WO2008037346A1 - Microscope à balayage laser muni d'un élément de manipulation de pupille - Google Patents

Microscope à balayage laser muni d'un élément de manipulation de pupille Download PDF

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Publication number
WO2008037346A1
WO2008037346A1 PCT/EP2007/007881 EP2007007881W WO2008037346A1 WO 2008037346 A1 WO2008037346 A1 WO 2008037346A1 EP 2007007881 W EP2007007881 W EP 2007007881W WO 2008037346 A1 WO2008037346 A1 WO 2008037346A1
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WO
WIPO (PCT)
Prior art keywords
pupil
scanning
manipulation
scanning element
objective
Prior art date
Application number
PCT/EP2007/007881
Other languages
German (de)
English (en)
Inventor
Matthias Wald
Original Assignee
Carl Zeiss Microlmaging 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 Microlmaging Gmbh filed Critical Carl Zeiss Microlmaging Gmbh
Publication of WO2008037346A1 publication Critical patent/WO2008037346A1/fr

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    • 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
    • G02B21/0036—Scanning details, e.g. scanning stages
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B13/00—Optical objectives specially designed for the purposes specified below
    • G02B13/0095—Relay lenses or rod lenses
    • 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

Definitions

  • the invention relates to a light-scanning microscope with a lens for sample imaging, wherein the lens has an objective pupil, a scanning device for biaxial beam deflection downstream of the objective in the imaging direction, wherein the scanning device has a first and a second, each uniaxially deflecting beam element which in Imaging direction behind each other, and a pupil manipulation element, which is arranged in a position associated with the objective pupil.
  • Laser scanning microscopes are known in the art. Reference is made, for example, to DE 197 02753 A1 or DE 10257237 A1, both of which describe a light scanning microscope designed as a laser scanning microscope. In this context, it should be noted that the term "light” here is understood to mean the entire region of the electromagnetic radiation which obeys the laws of optics.
  • Scanning microscopes or laser scanning microscopes usually acquire an object image by scanning the object with a spot or multispot arrangement.
  • the radiation recorded in the spot or multi-spot areas is detected with the highest possible depth resolution so that no structure of the spot or the multi-spot is resolved, for example by a so-called confocal detection.
  • Moving the spot or multispot area over the object then delivers the picture.
  • Confocal detection is a common way to achieve a very high depth resolution.
  • the signal evaluation is then restricted substantially to the focal plane, since areas lying outside the focal plane do not provide any significant signal information in the case of confocal detection; they are imaged in front of or behind the confocal aperture.
  • a scanning device that deflects the beam biaxially.
  • Commonly used are scanning devices that have two scanning elements, which deflect the beam uniaxially adjustable. Examples of scanning elements used in scanning microscopes are galvanometer mirrors or acoustically optical modulators. The deflection with the two scanning elements is conveniently carried out around mutually orthogonal axes. Based on the recorded image, it is therefore assumed that one scan element causes the deflection in the line direction, the other element in the image direction perpendicular thereto (the term "column direction" would also be conceivable).
  • the deflection of the light beam or light beam should ideally take place from one point, but this actually requires a single, then biaxial scan element.
  • the use of two scanning elements is much less expensive and also allows a faster adjustment of the deflection. It is therefore common in the art to arrange two scan elements as close to the pupil plane of the microscope objective. This has the further advantage that the two scanning elements can be kept small without fear of shading effects. Further, the problem otherwise encountered with adjustable mirrors is avoided, depending on the deflection angle, i. the mirror position, the light path by the stroke of the mirror deflection is different lengths, which can lead to defocusing effects.
  • the invention is based on the object, a light-scanning microscope of the type mentioned in such a way that the possibilities for pupil manipulation are extended and at the same time a pupil manipulation can be performed easily.
  • the invention solves this problem by a light-scanning microscope of the type mentioned, in which the first scanning element in the objective pupil or a pupil conjugate thereto, the first scanning element downstream of a relay optics for imaging the pupil contained in the first scan element in a second pupil is, the element for pupil manipulation in the second pupil generated by the relay optics and is formed reflective, so that the relay optics is traversed again in the imaging direction and these images the second pupil again into a third pupil, wherein the relay optics a beam deflector, so that the third pupil is spatially separated from the pupil plane containing the first scanning element, and the second scanning element is arranged in the third pupil.
  • a relay optic for pupil imaging is used, which is integrated in the beam path after the objective pupil or a conjugated pupil produced therefrom by intermediate imaging, so that three mutually associated pupils are formed.
  • the scanning elements are positioned so that the third pupil is automatically fixed relative to the scanning elements.
  • the pupil manipulation element is placed. It is further ensured that one of the three pupils coincides with the objective pupil or a pupil conjugated thereto.
  • the first scanning element is located in the objective pupil (or a pupil conjugated thereto), the pupil manipulation element is in a second pupil obtained by imaging the pupil with the first scanning element, and this is again imaged into a third pupil in which the pupil second scanning element is arranged.
  • the pupil manipulation element is in a second pupil obtained by imaging the pupil with the first scanning element, and this is again imaged into a third pupil in which the pupil second scanning element is arranged.
  • the relay optics is provided with a beam deflector and designed so that it is traversed twice in the imaging direction. From the first scanning element, the relay optics effects an image of the pupil with this scanning element on the second pupil. Since, according to the invention, a reflective element for pupil manipulation is arranged there, the relay optics, after reflection at the pupil manipulation element, produce a further pupil image on the third pupil which, due to the effect of the beam deflector, does not coincide with that of the pupil, in which the first scan element stands. The relay optics thus causes a double pupil image: once in the direction of the element for pupil manipulation and the other time away from it in the direction of the second scanning element.
