Microscope with at least one illuminating beam in the form of a light sheet

Abstract

A microscope includes at least one illuminating lens configured to guide at least one illuminating beam in the form of a light sheet for illuminating at least one specimen to be examined. The microscope also includes at least one detection lens configured to capture at least one detection beam issuing from the at least one specimen to be examined. The at least one illuminating lens has an optical axis at an angle α, which is not equal to 90°, to an optical axis of the at least one detection lens. The at least one illuminating beam enters the at least one illuminating lens at an entry angle β such that the light sheet lies within a focal plane of the at least one detection lens.

Claims

1. A microscope comprising: a first illuminating lens configured to guide at least one first illuminating beam in the form of a first light sheet for illuminating at least one specimen to be examined; a second illuminating lens configured to guide at least one second illuminating beam in the form of a second light sheet for illuminating the at least one specimen to be examined; and at least one detection lens configured to capture at least one detection beam issuing from the at least one specimen to be examined, wherein an optical axis of the first illuminating lens is at an angle α, which is not equal to 90°, to an optical axis of the at least one detection lens, wherein an optical axis of the second illuminating lens is at an angle α′, which is not equal to 90°, to an optical axis of the at least one detection lens, wherein the at least one first illuminating beam enters the first illuminating lens at a non-zero entry angle β to the optical axis of the first illuminating lens, as a result of which the first light sheet lies within a focal plane of the at least one detection lens and extends perpendicularly to the optical axis of the detection lens, and wherein the at least one second illuminating beam enters the second illuminating lens at a non-zero entry angle β′ to the optical axis of the second illuminating lens, as a result of which the second light sheet lies within a focal plane of the at least one detection lens and extends perpendicularly to the optical axis of the detection lens.

2. The microscope according to claim 1, wherein the at least one first illuminating beam enters the first illuminating lens outside the optical axis thereof.

3. The microscope according to claim 2, wherein the angle α has a value from 120° to 150°.

4. The microscope according to claim 3, wherein the angle α has a value of approximately 135°.

5. The microscope according to claim 1, wherein at least two detection lenses are provided and arranged in an objective revolver.

6. The microscope according to claim 1, wherein structurally identical lenses are used for the at least one detection lens and at least one of the first illuminating lens or the second illuminating lens.

7. The microscope according to claim 1, further comprising: at least two light sources, which radiate different wavelengths from one another; and at least two detection devices configured to detect the wavelengths that are different from one another.

8. The microscope according to claim 1, further comprising a first movement arrangement configured to generate a relative movement between a specimen illumination region and the specimen to be examined, wherein the specimen is placed on a stage.

9. The microscope according to claim 8, further comprising a second movement arrangement configured to move the stage in one plane.

10. The microscope according to claim 8, wherein the specimen to be examined is placed on the stage by means of a slide.

11. The microscope according to claim 8, wherein the first illuminating lens and the at least one detection lens are each designed as immersion lenses and are located at least in part in an immersion medium together with the specimen to be examined.

12. The microscope according to claim 1, wherein the specimen to be examined is placed on a fixed stage, further comprising: at least one device for changing the angle β, and wherein the focal plane of the at least one detection lens can be repositioned.

13. The microscope according to claim 1, wherein the at least one detection lens configured to capture at least one detection beam issuing from the at least one specimen to be examined comprises a first detection lens configured to capture a first detection beam issuing from the at least one specimen to be examined and a second detection lens configured to capture a second detection beam issuing from the at least one specimen to be examined, wherein the first detection lens has an optical axis and the second detection lens has an optical axis, and wherein the optical axis of the first detection lens is parallel to the optical axis of the second detection lens.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The present invention will be described in even greater detail below based on the exemplary figures. The invention is not limited to the exemplary embodiments. All features described and/or illustrated herein can be used alone or combined in different combinations in embodiments of the invention. The features and advantages of various embodiments of the present invention will become apparent by reading the following detailed description with reference to the attached drawings which illustrate the following:

(2) FIGS. 1a-1f are a schematic views of the construction and mode of operation of light sheet microscopes according to embodiments of the invention having one illuminating beam/lens and one detection beam/lens;

(3) FIG. 2 is a schematic view of the construction and mode of operation of a light sheet microscope according to an embodiment of the invention having two illuminating beams/lenses and two detection beams/lenses;

