EP2047313A1 - Procédé de microscopie à balayage laser et concentrateur de faisceaux - Google Patents

Procédé de microscopie à balayage laser et concentrateur de faisceaux

Info

Publication number
EP2047313A1
EP2047313A1 EP07786280A EP07786280A EP2047313A1 EP 2047313 A1 EP2047313 A1 EP 2047313A1 EP 07786280 A EP07786280 A EP 07786280A EP 07786280 A EP07786280 A EP 07786280A EP 2047313 A1 EP2047313 A1 EP 2047313A1
Authority
EP
European Patent Office
Prior art keywords
laser scanning
beam combiner
lasers
wavelengths
scanning microscopy
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Withdrawn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
EP07786280A
Other languages
German (de)
English (en)
Inventor
Jörg PACHOLIK
Dieter Huhse
Thomas Paatzsch
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
Cube Optics AG
Original Assignee
Carl Zeiss MicroImaging GmbH
Cube Optics AG
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, Cube Optics AG filed Critical Carl Zeiss MicroImaging GmbH
Publication of EP2047313A1 publication Critical patent/EP2047313A1/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Withdrawn legal-status Critical Current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
    • 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

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.
  • 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.
  • connection of the light source modules with the scan module is usually about
  • FIG. 1 The invention is shown schematically in FIG. 1
  • Tin encapsulated from telecommunication preferably in TTF
  • Thin-film technology is suitable for combining the light of, for example, eight light sources, which are brought in via fibers, and, advantageously via a polarization-maintaining glass fiber, for supplying the microscope (scan head) to an LSM.
  • Fig. 3 shows a possible embodiment is shown.
  • the solution is a compact, encapsulated, fully adjusted assembly that supports the
  • Beam combiner contains.
  • the laser sources are coupled via fibers and unified output via a fiber, the input and output fibers are fixed, so that no adjustment of a fiber to Strahlverlick as in the state of
  • the invention makes possible a compact construction of a beam combiner with the aid of, for example, one of the cubeo-fiber multiplexer or a comparable component.
  • the encapsulated assembly provides a rugged construction that is resistant to
  • Fiber connector realized, it is easily possible for the customer, a modular design

Landscapes

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

Abstract

L'invention concerne un procédé de microscopie à balayage laser, caractérisé par l'utilisation de multiplexeurs à fibres optiques blindés de télécommunication lors de la concentration de faisceaux de plusieurs lasers de différentes longueurs d'onde et de l'injection commune dans un microscope à balayage laser. L'invention concerne également un concentrateur de faisceaux correspondant, caractérisé de façon avantageuse en ce que des éléments de guidage de guides d'ondes optiques sortent d'un composant blindé, différents lasers pouvant être couplés à ces éléments de guidage, de préférence par l'intermédiaire de guides d'ondes optiques.
EP07786280A 2006-07-28 2007-07-24 Procédé de microscopie à balayage laser et concentrateur de faisceaux Withdrawn EP2047313A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102006034909A DE102006034909A1 (de) 2006-07-28 2006-07-28 Verfahren zur Laser-Scanning-Mikroskopie und Strahlvereiniger
PCT/EP2007/006549 WO2008012057A1 (fr) 2006-07-28 2007-07-24 Procédé de microscopie à balayage laser et concentrateur de faisceaux

Publications (1)

Publication Number Publication Date
EP2047313A1 true EP2047313A1 (fr) 2009-04-15

Family

ID=38610631

Family Applications (1)

Application Number Title Priority Date Filing Date
EP07786280A Withdrawn EP2047313A1 (fr) 2006-07-28 2007-07-24 Procédé de microscopie à balayage laser et concentrateur de faisceaux

Country Status (4)

Country Link
US (1) US20090303584A1 (fr)
EP (1) EP2047313A1 (fr)
DE (1) DE102006034909A1 (fr)
WO (1) WO2008012057A1 (fr)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3538941B1 (fr) 2016-11-10 2025-04-23 The Trustees of Columbia University in the City of New York Procédés d'imagerie rapide de grands échantillons à haute résolution
US12596243B2 (en) * 2023-09-06 2026-04-07 National Yang Ming Chiao Tung University Laser scanning microscope with electrical high-order modulation extraction module

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19633185A1 (de) * 1996-04-16 1997-10-23 Leica Lasertechnik Punktlichtquelle für ein Laserscanmikroskop und Verfahren zum Einkoppeln von mindestens zwei Lasern unterschiedlicher Wellenlänge in ein Laserscanmikroskop
US20010028031A1 (en) * 2000-04-04 2001-10-11 Johann Engelhardt Apparatus for combining light and confocal scanning microscope
US20060017001A1 (en) * 2004-07-23 2006-01-26 Paul Donders Method and apparatus for fluorescent confocal microscopy

Family Cites Families (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP3343276B2 (ja) * 1993-04-15 2002-11-11 興和株式会社 レーザー走査型光学顕微鏡
JP3258821B2 (ja) * 1994-06-02 2002-02-18 三菱電機株式会社 微小異物の位置決め方法、分析方法、これに用いる分析装置およびこれを用いた半導体素子もしくは液晶表示素子の製法
US5895915A (en) * 1997-07-24 1999-04-20 General Scanning, Inc. Bi-directional scanning system with a pixel clock system
JP2000199855A (ja) * 1998-11-02 2000-07-18 Olympus Optical Co Ltd 走査型光学顕微鏡装置
US6459484B1 (en) * 1999-10-21 2002-10-01 Olympus Optical Co., Ltd. Scanning optical apparatus
US6570659B2 (en) * 2001-03-16 2003-05-27 Lightlab Imaging, Llc Broadband light source system and method and light source combiner
JP2004029205A (ja) * 2002-06-24 2004-01-29 Olympus Corp レーザ走査型顕微鏡
US7218446B2 (en) * 2003-08-27 2007-05-15 Biomedical Photometrics Inc. Imaging system having a fine focus
US7280570B2 (en) * 2003-12-15 2007-10-09 Leica Microsystems Device for generating a light beam including multiple wavelengths
EP1760454A4 (fr) * 2004-06-24 2008-09-03 Olympus Corp Dispositif photométrique fluorescent
DE102004030669A1 (de) * 2004-06-24 2006-01-19 Leica Microsystems Cms Gmbh Mikroskop
US7413341B1 (en) * 2004-08-16 2008-08-19 The Research Foundation Of State University Of New York Imaging methods
US7352458B2 (en) * 2005-10-26 2008-04-01 President And Fellows Of Harvard College System and method for high sensitivity vibrational imaging with frequency modulation coherent anti-stokes Raman scattering analyses

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19633185A1 (de) * 1996-04-16 1997-10-23 Leica Lasertechnik Punktlichtquelle für ein Laserscanmikroskop und Verfahren zum Einkoppeln von mindestens zwei Lasern unterschiedlicher Wellenlänge in ein Laserscanmikroskop
US20010028031A1 (en) * 2000-04-04 2001-10-11 Johann Engelhardt Apparatus for combining light and confocal scanning microscope
US20060017001A1 (en) * 2004-07-23 2006-01-26 Paul Donders Method and apparatus for fluorescent confocal microscopy

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of WO2008012057A1 *

Also Published As

Publication number Publication date
WO2008012057A1 (fr) 2008-01-31
US20090303584A1 (en) 2009-12-10
DE102006034909A1 (de) 2008-01-31

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Effective date: 20120411