  • This design of the relay optics allows a very compact design. Forming the beam deflector as a beam splitter or prism, resulting in a total of a T-shaped beam path between the scanning elements. Such a construction is particularly space-saving and easy to adjust.
  • the element for pupil manipulation can now be chosen almost arbitrarily according to the desired microscopy method. It is particularly preferred to use an element which is adjustable by an electrical control signal. If one does not want to manipulate the pupil, the element is switched to a mode in which it reflects the beam path without any further manipulation.
  • an element which is adjustable by an electrical control signal. If one does not want to manipulate the pupil, the element is switched to a mode in which it reflects the beam path without any further manipulation.
  • One possible construction for such an element is a spatial light modulator or adaptive mirror. Also, a DMD array can be used.
  • the relay optics By means of the relay optics according to the invention, three pupils are created, which are in fixedly assigned positions. A pupil picks up the first scan element. The second pupil contains the element for pupil manipulation. The third pupil is provided for the second scanning element. The three pupils are conjugate to each other, so that an optimal effect of the element for pupil manipulation is achieved and there is no longer any restriction to certain elements.
  • the arrangement according to the invention makes it possible to arrange the first scanning element in a pupil conjugate to the objective pupil or in the objective pupil itself. As a result of the fixed assignment of the two other pupils mentioned, a fixed position of the objective pupil to the pupil with the pupil manipulation element is secured overall. A lateral Displacement of the beam path during scanning does not take place on the pupil manipulation element.
  • the two scanning elements are always exactly in the objective pupil or a pupil conjugated thereto.
  • negative effects that may occur in scan elements that are outside the pupil plane, avoided even without pupil manipulation operation in the microscope according to the invention.
  • the light path is not extended as a function of the deflection by impinging the light beam outside the deflection axis of the respective uniaxially deflecting scanning element.
  • the illumination and the imaging beam always strike the scanning element exactly on the deflection axis, regardless of the position of the other scanning element.
  • FIG. 1 shows a schematic representation of a light-scanning microscope
  • Figure 2 is an enlarged view of a scanning device of the light scanning microscope
  • FIGS 3 to 5 the beam path of the scanning device of Figure 2 with the corresponding optical elements in three different views.
  • FIG. 1 shows schematically a light scanning microscope designed as a laser scanning microscope (LSM) 1.
  • LSM laser scanning microscope
  • the LSM 1 is essentially subdivided into a microscope module 3, a detection module 4 and a lighting or excitation module 5.
  • the excitation module provides excitation radiation and feeds it into the microscope module 3, so that it is directed to the object 2 as spot-shaped illumination.
  • the spot-shaped illumination is guided by the microscope module 3 raster over the object 2.
  • the spot area illuminated with illumination radiation from the illumination module 5 on the object is transmitted via the microscope module 3 from the Detection module 4 detected confocally. If the illumination is designed as excitation radiation, an image of the fluorescence properties of the object 2 can be obtained.
  • the illumination or excitation module 5 has for illumination light sources 6 and 7, which may be formed for example as a laser.
  • the radiation from the light sources 6 and 7 is conducted via a deflecting mirror 8 or a beam splitter 9 and an illumination optical unit 10 to a beam splitter 11, referred to as a main color splitter, where it is coupled into the microscope module 3.
  • the specific embodiment of the coupling of the radiation, realized in the present embodiment by deflecting mirror 8, beam splitter 9 and illumination optics 10 is for the following invention without further meaning.
  • Other constructions are also possible, for example by means of fiber optics and suitable fiber optic couplers.
  • deviating from the two light sources shown in Figure 1 of course, only a single light source or a larger number of light sources can be used. Essential to the invention here is only that at the main color divider 11, an illumination beam 17 is coupled.
  • the main color splitter 11 may be constructed, for example, as in the already mentioned DE 197 02 753 A1. Instead of the dichroic main color splitter described therein, a color-neutral splitter can also be used, as it is e.g. is described in DE 10257237 A1.
  • the radiation arriving from the illumination or excitation module 5 on the main color splitter 11 in the form of the illumination beam 17 is then focused onto or into the object 2 by means of a scanning device 12 and a scanning optics 13 through a tube lens 15 and an objective 16.
  • the focusing is carried out in the embodiment in a diffraction-limited focus whose position in the object 2 along the optical axis by an adjustable sample table 18 can be adjusted.
  • the scanning device 12 deflects the coming of the main color splitter 11 illumination beam 17 biaxially, so that this falls differently deflected beam 19 through the tube lens 15 and the lens 16 on or in the object 2, thus in the object 2 at different locations transverse to the optical axis is focused.
  • a deflecting mirror 14 designed as a beam splitter is optionally provided between the scanning optics 13 and the tube lens 15, which optionally permits a visual inspection.
  • the scanning device 12 which, like the sample table 18, is also controlled by a control unit 26 via lines not designated or not shown, the focus of the deflected illumination beam 19 is placed at different locations in the object by the objective 16. Overall, there is a three-dimensional positioning.
  • the deflection by the scanning device 12 causes the recording of a two-dimensional image whose Depth position in the object 2 as a third dimension by the setting of the sample table 18, that is, the position of the focal plane in the object 2 is determined.
  • a focus adjustment by adjusting the lens 16 is possible.
