USRE45575E1

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Publication number: USRE45575E1
Application number: US13/932,475
Authority: US
Inventors: Helmut Lippert; Matthias Wald; Benno Radt
Original assignee: Carl Zeiss Microscopy GmbH
Current assignee: Carl Zeiss Microscopy GmbH
Priority date: 2007-03-29
Filing date: 2013-07-01
Publication date: 2015-06-23

Abstract

The invention is directed to an optical arrangement with a light source for emitting a light bundle and with optical elements for transforming this light bundle into the shape of a light sheet, particularly suitable for illuminating individual planes of a three-dimensional specimen in selective plane illumination microscopy (SPIM). According to the invention, means are provided for varying the cross section of the light sheet, for varying the length of the light sheet and/or for influencing the direction in which individual beam components extending within the light sheet are directed to the specimen substance. This makes it possible to adapt the geometry of the light sheet to the illumination requirements for observing one and the same specimen plane with a plurality of different objectives and, if required, to reduce shadows occurring within the observed specimen plane as a result of the illumination.

Description

CROSS-REFERENCE TO RELATED APPLICATION

Notice: More than one reissue application has been filed for the reissue of U.S. Pat. No. 7,787,179. The reissue applications are U.S. patent application Ser. No. 13/600,663 (now abandoned), filed on Aug. 31, 2012, which is a reissue patent application of U.S. Pat. No. 7,787,179, filed as U.S. patent application Ser. No. 11/946,232 on Nov. 28, 2007; and U.S. patent application Ser. No. 13/932,475 (the present application), filed on Jul. 1, 2013, which is a divisional reissue patent application of U.S. patent application Ser. No. 13/600,663.

This application claims priority of German Application No. 10 2007 015 08.8, filed Mar. 29, 2007, the complete disclosure of which is hereby incorporated by reference.

BACKGROUND OF THE INVENTION

a) Field of the Invention

The invention is directed to an optical arrangement with a light source for emitting a light bundle and with optical elements for transforming this light bundle into the shape of a light sheet, particularly suitable for illuminating individual planes of a three-dimensional specimen in selective plane illumination microscopy (SPIM).

b) Description of the Related Art

In contrast to confocal laser scanning microscopy (LSM), in which a three-dimensional specimen is scanned point by point in individual planes of different depth and the image information thus acquired is then combined to form a three-dimensional image of the specimen, the SPIM technique relies on widefield microscopy and makes it possible to display an image of the specimen based on optical sections through individual planes of the specimen.

The advantages of the SPIM technique include the greater speed at which image information is acquired, the reduced risk of bleaching biological specimens, and an expanded penetration depth of the focus in the specimen.

In principle, in the SPIM technique fluorophores which are by themselves contained in the specimen or have been introduced into the specimen for contrasting are excited by laser light, and the laser radiation is formed into a light sheet, as it is called. A selected plane in the depth of the specimen is illuminated by the light sheet and an image of this specimen plane is acquired in the form of an optical section by imaging optics.

To illustrate the geometry of the light sheet more clearly, it will be assumed in the context of the present invention that the light sheet has a cross section which extends in the X and Y coordinate directions perpendicular to the beam direction of the laser light and a length which extends in the Z coordinate direction along the beam direction.

The optical axis of the objective by which the illuminated specimen plane is to be imaged or observed is oriented perpendicular to the Z coordinate direction.

Optical arrangements for generating a light sheet in connection with the SPIM technique are described in DE 102 57 423 A1 and DE 10 2005 027077 A1.

These arrangements produce only a rigid light sheet which is not variable with respect to its thickness, which should correspond to the extension in the X coordinate. This circumstance is particularly disadvantageous when one and the same plane of a specimen is to be imaged successively in time with different objectives whose optical imaging characteristics differ from one another.

In such cases, it is desirable to have the possibility of adapting the geometry of the light sheet, above all, its thickness, to the respective objective so that only the plane of interest is actually illuminated in the specimen and, accordingly, an unwanted bleaching of the specimen substance outside this plane is prevented. Also, the depth of focus of the light sheet can be adapted in this way to the respective object field being observed.

