WO2009043471A1 - Dispositif de détection optique d'un rayonnement lumineux excité et/ou réfléchi dans un échantillon - Google Patents

Dispositif de détection optique d'un rayonnement lumineux excité et/ou réfléchi dans un échantillon Download PDF

Info

Publication number
WO2009043471A1
WO2009043471A1 PCT/EP2008/007685 EP2008007685W WO2009043471A1 WO 2009043471 A1 WO2009043471 A1 WO 2009043471A1 EP 2008007685 W EP2008007685 W EP 2008007685W WO 2009043471 A1 WO2009043471 A1 WO 2009043471A1
Authority
WO
WIPO (PCT)
Prior art keywords
light
detection
arrangement according
excitation
step element
Prior art date
Application number
PCT/EP2008/007685
Other languages
German (de)
English (en)
Inventor
Ralf Wolleschensky
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 WO2009043471A1 publication Critical patent/WO2009043471A1/fr

Links

Classifications

    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B27/00—Optical systems or apparatus not provided for by any of the groups G02B1/00 - G02B26/00, G02B30/00
    • G02B27/10—Beam splitting or combining systems
    • G02B27/14—Beam splitting or combining systems operating by reflection only
    • G02B27/143—Beam splitting or combining systems operating by reflection only using macroscopically faceted or segmented reflective surfaces
    • 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
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B27/00—Optical systems or apparatus not provided for by any of the groups G02B1/00 - G02B26/00, G02B30/00
    • G02B27/10—Beam splitting or combining systems
    • G02B27/1006—Beam splitting or combining systems for splitting or combining different wavelengths
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B27/00—Optical systems or apparatus not provided for by any of the groups G02B1/00 - G02B26/00, G02B30/00
    • G02B27/10—Beam splitting or combining systems
    • G02B27/1086—Beam splitting or combining systems operating by diffraction only
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B5/00—Optical elements other than lenses
    • G02B5/18—Diffraction gratings
    • G02B5/1876—Diffractive Fresnel lenses; Zone plates; Kinoforms