  • the radiation generated in object 2 e.g. Fluorescence radiation is detected by imaging the focus in the object 2 by means of the objective 16 and the tube lens 15 as well as the scanning optics 13 into the detection module 4 for each point of the image to be recorded.
  • the main color splitter 11 guides the beam 21, which, after passing through the scanning device 12, is again stationary in the imaging direction to the detection module 4, which has confocal detector elements.
  • Confocal filtering of the radiation from the focus in the object 2 takes place via an output coupler 22 and a pinhole optics 23 at a pinhole 24.
  • the plane of the confocal diaphragm 24 is conjugate to the focal plane in the object 2.
  • a detector 25 picks up the confocal filtered radiation. He is also connected to the controller 26.
  • the control unit 26 thus generates for each position of the focus in the object 2 a corresponding pixel, which is characterized by the intensity information from the detector 25 and its position in the image by the position of the scanning device 12.
  • the detection module 4 can have a plurality of spectral channels. There are then the corresponding elements 22 to 25 multiple times. In Figure 1, this is symbolized schematically by a second detection channel, the reference numerals are provided with an apostrophe. A dot-dash line indicates that even more detection channels are possible.
  • the output couplers 22 and 22 'thus act as so-called secondary color splitter. Its spectral characteristic determines which spectral range has the radiation detected by the associated detector.
  • the construction of the detection module 4 is of no further interest for the invention described here, in particular it does not determine whether or how confocal filtering takes place. It is only essential for the invention here that after the scanning device 12, a stationary beam 21 is present, which is referred to in the literature as a de-scanned beam.
  • the scanning device 12 ensures that the beam is deflected biaxially to the deflected beam 19, whereby the focus of the illumination radiation in the object 2 is perpendicular to the
  • dot-shaped illumination is of no further concern to this illustration, and therefore of no further importance to the present invention, which is primarily dedicated to scanning device 12 is (it also comes as a wide-field lighting in question), the scanning device 12 is considered below in the imaging direction.
  • the scanning device 12 is shown schematically in more detail in FIG. It consists of two scanning mirrors 40 and 41, which in this embodiment are examples of uniaxially deflecting scanning elements. Each scanning mirror deflects about a deflection axis, wherein the deflection axes of the scanning mirrors 40 and 41 are preferably orthogonal to one another. In principle, however, any inclination is sufficient to be able to cover a two-dimensional field with the focus.
  • a first scanning mirror 40 and this following a second scanning mirror 41 so that after the second scanning mirror 41 of the stationary beam 21 is given.
  • a pupil manipulation unit 42 which is shown only schematically in FIG.
  • FIGS. 3-5 show the scanning device 12 with the pupil manipulation unit 42 in detail with its beam path.
  • FIG. 4 shows the structure of FIG. 3 in this case from the direction designated A.
  • FIG. 5 shows the same beam path from the direction designated B. The description follows the de-scanning of the biaxially deflected beam 19 to the stationary beam 21.
  • the biaxially deflected beam 19 first falls on the first scanning mirror 40, whose uniaxial motion thus uniaxially de-scans.
  • the first scanning mirror 40 is located in a pupil 48 of the objective 16, which is shown schematically in FIG. 3 by a dashed line.
  • the originally biaxially deflected i. a two-dimensional field sweeping deflected beam 19 is after the scanning mirror 40 only in one spatial direction, ie line-shaped, deflected. It then falls on a prism 47, which is part of the pupil manipulation unit 42. From the prism 47, the beam passes through a first optical group 44 and a second optical group 45, which together form the pupil 48, in which the first scanning mirror 40 is located, in a second pupil 49. In this second pupil 49, a reflective element for pupil manipulation is arranged. In the exemplary embodiment, this is an adaptive mirror 43, which is controlled by the control unit 26.
  • the second pupil 49 becomes the prism 47 again through the second optical group 45 and the first optical group 44 and from there into a third one Pupil 50 shown.
  • the second scanning mirror 41 In this is the second scanning mirror 41.
  • the objective pupil 48, the second pupil 49 and the third pupil 50 are thus conjugate to each other, so that the first scanning mirror 40, the adaptive mirror 43 and the second scanning mirror 41 are in mutually conjugate layers.
  • the second scanning mirror 41 then causes a de-scanning about the remaining axis, so that thereafter the stationary beam 21 is present.
  • the stationary beam 21, which is generated by the illumination beam 17 in this approach, is first uniaxially deflected by the second scanning mirror 41, guided by the prism 47 through the first and second optical groups 44, 45 toward the adaptive mirror 43 depending on the setting pupil manipulating reflected so that it passes through the second optical group and the first optical group 45, 44 and the prism 47 to the first scanning mirror 40, which makes a biaxial deflection from the hitherto existing uniaxial deflection and the deflected beam 19 provides.
  • the pupil manipulation unit 42 comprises the prism 47, the first optical group 44 and the second optical group 45, between which an intermediate image 46 is formed.
  • the prism 47 guides (in the imaging direction) the radiation into the first optical group 44 and the second optical group 45 so at an offset to the optical axis of the optical groups 44 and 45, that after reflection on the adaptive mirror 43 of the received beam from the prism 47 is not in the direction the objective pupil 48 is reflected, but reaches the third pupil 50.
  • the prism 47 is designed here as a roof prism. In principle, however, a single mirror surface is sufficient so that only one deflection by the prism 47 has to be carried out. The beam path would then continue without deflection so that the position of the third pupil 50 would be to the left of the pupil manipulation unit (as viewed in the direction of the adaptive mirror).
  • the construction shown in Figure 3 and in Figures 4 and 5 has the advantage that the first and second scanning mirrors 41, 41 lie on a common optical axis, i. the optical axis of the deflected beam 19 and the stationary beam 21 can coincide. This facilitates the adjustment of the arrangement.