Another disadvantage in the arrangements mentioned above is that the light sheet that is generated with them has a Gaussian intensity profile in cross section so that a uniform illumination of the specimen plane being examined is impossible.

WO 2004/0530558 A1 describes a method in which a light-sheet type illumination is generated based on a relative movement between a line-shaped light field and the specimen to be observed. The light-sheet type illumination is carried out in that the light field is lined up multiple times successively in time due to the relative movement. This has the disadvantage that shadows result within the plane of the specimen being examined because of portions of the specimen substance which are not transparent for the illumination light and which lie in the illumination direction.

EP 0 248 204 B1 describes the generation of a line-shaped illumination with a linear fiber array and cylindrical lenses arranged downstream. However, again, the geometry of the light sheet cannot be varied.

The publication U.S. Pat. No. 4,826,299 describes the shaping of a light sheet with a Powell lens. The Powell lens has an aspherical profile in one coordinate direction and is flat in the coordinate orthogonal to it so that a virtually homogenized line-shaped light field is formed from a light bundle and can be used as a light sheet. However, again the geometry of this light sheet can not be varied, specifically with respect to its thickness and length, so that an optimal illumination through the specimen plane to be examined is also impossible in this case when using different objectives whose optical characteristics differ from one another.

All of the arrangements mentioned above have the disadvantage of unwanted shadows within the plane of the specimen being examined which are caused by portions of the specimen substance in the illumination direction which are not transparent for the illumination light.

OBJECT AND SUMMARY OF THE INVENTION

Proceeding from this prior art, it is the primary object of the invention to provide an optical arrangement for generating a light sheet which makes it possible to observe individual planes of a specimen with a greater efficiency than in previously known arrangements.

According to the invention, in an optical arrangement for generating a light sheet of the type mentioned above, means are provided for varying the cross section of the light sheet, for varying the length of the light sheet and/or for influencing the direction in which individual beam components extending within the light sheet are directed to the specimen substance.

This makes it possible to adapt the geometry of the light sheet to the illumination requirements for observing one and the same specimen plane with a plurality of different objectives and, if required, to achieve a reduction in shadows occurring within the observed specimen plane as a result of the illumination.

In a first construction of the arrangement according to the invention, a light source is provided which emits a bundle of coherent light in whose path are provided:

- a collimator,
- an aspherical optical element,
- a lens or lens group for realizing a field diaphragm plane, and
- a lens or lens group for realizing an aperture diaphragm plane.

In this connection, a bundle of parallel light is initially generated by the collimator and is then transformed into the shape of the light sheet by means of the aspherical optical element. A Powell lens, for example, can be used as an aspherical optical element.

The cross section of the light sheet is adapted to the illumination requirements of a particular objective with a field diaphragm which is placed in the field diaphragm plane and whose diaphragm opening is adequate for the desired cross section. If the objective is exchanged for an objective with a different aperture or different imaging scale, for example, the field diaphragm is exchanged for a field diaphragm whose diaphragm opening corresponds to the desired cross section. By exchanging the diaphragms, a change in cross section with respect to the width of the light sheet is achieved, that is, its extension in the Y coordinate is varied.

The diaphragms can be arranged on a changer wheel, for example. Changing the diaphragms can be carried out manually or automatically, and the respective field diaphragm is chosen depending on the characteristics of the objective that is used.

In an alternative construction, the influencing of the cross section is not carried out, as was described, by exchanging diaphragms, but by placing one or two zoom optics in the illumination beam path.

For this purpose, the lens group for realizing a field diaphragm and/or the lens group for realizing an aperture diaphragm are/is designed in such a way that they have a variable focal length.

Further, a device for reducing shadows in the specimen is provided in this first embodiment of the arrangement according to the invention.

To this end, a wobble plate is arranged in a pupil plane of the beam path which has already been shaped to form the light sheet, or an oscillating mirror or a polygon scanner is positioned in a plane conjugate to the field diaphragm plane.