(4) FIG. 3 shows a series of specimens to be examined on a stage that can be moved in a plane under a light sheet microscope according to an embodiment of the invention having one illuminating beam/lens and one detection beam/lens;

(5) FIG. 4 shows typical biological specimens on a slide;

(6) FIG. 5 is a schematic view of the construction and the mode of operation of a light sheet microscope according to an embodiment of the invention having two illuminating beams/lenses and one detection beam/lens having a fixed stage;

(7) FIG. 6 shows biological specimens to be examined a) in the form of a two-dimensional in vitro cell culture on a layer of agarose, and b) in the form of a three-dimensional cell culture in a medium;

(8) FIG. 7 is a schematic view of the construction and mode of operation of a light sheet microscope according to an embodiment of the invention having two illuminating beams/lenses and one nosepiece for capturing the detection beam.

DETAILED DESCRIPTION

(9) In an embodiment the present invention provides a microscope having at least one illuminating beam in the form of a light sheet and which overcomes certain disadvantages and restrictions of prior art microscopes.

(10) A light sheet microscope according to an embodiment of the invention has at least one illuminating beam in the form of a specimen illumination region (light sheet) expanded transversely to the axis of the illuminating beam and simultaneously in the focal plane of the detection lens.

(11) A light sheet microscope according to an embodiment of the invention has at least one illuminating lens to guide the at least one illuminating beam. In a particular embodiment there are at least two illuminating lenses, which are preferably arranged in the form of a nosepiece. The illuminating lenses are preferably either immersion or air lenses.

(12) A light sheet microscope according to an embodiment of the invention has at least one detection device, which captures at least one detection beam issuing from a specimen to be examined, which is wholly or partially inside the specimen illumination region and is therefore illuminated by the at least one illuminating beam.

(13) A light sheet microscope according to an embodiment of the invention has at least one detection lens to guide the at least one detection beam. In the process the at least one detection beam is particularly preferably guided in the optical axis of the at least one detection lens. In a particular embodiment there are at least two detective lenses, which are arranged in the form of a nosepiece.

(14) A light sheet microscope according to an embodiment of the invention has an optical axis of at least one detection lens that is not perpendicular to the optical axis of the at least one illuminating lens, but rather assumes an angle α thereto, which preferably has a value of from 100° to 170°, more preferably a value of from 120° to 150°, in particular a value of approximately 135°.

(15) In order to ensure that the at least one illuminating beam comes from the at least one illuminating lens at such an initial angle in relation to the optical axis of the at least one illuminating lens that the specimen illumination region (light sheet) comes to be located in the focal plane of the at least one detection lens, it is necessary for the at least one illuminating beam to enter the at least one illuminating lens outside the optical axis of the at least one illuminating lens at an entry angle β. Only in this manner can it be ensured that, despite the construction of the present light sheet microscope, which differs from the formerly standard right-angled arrangement of the at least one illuminating lens relative to the at least one detection lens, the light sheet is perpendicular to the optical axis of the at least one detection lens and aligns precisely with the focal plane of the at least one detection lens.

(16) In an embodiment, structurally identical lenses are used for the at least one illuminating lens and for the at least one detection lens respectively. In particular, this makes it possible to reverse the light path and to use the at least one detection lens as an illuminating lens and vice versa, as a result of which—if a corresponding number of detection devices is present—an increased imaging speed and/or an increased light yield are facilitated.

(17) In an embodiment there are at least two light sources, in particular pump lasers, which radiate different wavelengths that are detected by at least two detection devices, which are each sensitive to one of these wavelengths.

(18) The specimen to be examined is located on a microscope stage. In an embodiment there is a slide (mount) located in between the specimen and the stage.

(19) In an embodiment there is a first movement arrangement, which acts to generate a relative movement between the specimen illumination region and the specimen to be examined. In this way, the specimen is moved through the light sheet in order to obtain a series of two-dimensional images and thus a three dimensional image of the specimen.

(20) In an embodiment, the specimen to be examined is placed on a fixed stage. In order to nevertheless move the specimen through the light sheet, in order to obtain a series of two-dimensional images and thus a three-dimensional image of the specimen, there is at least one device, which acts to change the angle β, and the focal plane of the at least one detection lens can be repositioned.