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

Abstract

L'invention concerne un microscope à balayage lumineux comprenant un objectif (16) de représentation d'échantillon. Selon l'invention, l'objectif (16) présente une pupille (48) d'objectif. Le microscope à balayage lumineux comprend également un dispositif (12) de balayage disposé à la suite de l'objectif (16) dans le sens de la représentation pour dévier le rayon dans deux axes et le dispositif (12) de balayage présente un premier et un deuxième éléments de balayage réalisant chacun une déviation dans un axe et qui sont disposés l'un derrière l'autre dans le sens de la représentation, ainsi qu'un élément (43) de manipulation (40, 41) de pupille qui est disposé dans une position associée à la pupille (48) d'objectif. Toujours selon l'invention, le premier élément (40) de balayage se trouve dans la pupille (48) d'objectif ou dans une pupille conjuguée avec celle-ci, une optique (42) relais est disposée à la suite du premier élément (40) de balayage pour représenter la pupille (48) contenant le premier élément (40) de balayage dans une deuxième pupille (49), l'élément (43) de manipulation de pupille se trouve dans la deuxième pupille (49) générée par l'optique (42) relais et il est réalisé réfléchissant de sorte que l'optique (42) relais soit encore une fois traversée dans le sens de la représentation et que celle-ci représente une nouvelle fois la deuxième pupille (49) dans une troisième pupille (50). Selon l'invention, l'optique (42) relais présente un dispositif (47) de déviation de rayon qui sépare la troisième pupille (50) de la pupille contenant le premier élément (40) de balayage et le deuxième élément (41) de balayage est disposé dans la troisième pupille (50).
PCT/EP2007/007881 2006-09-27 2007-09-10 Microscope à balayage laser muni d'un élément de manipulation de pupille WO2008037346A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102006045839.7A DE102006045839B4 (de) 2006-09-27 2006-09-27 Laserscanningmikroskop mit Element zur Pupillenmanipulation
DE102006045839.7 2006-09-27

