors. Due to the compact arrangement in the housing G and the possible flexible coupling and uncoupling of light on the pre-adjusted light guides LF1, LF2, LF3, the incoming radiation and the choice of external detectors on LF2, LF3 can be done very flexible and also fast and quasi adjustment-free. In the context of the invention, for example, at the location of the optical fiber LF1 also a direct coupling of a part of the detection light take place, which advantageously does not change the compactness and flexibility of the invention.
  • the invention consists essentially of a waveguide splitting module coupled to optical fibers (e.g., glass fibers).
  • optical fibers e.g., glass fibers
  • all detectors with glass fibers could also be coupled to the scan module.
  • the light is thus collected in a glass fiber after passing through the confocal pinhole.
  • the light coming from an optical fiber is advantageously collimated into a parallel beam path, split spectrally (by prism or grating), and then a part of the light is projected into an output fiber via a movable mirror, while the other part of the light is directly into a second optical path Output fiber is mapped.
  • the fiber may also be firmly connected (with one end inside) to the housing so that one end of the fiber dangles from the housing and can be connected to a detector.
  • Fig. 3 again shows the invention, in the upper part in plan view (LF3 hidden) and below (schematically) in side view, in the direction of arrow in plan view.
  • the folding of the optical beam path through the reflection at the grating was ignored here in order to be able to clearly display the whole.

Landscapes

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

Abstract

L'invention concerne un microscope à balayage laser comprenant un faisceau d'éclairage servant à éclairer un échantillon et un faisceau de détection servant à détecter la lumière de cet échantillon. Un élément dispersif (P) est placé dans le trajet du faisceau de détection pour décomposer la lumière de l'échantillon en fonction de la longueur d'onde et au moins un premier et un deuxième détecteur permettent de détecter des domaines de longueurs d'onde différents, au moins un domaine de longueurs d'onde de la lumière décomposée de l'échantillon étant dévié en direction de la détection par l'intermédiaire d'un élément réfléchissant (SP) réglable. Une première et une deuxième fibre optique (LF2, LF3) servent à transmettre la lumière de l'échantillon aux premier et deuxième détecteurs et l'élément dispersif (P) est disposé dans un boîtier (G) pour former un trajet de faisceau préréglé avec l'élément réfléchissant, les fibres optiques (LF1, LF2, LF3) étant couplées à ce boîtier (G).
PCT/EP2007/006547 2006-07-28 2007-07-24 Microscope à balayage laser WO2008012056A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102006034907.5 2006-07-28
DE200610034907 DE102006034907A1 (de) 2006-07-28 2006-07-28 Laser-Scanning-Mikroskop

Publications (1)

Publication Number Publication Date
WO2008012056A1 true WO2008012056A1 (fr) 2008-01-31

Family

ID=38561779

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2007/006547 WO2008012056A1 (fr) 2006-07-28 2007-07-24 Microscope à balayage laser

Country Status (2)

Country Link
DE (1) DE102006034907A1 (fr)
WO (1) WO2008012056A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7932268B2 (en) 2004-03-05 2011-04-26 The Trustees Of The University Of Pennsylvania Methods for treating disorders or diseases associated with hyperlipidemia and hypercholesterolemia while minimizing side effects
US8530824B2 (en) 2010-06-09 2013-09-10 Olympus Corporation Scanning microscope
US20140211305A1 (en) * 2011-09-29 2014-07-31 Carl Zeiss Microscopy Gmbh Laser scanning microscope
CN113557445A (zh) * 2019-03-12 2021-10-26 法雷奥开关和传感器有限责任公司 用于检测物体的光学测量系统的光信号偏转装置、测量系统以及用于操作光信号偏转装置的方法

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102019106266A1 (de) * 2019-03-12 2020-09-17 Valeo Schalter Und Sensoren Gmbh Lichtsignalumlenkeinrichtung für ein optisches Messsystem zur Erfassung von Objekten, Messsystem und Verfahren zum Betreiben einer Lichtsignalumlenkeinrichtung