Definitions

  • the invention relates to an achromatic main color splitter for laser scanning microscopy, which allows efficient excitation and detection in preferential confocal imaging and the integration thereof into a microscope, in particular a laser scanning microscope.
  • Such beam splitters are described for example in DE 19702753 A1. From the beach of technology, the use of the polarization state of the light is known for beam splitting (MDB). For this purpose, the excitation radiation is linearly polarized. However, the fluorescence is usually unpolarized. By forming the MDB as polarization splitter, however, only 50% of the fluorescence radiation can be separated from the excitation radiation achromatically.
  • MDB beam splitting
  • the object of the invention is to achieve an achromatic beam separation of the excitation light from the detection light with high efficiency both for the excitation light and for the detection light. This is realized by the arrangements explained below.
  • FIGS. 1-6 and 9-11 each describe arrangements for separating the common excitation and detection beam path.
  • the basic structure of the beam splitting is shown schematically in Fig. 1 based on the prior art.
  • the excitation beam path here comprises beam paths (3) from the direction of the light source (LQ), which are reflected by the beam splitter (MDB) in the direction of the sample (PR).
  • the beam path from the beam splitter (MDB) to the sample (PR) is referred to as a common excitation and detection beam path (1).
  • fluorescent light (FL) is excited in the sample.
  • the sample reflects a portion of the excitation light (AL).
  • the fluorescent light is preferably transmitted by the beam splitter (MDB) and enters the detection beam path (2), which includes the beam paths from MDB to the detector (DE).
  • Excitation light reflected by the sample (PR) predominantly passes through the MBD in the direction of the excitation beam path (3) and thus in the direction of the light source (LQ).
  • the detection light for example
  • Fluorescent light has a larger spectral bandwidth than the excitation light. This is because the excitation occurs in bands of approximately up to 20 nm spectral width. The spectral bandwidth influences the temporal coherence length of the light.
  • Fluorescent light thus has a shorter coherence length than that
  • the beam splitter according to the invention advantageously does not change the imaging properties as compared to the prior art, or only insignificantly.
  • Fig. 2a shows an optically transparent step element (PM).
  • This element is characterized in that it has along the optical axis (o.A.) steps with a width (B) and a height (A).
  • the steps can, for example, by successive set glass plates with a lateral offset, by etching processes
  • the height (A) is chosen so that it is greater than the temporal
  • Coherence length of the excitation light For example, light having a fluorescence in the range of 500 to 550 nm has a coherence length of 3.6 ⁇ m.
  • Laser light in contrast, has a narrow band depending on the laser medium
  • the step height could therefore preferably be greater than about 5-10 microns.
  • the width (B) of the step element (PM) is chosen so that it is greater than the spatial coherence length, so that the step element is not light-bending for the
  • Fig. 2b shows the behavior of the element explained in Fig. 2a) in the beam path.
  • Detection and excitation light (1) reach along the optical axis oA on the step element (PM), which is perpendicular (the end face SF of Figure 2a) is arranged to the optical axis in the beam path.
  • the light is split into sub-beams (TrT n ) at the stages, with adjacent sub-beams giving a discrete phase shift to the height of stage (A) to each other. Since the height (A) is greater than the coherence length of the detection light for the detection light, the partial beams generated at the stages can no longer interfere.
  • the partial beams of the detection light FL pass without angle change (2) through the step element.
  • the excitation light AL has a longer coherence length than the step height (A).
  • the partial beams can interfere with each other. This results in a deflection of the excitation light out of the optical axis according to the well-known Snell's law.
  • the first diffraction order is considered.
  • Fig. 3a shows a further behavior of the explained with reference to Fig. 2a) element in the beam path.
  • Detection and excitation light (1) illuminate the step element (PM) in a circular manner along the optical axis (O in FIG. 3b), wherein the step element is again arranged perpendicular to the optical axis in the beam path.
  • the light is decomposed at the stages into sub-beams (Ti-T n ) (FIG. 3 c), with adjacent sub-beams giving a discrete phase shift to the height of stage (A) relative to each other (pupil P).
  • Downstream of the step element is a lens (L).
  • each sub-beam is imaged individually and independently of the other sub-beams through the lens.
  • Decisive for the imaging by the lens is the extension of the partial beams, ie the width of the step (B) and the beam diameter. Since the beam diameter is larger than the width (B), a line-shaped distribution along the x-axis (a in FIG. 3d) arises in the focus of the lens (image) for the fluorescent light.
  • the excitation light AL Since the excitation light AL has a greater coherence length than the fluorescent light, it experiences a deflection in the x direction (FIG. 3).
  • the lens continues to focus in both axes, resulting in a point focus (b in Figure 3d). This in turn creates a spatial separation of the beam paths for the excitation and detection light due to the different coherence properties of the light.
  • a simplified spatial separation of excitation and detection light is made possible.
  • Fig. 4a shows a further arrangement with which the coherence properties of the excitation and detection light can be used for efficient beam separation.
  • the step element PM in this case has a rotationally symmetrical structure about the optical axis (o. A.) On.
  • the envelope of the step structure (a) preferably describes a convex lens shape.
  • the step height is again larger for the detection light and smaller for the excitation light than the coherence length of the radiation. Due to the structure, light is split into annular partial beams with the same phase. For the detection light, these partial beams do not interfere. Thereby, the beam shape and direction of the detection light FL is not affected. For the excitation light, the partial beams interfere and produce a focal point at the focal point of the phase element PM.