(21) In a further embodiment, at least one of the present illuminating lenses and at least one of the present detection lenses are each constructed as immersion lenses and are located at least in part together with the specimen to be examined in an immersion medium.

(22) In an embodiment there is a second movement arrangement, which allows the stage to be moved in a plane.

(23) The construction of the light sheet microscope according to an embodiment of the invention overcomes the formerly standard right-angled arrangement of illuminating and detection lenses, which is associated with spatial limitations. This opens up new possibilities for the lenses in relation to capture technology and also for placing the specimen under examination on a stage and/or moving the specimen together with the stage. In this manner new possible applications come about.

(24) A light sheet microscope according to an embodiment of the invention enables a simple positioning of the specimen on a stage. When the specimen is changed over, the specimen remains in the medium and does not leave it. A quick and easy changeover between various lenses for both illumination and also for detection is possible.

(25) A light sheet microscope according to an embodiment of the invention enables an increased capture speed and/or an increased light yield as a consequence of simultaneous illumination and detection in two structurally identical lenses.

(26) A light sheet microscope according to an embodiment of the invention enables acombination with conventional inverse microscopes and creates synergies therebetween. A combination with other microscope technologies, in particular epifluorescence or PALM, becomes easily possible, since the construction according to an embodiment of the invention allows the quick and easy changeover of lenses.

(27) A light sheet microscope according to an embodiment of the invention enables a simple change of lens together with a simple positioning on a stage and makes possible a very versatile microscope for very different applications both for large and also for small specimens, from normal to high resolution, from the examination of single specimens right through to a so-called high throughput screening.

(28) In the three embodiments shown in FIGS. 1a), 1c) and 1e), two lenses 10, 20 are arranged such that the optical axes 11, 21 of the two lenses 10, 20 adopt an angle α of approximately 135° relative to one another. The specimen (sample) 1 is on a suitable mount, for example agarose, as the slide 2, which can be moved in all spatial directions on a stage 3 by means of a movement arrangement 4. The two lenses 10, 20 are permanently installed.

(29) According to the embodiment shown in FIG. 1a), a light sheet is generated in the first lens, which acts as an illuminating lens 10, by means of devices known from the prior art. This light sheet does not, however, extend parallel to the optical axis of the illuminating lens 10, as described in the prior art, but rather enters the illuminating lens 10 at a wide entry angle β and outside the optical 11 axis thereof. This arrangement ensures that even at an angle α of approximately 135° of the optical axes 11, 21 of the lenses 10, 20 relative to one another, the light sheet nevertheless extends perpendicularly to the optical axis 21 and within the focal plane of the second lens, which acts as a detection lens 20. Detection of the detection beam 35, which issues from the specimen 1 and passes through the detection lens 20 along the optical axis 21 thereof, is performed by means of a detector 36, for which purpose a fast CCD camera or a sCMOS camera in can be used.

(30) In the second embodiment shown in FIG. 1c), a light sheet is generated in the second lens, which now acts as an illuminating lens 10′, by means of devices known from prior art. Here too, the light sheet does not extend parallel to the optical axis of the illuminating lens 10′ as described in the prior art, but rather enters the illuminating lens 10′ at a wide entry angle β′. This arrangement ensures here, too, that at an angle α′ of approximately 135° of the optical axes 11′, 21′ of the lenses 10′, 20′ relative to one another, the light sheet nevertheless extends perpendicularly to the optical axis 21′ of the first lens, which acts here as a detection lens 20′. Detection of the detection beam 35′, which issues from the specimen 1 and passes through the detection lens 20′ along the optical axis 21′ thereof is also performed here by means of a detector 36′.

(31) According to the third embodiment shown in FIG. 1e), a light sheet is generated in the first lens, which acts as an illuminating lens 10, which light sheet here too does not extend parallel to the optical axis of the illuminating lens 10, but rather enters the illuminating lens 10 at a wide entry angle β and outside the optical 11 axis thereof. As a result of this, even at an angle α of approximately 135° of the optical axes 11, 21 of the lenses 10, 20 relative to one another, the light sheet extends perpendicularly to the optical axis 21 and within the focal plane of the second lens, which acts as a first detective lens 20. The first detection beam 35 issuing from the specimen 1 passes through the first detection lens 20 along the optical axis 21 thereof and is guided onto a detector 36 by means of a first deflection mirror 37. At the same time, a second detection beam 35′ issuing from the specimen 1 is guided through the first lens, which acts as a second detection lens 20′ for this purpose, along the optical axis 21′ thereof by means of a second deflection mirror 37 and a reverse beam splitter 39 onto the same detector 36. Since the light sheet in this arrangement does not extend perpendicularly to the optical axis 21′ and therefore not within the focal plane of the second detective lens 20′ it is, however, necessary to send the second detection beam 35′ through a device 38 for oblique plane microscopy (OPM) beforehand in order to overlay the two images clearly according to Cutrale et al., loco citato. Using the arrangement according to FIG. 1e) it is possible to reverse the light path and to use the illuminating lens 10 simultaneously as a second detection lens 20′, as a result of which a higher light yield is achieved here.