Owing to the deflecting movement generated in this way, the direction of the beam components of the light sheet is influenced in such a way that these beam components strike the specimen substance successively in time in alternating directions so that shadows caused by opaque specimen substances within the illuminated specimen plane are prevented or at least substantially reduced.

In a second construction of the arrangement according to the invention, a light source is provided which emits a bundle of spatially partially coherent light, and one or two cylindrical-lens arrays and fixed or variable collimating optics are arranged in the path of this light bundle, the lateral coherence length being less than the period of the cylindrical-lens arrays.

The cylindrical-lens arrays take over the function of a honeycomb condenser in a plane, for example, the Y-Z plane, wherein the individual beam components or partial apertures generated by the first array are spatially superimposed in the field diaphragm plane with or without the help of a second array and the collimating optics. When a second array is present, the intensity distribution in the field diaphragm plane is more homogeneous than when a second array is not present. The geometry of the light sheet can be adapted by means of variable collimating optics.

The spatially partially coherent light is generated, for example, by means of a temporally partially coherent light source such as a broadband laser, a dispersive optical element being placed in its beam path for purposes of reducing the spatial coherence. A grating, a prism or a stepped mirror can be used as a dispersive optical element.

In a third construction of the arrangement according to the invention, the light source comprises an array of individual laser light sources. Located downstream of these light sources are a cylindrical lens or GRIN lens extending over the entire array and collimating optics. Every laser light source completely illuminates the entire desired light sheet. The width of the light sheet in the Y-Z plane is determined by the emitted aperture of the laser light sources and the focal length of the collimating optics. The thickness of the light sheet in the X-Z plane is determined by the emitted aperture of the light sources and the focal lengths of the cylindrical optics and collimating optics. Because of the spatial arrangement of the laser light sources, every light source illuminates the specimen plane at a different angle so that shadows within the specimen plane are prevented or at least substantially reduced as was described above.

With GRIN lenses, in contrast to conventional collector lenses, the focal length is influenced by a continuous variation of the refractive index in the lens material.

In a fourth construction of the arrangement according to the invention, a micro-optical element for transforming the light bundle into the shape of a light sheet is arranged in the light bundle coming from a coherent light source and exiting from a light-conducting fiber.

This micro-optical element can be micro-optics provided with optically active free-form surfaces or micro-optics which are constructed in the form of a GRIN lens and which have simultaneously a homogenizing effect in one cross-sectional direction and a focusing effect in the orthogonal cross-sectional direction.

A fifth construction of the arrangement according to the invention provides a light source which emits a bundle of coherent light within whose path the following components are provided:

- anamorphotic optics, for example, in the form of a cylindrical lens telescope, for generating a light bundle with an elliptic cross section,
- an optical deflection device, for example, in the form of an oscillating mirror, a polygon scanner or a digital micromirror device (DMD) for generating a scanning movement of this light bundle, and
- a focusing lens or lens group through which the scanned light bundle is directed to the specimen.

In a manner known per se, the DMD comprises about 500,000 microscopically small mirrors which can be tilted very quickly individually.

A light-sheet-like illumination is generated by this arrangement in that the light bundle which has an elliptic cross section and is focused in the specimen is lined up multiple times successively with respect to time due to the scanning movement. The lining up of the individually focused light bundles yields the geometry of the light sheet.

Shadows due to non-transparent specimen substances within the illuminated specimen plane are prevented or reduced in this construction of the arrangement according to the invention by the cylindrical lens telescope through which the direction of individual beam components is influenced as was described above.