  • the focal point results from the convex shape of the envelope of the step element.
  • the figure shows a simplified representation of the lens with a straight end surface. This surface can be shaped accordingly.
  • a spatial separation of the excitation and detection light can take place with a small mirror R in the otherwise transmissive beam splitter (MDB), the excitation light AL being reflected in the direction of the output (3).
  • the shape of the MDB is illustrated in part 4b). It has a mirrored region (R) and a transparent region (T). This in turn creates a spatial separation of the beam paths for the excitation and detection light due to the different coherence properties of the light.
  • Fig. 5 shows an arrangement with which the detection light can be refocused after separation from the excitation light.
  • a compensation element cPM
  • the cPM is such that the modified phase front introduced by PM is compensated again.
  • the cPM must have exactly the inverted (mirror-inverted) form of the PM. Substituting both elements PM and cPM together, then results in a plane-parallel plate.
  • Fig. 6 shows an advantageous combination of the arrangements of Figs. 4 and 2b.
  • Fig. 4 uses a special mirror (MBD) for the spatial splitting of the excitation (3) and detection light (2), whereby the MDB shadows a part of the detection light so that efficiency losses occur.
  • MBD mirror
  • PM1 deflecting
  • PM2 focusing
  • the focusing of only the excitation light outside the detection beam path takes place on a reflector M, so that a particularly efficient beam separation is made possible.
  • compensation elements cPM1 and cPM2 are provided in the further beam path of the fluorescent light, cPM1 for compensation of PM2 is designed as hollow element HS with cavities H mirrored to PM2, otherwise all formed as a transmissive element, while cPM2 is mirror-inverted to PM1.
  • the phase offset of the elements PM1, PM2 is hereby canceled and at the output of cPM2 fully focusable detection light FL is present.
  • the step elements (PM) can also be designed to be reflective without restriction.
  • An example of a step element is shown in Fig. 7).
  • the common excitation and detection light is directed from direction (1) onto the step element.
  • partial beams of different phases are generated.
  • the step difference (h) is chosen so that the coherence length of the fluorescent light is smaller than the step difference.
  • the detection light (2) thereby experiences a classical reflection, the incidence (a1) and the angle of departure (a2) being equal, since the partial beams (determined by the width of the steps (b)) can not interfere.
  • the step difference (h) is smaller than the coherence length of the excitation light.
  • the sub-beams of the excitation light can not interfere.
  • lambda is the wavelength of the excitation light.
  • a spatial separation of the excitation from the detection light can be done by a suitable choice of the number of stripes (step width b) or the angle of incidence on (PM) (a1).
  • the use of reflective devices has the additional advantage of being achromatic, at least for the detection light.
  • Fig. 8 shows two exemplary schematic arrangements in a laser scanning microscope, wherein a spatial separation of the beam paths for the excitation and detection light due to the different coherence properties of the light is realized.
  • the excitation light of the light source is coupled in the same direction via the step element as it is decoupled after returning from the sample.
  • Partial image a) shows an arrangement according to the Fig. 1, wherein the beam splitter element (MDB) has been replaced by a beam splitter (S) according to the invention.
  • the beam splitter (S) is designed so that the phase front modified by the step element (PM) is again smoothed by the element cPM.
  • the light can be focused on the output side through the confocal diaphragm (PH).
  • Partial image b) shows a further arrangement, wherein the confocal diaphragm (PH) is arranged in the common excitation and detection beam path (1). Compensation of the phase front modified by the step element (PM) is not required in the detection beam path (2). This has the advantage that the adjustment is simplified because no compensating element (cPM) is used.
  • the compensation element CPM shown in partial image a) can also be dispensed with if a rectangular diaphragm is used as the confocal diaphragm PH.
  • a rectangular diaphragm is used as the confocal diaphragm PH.
  • the aspect ratio of the length to the width of the rectangle will be preferably chosen such that it corresponds to the ratio of the width of the steps of the step element (B) to the beam diameter at the entrance of the step element.
  • Fig. 9 shows an example of a detailed representation of the arrangement according to Fig. 8a) with the beam splitter element of Fig. 6).
  • the beam splitting i. the beam paths for the excitation and detection light are identical for the outward and return paths.
  • an irradiation of the light source LQ or a decoupling of the excitation light (which was reflected at the sample) takes place in the direction of the light source - see also explanation above, in particular to FIG. 8a.
  • Fig. 10 shows an example of a detailed representation of the arrangement of Fig. 8a) with the beam splitter element of Fig. 5).
  • Fig. 11 shows an example of a detailed representation of the arrangement for a light microscope with a CCD detector, wherein the beam splitter element of Fig. 5) is used for beam splitting. Plotted drawn is the pupil beam, pulled through the object beam path.
  • the detection light has a shorter coherence length than the excitation light. If excitation light with a shorter coherence length compared to the detection light is used (white light source), then the step element is designed so that the step height is smaller than the coherence length of the detection light but greater than that of the excitation light. This means that with respect to the arrangements described, the excitation light would pass through the step element and the detection light (fluorescence) would be interfered and diffracted out of the optical axis. In the described arrangements, only the excitation and detection beam path is then interchanged.