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19702753A1 (de) * 1997-01-27 1998-07-30 Zeiss Carl Jena Gmbh Laser-Scanning-Mikroskop
DE19902625A1 (de) * 1998-01-28 1999-09-30 Leica Microsystems Vorrichtung zur gleichzeitigen Detektion mehrerer Spektralbereiche eines Lichtstrahls
JP2002221663A (ja) * 2001-01-29 2002-08-09 Nikon Corp 走査型共焦点顕微鏡
WO2003090613A1 (fr) * 2002-04-26 2003-11-06 Optiscan Pty Ltd Microscope confocal a balayage laser avec retour par faisceau de fibres
US20060017920A1 (en) * 2004-07-26 2006-01-26 Atsuhiro Tsuchiya Laser-scanning examination apparatus
DE102004049770A1 (de) * 2004-10-12 2006-04-13 Leica Microsystems Cms Gmbh Vorrichtung zur Selektion und Detektion mindestens zweier Spektralbereiche eines Lichtstrahls

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19702753A1 (de) * 1997-01-27 1998-07-30 Zeiss Carl Jena Gmbh Laser-Scanning-Mikroskop
DE19902625A1 (de) * 1998-01-28 1999-09-30 Leica Microsystems Vorrichtung zur gleichzeitigen Detektion mehrerer Spektralbereiche eines Lichtstrahls
JP2002221663A (ja) * 2001-01-29 2002-08-09 Nikon Corp 走査型共焦点顕微鏡
WO2003090613A1 (fr) * 2002-04-26 2003-11-06 Optiscan Pty Ltd Microscope confocal a balayage laser avec retour par faisceau de fibres
US20060017920A1 (en) * 2004-07-26 2006-01-26 Atsuhiro Tsuchiya Laser-scanning examination apparatus
DE102004049770A1 (de) * 2004-10-12 2006-04-13 Leica Microsystems Cms Gmbh Vorrichtung zur Selektion und Detektion mindestens zweier Spektralbereiche eines Lichtstrahls

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10555938B2 (en) 2004-03-05 2020-02-11 The Trustees Of The University Of Pennsylvania Methods for treating disorders or diseases associated with hyperlipidemia and hypercholesterolemia while minimizing side effects
US8618135B2 (en) 2004-03-05 2013-12-31 The Trustees Of The University Of Pennsylvania Methods for treating disorders or diseases associated with hyperlipidemia and hypercholesterolemia while minimizing side effects
US7932268B2 (en) 2004-03-05 2011-04-26 The Trustees Of The University Of Pennsylvania Methods for treating disorders or diseases associated with hyperlipidemia and hypercholesterolemia while minimizing side effects
US9265758B2 (en) 2004-03-05 2016-02-23 The Trustees Of The University Of Pennsylvania Methods for treating disorders or diseases associated with hyperlipidemia and hypercholesterolemia while minimizing side-effects
US9364470B2 (en) 2004-03-05 2016-06-14 The Trustees Of The University Of Pennsylvania Methods for treating disorders or diseases associated with hyperlipidemia and hypercholesterolemia while minimizing side-effects
US11554113B2 (en) 2004-03-05 2023-01-17 The Trustees Of The University Of Pennsylvania Methods for treating disorders or diseases associated with hyperlipidemia and hypercholesterolemia while minimizing side-effects
US9433617B1 (en) 2004-03-05 2016-09-06 The Trustees Of The University Of Pennsylvania Methods for treating disorders or diseases associated with hyperlipidemia and hypercholesterolemia while minimizing side-effects
US9861622B2 (en) 2004-03-05 2018-01-09 The Trustees Of The University Of Pennsylvania Methods for treating disorders or diseases associated with hyperlipidemia and hypercholesterolemia while minimizing side-effects
US10016404B2 (en) 2004-03-05 2018-07-10 The Trustees Of The University Of Pennsylvania Methods for treating disorders or diseases associated with hyperlipidemia and hypercholesterolemia while minimizing side effects
US8530824B2 (en) 2010-06-09 2013-09-10 Olympus Corporation Scanning microscope
US20140211305A1 (en) * 2011-09-29 2014-07-31 Carl Zeiss Microscopy Gmbh Laser scanning microscope
US9389402B2 (en) * 2011-09-29 2016-07-12 Carl Zeiss Microscopy Gmbh Laser scanning microscope
CN113557445A (zh) * 2019-03-12 2021-10-26 法雷奥开关和传感器有限责任公司 用于检测物体的光学测量系统的光信号偏转装置、测量系统以及用于操作光信号偏转装置的方法

Also Published As

Publication number Publication date
DE102006034907A1 (de) 2008-01-31

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