The invention will be described more fully in the following with reference to embodiment examples.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings:

FIG. 1 shows the principle of a first construction of the arrangement according to the invention with diaphragms for influencing the geometry of the light sheet;

FIG. 2 shows the principle of the first construction of the arrangement according to the invention, but in this case with zoom optics for influencing the geometry of the light sheet;

FIG. 3 shows the principle of the first construction of the arrangement according to the invention with a wobble plate for reducing shadows;

FIG. 4 shows the principle of the first construction of the arrangement according to the invention, but with an oscillating mirror for reducing shadows;

FIG. 5 shows the principle of a second construction of the arrangement according to the invention with cylindrical-lens arrays for shaping the light sheet and for reducing shadows;

FIG. 6 shows an example for generating spatially partially coherent light for realizing the construction according toFIG. 5;

FIG. 7 shows the principle of a third construction of the arrangement according to the invention with an array of individual laser light sources for shaping the light sheet;

FIG. 8 shows the principle of a fourth construction of the arrangement according to the invention in which the light sheet is shaped by a micro-optical element;

FIG. 9 shows the principle of a fifth construction of the arrangement according to the invention in which the light sheet is shaped by one-dimensional scanning of a light bundle; and

FIG. 10 shows a first illustration of the prior art.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

InFIG. 10, which will serve in the following for a brief explanation of the principle of selective plane illumination microscopy (SPIM) in the prior art, a specimen1, for example, a biological substance, is surrounded by a transparent gel2. This specimen1 is a three-dimensional specimen1 which extends in the drawing plane and perpendicular to the drawing plane. It will be assumed that the gel2 is shaped to form a circular cylinder with an axis of rotation oriented perpendicular to the drawing plane.

As was already slated above, three-dimensional images of the specimen1 are obtained on the basis of a plurality of optical sections through individual specimen planes by the SPIM technique. It will be assumed that the plane of the specimen1 to be observed has the thickness d in the drawing plane.

In order to observe this plane, alight sheet3 having a length1 and a thickness d in the drawing plane which remains as uniform as possible along the entire length1 and which extends

perpendicular to the drawing plane at least over the entire dimensioning of the specimen1 is required.

In the context of the present description of the invention, it will be assumed that the length1 of thelight sheet3 extends in the Z coordinate direction, its thickness d extends in the X coordinate direction, and its width b extends perpendicular to the drawing plane in the Y coordinate direction. The Z coordinate direction also corresponds to the direction of the illumination beam path.

FIG. 1 shows a first construction of the arrangement according to the invention. A light source (not shown) emits a coherent light which exits from a light-conducting fiber4 as alight bundle5 and is directed through a collimator6 to anaspherical element7.

By passing through theaspherical element7, thelight bundle5 is expanded in the Y-Z plane shown inFIG. 1a, while thelight bundle5 passes theaspherical element7 in the X-Z plane, shown inFIG. 1b, substantially unchanged.

This reshaping yields the thickness d of thelight sheet3 in the X coordinate direction and its width b in the Y coordinate direction.

As is further shown inFIG. 1a andFIG. 1b, the illumination beam path which is shaped to form thelight sheet3 first passes a lens group8, for example, an achromat, to realize afield diaphragm plane9, then anotherlens group10 to realize anaperture diaphragm plane11 and, finally, athird lens group12 constructed as focusing optics through which thelight sheet3 is directed into the specimen1.

When afield diaphragm13 is placed in thefield diaphragm plane9, the geometry of thelight sheet3 is defined with respect to its width b by the diaphragm opening of thefield diaphragm13. The diaphragm opening of anaperture diaphragm14 placed in theaperture diaphragm plane11 determines the thickness d and the length1 of thelight sheet3.

In this way, with afield diaphragm13 of a determined diaphragm opening and anaperture diaphragm14 of a determined diaphragm opening, the cross section and the length1 of thelight sheet3 can be adapted to an objective used to observe the selected plane of the specimen1.

If this objective is exchanged for a different objective to observe the same plane of the specimen1, for example, to observe the same plane with another imaging scale, the invention provides that:

- thecurrent field diaphragm13 is exchanged for afield diaphragm13 whose diaphragm opening is adapted to this second objective, or
- thecurrent aperture diaphragm14 is exchanged for anaperture diaphragm14 whose diaphragm opening is adapted to this substituted objective, or
- bothdiaphragms13,14 are exchanged at the same time to adapt the cross section and the length1 of the light sheet to the substituted objective and, therefore, to make possible an efficient observation of the specimen1 in the selected plane.