Landscapes

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

Abstract

Dispositif de détection optique d'un rayonnement lumineux excité et/ou réfléchi dans un échantillon, selon lequel : l'éclairage de l'échantillon présente une plus grande longueur de cohérence dans le temps que le rayonnement de détection et il est prévu un élément étagé essentiellement sous forme de gradins avec des hauteurs de gradins supérieures à la longueur de cohérence de la lumière de détection et inférieures à celle de la lumière d'excitation, sachant qu'un faisceau partiel de la lumière est produit à chaque gradin et qu'une modification des propriétés de rayonnement de la lumière d'excitation s'effectue par interférence des faisceaux partiels de la lumière d'excitation; ou bien l'éclairage de l'échantillon présente une plus petite longueur de cohérence dans le temps que le rayonnement de détection et il est prévu un élément étagé essentiellement sous forme de gradins avec des hauteurs de gradins inférieures à la longueur de cohérence de la lumière de détection et supérieures à celle de la lumière d'excitation, sachant qu'un faisceau partiel de la lumière est produit à chaque gradin et qu'une modification des propriétés de rayonnement de la lumière de détection s'effectue par interférence des faisceaux partiels de la lumière de détection.
PCT/EP2008/007685 2007-09-28 2008-09-16 Dispositif de détection optique d'un rayonnement lumineux excité et/ou réfléchi dans un échantillon WO2009043471A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102007047467.0 2007-09-28
DE200710047467 DE102007047467A1 (de) 2007-09-28 2007-09-28 Anordnung zur optischen Erfassung von in einer Probe angeregter und/oder rückgestreuter Lichtstrahlung

Publications (1)

Publication Number Publication Date
WO2009043471A1 true WO2009043471A1 (fr) 2009-04-09

Family

ID=40090068

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2008/007685 WO2009043471A1 (fr) 2007-09-28 2008-09-16 Dispositif de détection optique d'un rayonnement lumineux excité et/ou réfléchi dans un échantillon

Country Status (2)

Country Link
DE (1) DE102007047467A1 (fr)
WO (1) WO2009043471A1 (fr)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4281896A (en) * 1978-08-02 1981-08-04 The Charles Stark Draper Laboratory, Inc. Shared aperture separator for reciprocal path optical beams
WO1999042884A1 (fr) * 1998-02-19 1999-08-26 Leica Microsystems Heidelberg Gmbh Systeme optique a element spectroselectif
US6310733B1 (en) * 1996-08-16 2001-10-30 Eugene Dolgoff Optical elements and methods for their manufacture
US20020027716A1 (en) * 2000-09-01 2002-03-07 Koichiro Tanaka Method of processing beam, laser irradiation apparatus, and method of manufacturing semiconductor device
EP1420281A2 (fr) * 2002-11-15 2004-05-19 CARL ZEISS JENA GmbH Méthode et dispositif pour l'acquisition optique à vaste profondeur de champ

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19758744C2 (de) 1997-01-27 2003-08-07 Zeiss Carl Jena Gmbh Laser-Scanning-Mikroskop
US6888148B2 (en) 2001-12-10 2005-05-03 Carl Zeiss Jena Gmbh Arrangement for the optical capture of excited and /or back scattered light beam in a sample