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WO2008037346A1 true WO2008037346A1 (fr) 2008-04-03

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102014005880A1 (de) 2014-04-17 2015-11-05 Carl Zeiss Ag Lichtrastermikroskop mit vereinfachter Optik, insbesondere mit veränderlicher Pupillenlage
DE102014017001A1 (de) 2014-11-12 2016-05-12 Carl Zeiss Ag Mikroskop mit geringem Verzeichnungsfehler
US9989754B2 (en) 2012-03-09 2018-06-05 Carl Zeiss Microscopy Gmbh Light scanning microscope with spectral detection
US11317798B2 (en) * 2016-09-06 2022-05-03 Nikon Corporation Catadioptric unit-magnification afocal pupil relay and optical imaging system employing the same

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012046257A1 (fr) * 2010-10-05 2012-04-12 Fabio Mammano Objectif grin corrigé de façon adaptative pour microscopie
DE102014017003A1 (de) 2014-11-12 2016-05-12 Carl Zeiss Ag Scaneinrichtung zur 3D-Positionierung von Laserspots
DE102016119727A1 (de) 2016-10-17 2018-04-19 Carl Zeiss Microscopy Gmbh Vorrichtung zur Strahlmanipulation für ein Scanning-Mikroskop und Mikroskop

Citations (2)

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Publication number Priority date Publication date Assignee Title
WO2003020121A1 (fr) * 2001-08-30 2003-03-13 University Of Rochester Optique adaptative dans un ophtalmoscope laser a balayage
EP1372011A2 (fr) * 2002-06-15 2003-12-17 CARL ZEISS JENA GmbH Microscope, en particulier microscope à balayage laser avec dispositif optique adaptif

Family Cites Families (1)

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Publication number Priority date Publication date Assignee Title
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 (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003020121A1 (fr) * 2001-08-30 2003-03-13 University Of Rochester Optique adaptative dans un ophtalmoscope laser a balayage
EP1372011A2 (fr) * 2002-06-15 2003-12-17 CARL ZEISS JENA GmbH Microscope, en particulier microscope à balayage laser avec dispositif optique adaptif

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9989754B2 (en) 2012-03-09 2018-06-05 Carl Zeiss Microscopy Gmbh Light scanning microscope with spectral detection
DE102014005880A1 (de) 2014-04-17 2015-11-05 Carl Zeiss Ag Lichtrastermikroskop mit vereinfachter Optik, insbesondere mit veränderlicher Pupillenlage
US10551606B2 (en) 2014-04-17 2020-02-04 Carl Zeiss Microscopy Gmbh Light-scanning microscope with simplified optical system, more particularly with variable pupil position
US11086114B2 (en) 2014-04-17 2021-08-10 Carl Zeiss Microscopy Gmbh Light-scanning microscope with simplified optical system, more particularly with variable pupil position
DE102014017001A1 (de) 2014-11-12 2016-05-12 Carl Zeiss Ag Mikroskop mit geringem Verzeichnungsfehler
WO2016075195A1 (fr) 2014-11-12 2016-05-19 Carl Zeiss Microscopy Gmbh Microscope à faible distorsion
CN107003506A (zh) * 2014-11-12 2017-08-01 卡尔蔡司显微镜有限责任公司 具有低畸变像差的显微镜
US10254524B2 (en) 2014-11-12 2019-04-09 Carl Zeiss Microscopy Gmbh Microscope having low distortion aberration
CN107003506B (zh) * 2014-11-12 2020-05-05 卡尔蔡司显微镜有限责任公司 具有低畸变像差的显微镜
US11317798B2 (en) * 2016-09-06 2022-05-03 Nikon Corporation Catadioptric unit-magnification afocal pupil relay and optical imaging system employing the same

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DE102006045839A1 (de) 2008-04-03

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