The thickness d and length1 of thelight sheet3 cannot be adjusted independently from one another because, as was already stated, the depth of focus range depends upon the inverse square of the numerical aperture of the focused light bundle when thelight sheet3 is generated anamorphotically by the asphericaloptical element7.

It is conceivable to change the

diaphragms

13,14 either manually or automatically. In the latter case, the

diaphragms

13,14 are arranged, for example, on changer wheels coupled with the drives and the drives are coupled with a control unit from which actuating commands are issued which are generated depending on the characteristics of the substituted objective.

FIG. 2 shows the first construction of the arrangement according to the invention in a variant in which zoom optics, which in this instance comprise

lens groups

15,16 and17, for example, are provided for influencing the geometry of thelight sheet3 instead of the Held diaphragm and aperture diaphragm.

For the sake of clarity, the reference numbers used inFIG. 2 are the same as those used inFIG. 1 for identical optical components. As inFIG. 1, the Y-Z plane is shown inFIG. 2a and the X-Z plane is shown inFIG. 2b.

To reshape thelight bundle5 into alight sheet3, anaspherical element7 similar to that shown inFIG. 1 is again provided in the variant according toFIG. 2, a lens group8, preferably, an achromat, being arranged downstream of theaspherical element7 in order to realize a field diaphragm plane. Alight sheet3 with a homogenized radiation intensity in cross section is formed in the field diaphragm plane. The field diaphragm plane is located, for example, directly in front of alens18. Thelight sheet3 is imaged in the object plane, and accordingly in the specimen1, by means of a lens19 and the

lens groups

15,16 and17 of the zoom optics.

Influencing of the geometry of thelight sheet3 with the zoom optics is carried out in that the focal length of the zoom is varied, for example, by means of axial displacement of the

lens groups

15 and17. Thelenses18 to21 ensure the correct pupil position within the zoom optics and guarantee a substantially collimated beam path in the field diaphragm plane in the Y-Z section. In this constructional variant, the lenses20 and21 are constructed as cylindrical lenses.

FIG. 3 shows a modified variant of the first construction of the arrangement according to the invention which was mentioned above with reference toFIG. 1 andFIG. 2. The identical reference numbers are again used to designate the same components.

Only the section in the Y-Z plane is shown; the X-Z plane is omitted since it follows analogously fromFIG. 1 andFIG. 2.

In contrast to and in addition to the variants according toFIG. 1 andFIG. 2, a device for reducing the formation of shadows in the specimen1 is provided in the variant according toFIG. 3. By reducing the formation of shadows, shadows are prevented on the specimen substance that lies behind specimen particles not transparent to the illumination light in the direction of the illumination beam within the observed specimen plane.

In this regard, it can be seen fromFIG. 3 that after passing thefield diaphragm13 and thelens group10 thelight sheet3 strikes a wobble plate22 which is located in a pupil plane of the illumination beam path that is shaped to form thelight sheet3. Because of the oscillating movements of the wobble plate, the direction of the beam components of thelight sheet3 is influenced in such a way that they strike the specimen substance successively in time in alternating directions and at different angles so that non-transparent specimen substances are illuminated from behind and shadows caused by these specimen substances within the illuminated specimen plane are prevented or at least substantially reduced.

FIG. 4 shows another variant for reducing shadows relating to the first construction of the arrangement according to the invention which was described above with reference toFIGS. 1 to 3. In this case, anoscillating mirror23 is positioned in a plane conjugate to thefield diaphragm plane9. Because of its oscillating motion, theoscillating mirror23, similar to the wobble plate inFIG. 3, causes the direction of the beam components of thelight sheet3 to be influenced in such a way that they strike the specimen substance in different directions.

In a second construction of the arrangement according to the invention shown inFIG. 5, a light source which radiates a bundle of spatially partially coherent light is required. Two cylindrical-

lens arrays

24 and25 are arranged in the path of the light coming from this light source (not shown in the drawing). Together with collimating optics37, these two cylindrical-

lens arrays

24 and25 form a honeycomb condenser which acts in the Y-Z plane and through which an homogenization of the radiation intensity is achieved in this Y-Z plane. Only collimating optics37 which generate the thickness d of the light sheet in the specimen plane acts in the X-Z plane. The Y-Z plane is again shown inFIG. 5a and the X-Z plane is shown inFIG. 5b.