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4281896A (en) * 1978-08-02 1981-08-04 The Charles Stark Draper Laboratory, Inc. Shared aperture separator for reciprocal path optical beams
US6310733B1 (en) * 1996-08-16 2001-10-30 Eugene Dolgoff Optical elements and methods for their manufacture
WO1999042884A1 (fr) * 1998-02-19 1999-08-26 Leica Microsystems Heidelberg Gmbh Systeme optique a element spectroselectif
US20020027716A1 (en) * 2000-09-01 2002-03-07 Koichiro Tanaka Method of processing beam, laser irradiation apparatus, and method of manufacturing semiconductor device
EP1420281A2 (fr) * 2002-11-15 2004-05-19 CARL ZEISS JENA GmbH Méthode et dispositif pour l'acquisition optique à vaste profondeur de champ

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
POPOV E K ET AL: "TRANSMISSION GRATINGS FOR BEAM SAMPLING AND BEAM SPLITTING", APPLIED OPTICS, OSA, OPTICAL SOCIETY OF AMERICA, WASHINGTON, DC, vol. 35, no. 16, 1 June 1996 (1996-06-01), pages 3072 - 3075, XP000594924, ISSN: 0003-6935 *

Also Published As

Publication number Publication date
DE102007047467A1 (de) 2009-04-02

Similar Documents

Publication Publication Date Title
EP2860566B1 (fr) Microscopie à balayage haute résolution
DE10004191B4 (de) Fluoreszenz-Scanmikroskop
EP2823347B1 (fr) Microscope optique à balayage, à détection spectrale
EP1248132B1 (fr) Méthode et dispositif de détection optique à résolution de la profondeur d'un échantillon
DE10227120A1 (de) Mikroskop, insbesondere Laserscanningmikroskop mit adaptiver optischer Einrichtung
DE102006047913B4 (de) Hochempfindliche spektralanalytische Einheit
EP3507642B1 (fr) Procede d'ajustement d'un microscope a fluorescence a balayage laser et microscope a fluorescence a balayage laser comprenant un appareil d'ajustement automatique
EP1845349B1 (fr) Unité d'analyse spectrale dotée d'un réseau de diffraction
DE102005055679A1 (de) Spektroskop und damit ausgerüstetes Mikrospektroskop
EP3948392B1 (fr) Procédé et dispositif de détection de déplacements d'un échantillon par rapport à un objectif
DE102020209889A1 (de) Mikroskop und Verfahren zur mikroskopischen Bildaufnahme mit variabler Beleuchtung
EP1122574B1 (fr) Arrangement de microscope
EP1141763A1 (fr) Agencement servant a separer la lumiere d'excitation et la lumiere d'emission dans un microscope
EP1651992B1 (fr) Microscope a balayage
DE102009012874B4 (de) Mikroskop, insbesondere Laser Scanning Mikroskop
LU93022B1 (de) Verfahren und Mikroskop zum Untersuchen einer Probe
WO2024012878A1 (fr) Appareil de mesure confocale chromatique de distances
WO2008012056A1 (fr) Microscope à balayage laser
WO2004057401A1 (fr) Procede et ensemble pour l'analyse et/ou le traitement optique d'un echantillon
WO2012069443A1 (fr) Microscope confocal à balayage laser et procédé d'examen d'un échantillon
EP3475750B1 (fr) Dispositif d'éclairage pour un microscope
DE19851240C1 (de) Fluoreszenzmikroskop mit Mehrphotonenanregung
WO2008043459A2 (fr) Dispositif de division de lumière de détection
WO2009043471A1 (fr) Dispositif de détection optique d'un rayonnement lumineux excité et/ou réfléchi dans un échantillon
WO2003069391A1 (fr) Dispositif de microscopie optique confocale

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 08802220

Country of ref document: EP

Kind code of ref document: A1

122 Ep: pct application non-entry in european phase

Ref document number: 08802220

Country of ref document: EP

Kind code of ref document: A1