The spatially partially coherent light required for the arrangement according to the invention shown inFIG. 5 can be generated, for example, as inFIG. 6.

In this case, abroadband laser26 is provided. A temporally partially coherent beam proceeds from thisbroadband laser26 and is directed to agrating27. The grating27 acts as a dispersive optical element so that the spatial coherence of the light reflected by the grating27 is reduced. This light strikes the cylindrical-

leas arrays

24 and25 as is shown inFIG. 5. Alternatively, dispersive optical elements in the form of prisms or stepped mirrors can also be used instead of the grating27.

FIG. 7 shows a third construction of the arrangement according to the invention for generating alight sheet3. In this case, the light source comprises an array of individuallaser light sources28. Each light source generates acomplete light sheet3 by means of thecylindrical lens29 and thelens group12. The spatial arrangement of thelaser light sources28 influences the direction of individual radiation components in such a way that shadows due to non-transparent specimen substances within the illuminated specimen plane are prevented or substantially reduced.

It is also possible to use a GRIN lens instead of thecylindrical lens29.

FIG. 8 shows a fourth construction of the arrangement according to the invention. In this case, alight bundle5 coming from a coherent light source and exiting from a light-conducting fiber (not shown) is directed to amicro-optical element30 which provides for the transformation of thelight bundle5 into the shape of alight sheet3. Thismicro-optical element30 can be, for example, micro-optics provided with optically active free-form surfaces, or it can be constructed in the form of a GRIN lens.

Aside from transforming thelight bundle5 into the shape of thelight sheet3, amicro-optical element30 of the kind mentioned above also achieves a homogenizing action in the Y-Z plane (shown inFIG. 8a) and a focusing action in the orthogonal plane X-Z (shown inFIG. 8b).

Another, fifth construction of the arrangement according to the invention is shown inFIG. 9. Realization of this construction again requires a light source from which alight bundle5 of coherent light proceeds. Thelight bundle5 is directed to anamorphotic optics, for example, in the form of acylindrical telescope31 which causes an independent cross-sectional change in thelight bundle5 in the X-axis and Y-axis so that the light bundle obtains an elliptical cross section. An optical deflecting device, in this case, for example, in the form of anoscillating mirror33, is arranged further along the path. It is also conceivable to use a polygon scanner or a DMD instead of theoscillating mirror33 to generate a scanning movement.

The light bundle which is deflected in a scanning manner by theoscillating mirror33 is directed into the specimen1 through thelens group12.

As a result of the scanning movement of theoscillating mirror33, the elliptical light bundle that is focused in the specimen is located successively in time at

positions

34,35 and36. The lining up of the respective illuminated areas in the specimen1 yields the desiredlight sheet3.

In this construction, shadows are prevented or reduced within the observed specimen plane due to the radiating angle occurring when focusing. The beam angles can be adjusted through the characteristics of theanamorphotic optics31.

While the foregoing description and drawings represent the present invention, it will be obvious to those skilled in the art that various changes may be made therein without departing from the true spirit and scope of the present invention.

REFERENCE NUMBERS

1 specimen
2 gel
3 light sheet
4 light-conducting fiber
5 light bundle
6 collimator
7 aspherical optical element
8 lens group
9 field diaphragm plane
10 lens group
11 aperture diaphragm plane
12 lens group
13 field diaphragm
14 aperture diaphragm
15,16,17 lens groups
18,19,20,21 lenses
22 wobble plate
23 oscillating mirror
24 cylindrical-lens array
25 cylindrical-lens ray
26 broadband laser
27 grating
28 laser light sources
29 cylindrical lens
30 micro-optical element
31 cylindrical-lens telescope
32 light bundle with elliptical cross section
33 oscillating mirror
34,35,36 positions
37 collimating optics