(32) The embodiments of the arrangement of light sheet microscopes according to the embodiments of the invention shown in FIG. 1 can facilitate certain examination methods described hereinafter.

(33) A. Light Sheet Microscopy Using the Same Lenses

(34) For light sheet microscopy of large specimens, for example of fish embryos or plants, and of small specimens, which include cells and sub-cellular structures, two identical lenses having a mid to high numerical aperture are used with the same lenses and are arranged as in FIG. 1 at an angle α of approximately 135° relative to one another, as a result of which two channels can be excited and detected simultaneously.

(35) In one embodiment, the excitation is performed using a first illuminating beam 33 having a first wavelength (here 488 nm) in the first lens 10, while the fluorescence signal, as the first detection signal 35, is captured by the two lenses 10, 20, merged and detected by means of a first detection device 36. To achieve this, a second excitation is performed simultaneously using a second illuminating beam 33′ having a second wavelength (here: 561 nm) in the second lens 20, while the fluorescence signal resulting in the process, as a second detection signal 35′, is again captured by the two lenses 10, 20, merged and detected by a second detection device 36′.

(36) If a time series is recorded, the assignment of both excitation wavelengths to the lenses can be retained in a first embodiment. In a second embodiment in contrast, the assignment of the two excitation wavelengths to the lenses is defined alternately. The alternating nature of the illumination ensures improved illumination from different angles.

(37) The simultaneous excitation and detection of two wavelengths allows a considerable time gain for light sheet microscopy. As a result of the simultaneous detection by means of two lenses 10, 20 using a detection device 36, the detectable signal is increased. A further advantage of this arrangement is the illumination of specimens, which are difficult to penetrate optically on one side. The described arrangement is suitable for optimal illumination and for the detection of such structures, for example in fish embryos which are near the yolk.

(38) B. Light Sheet Microscopy Using Lenses of Different Numerical Aperture and Magnification

(39) FIGS. 2 to 4 show how light sheet microscopy of large specimens, for example of fish embryos or plants, can be arranged using lenses of different numerical aperture and magnification, having the possibility and device for observing a plurality of samples in a time lapse image.

(40) According to FIG. 2 the sample is illuminated by a first illuminating lens 10 having a low numerical aperture (NA<0.2) and a very large free working distance (>3 mm). The light sheet is generated at an entry angle β of the first illuminating beam 33 into the first illuminating lens 10 and detected using a detection lens 20. In order to obtain illumination from both sides, a second illuminating lens 10′ is added, into which a second illuminating beam 33′ enters at an entry angle β′.

(41) If the free working distance between the illuminating lenses 10, 10′ is sufficient, a second detection lens 20′ can optionally be added in order to achieve detection by means of the second detection lens 20′ from two sides each having their own detection device 36, 36′. In order to move the specimen through the light sheet, all lenses 10, 10′, 20, 10′ remain fixed, only the stage 3 is moved in a manner that is accurate to the micron.

(42) In FIG. 2 the use of immersion lenses as illuminating lenses 10, 10′ is shown by way of example. If, on the other hand, air lenses are used, then it is advantageous to install a thin glass plate in between the specimen (which is not shown in FIG. 2) and the illuminating lenses 10, 10′ respectively.

(43) The construction selected in FIG. 1 is very suitable for detecting a plurality of large specimens in a time lapse image, as shown in FIG. 3. The specimens 1, 1′, 1″, 1′″ are positioned in series at an appropriate distance from one another on the stage 3. In this case it is advantageous for the largest axis of the specimens 1, 1′, 1″, 1′″ not to point in the direction of the lenses 10, 10′, . . . , 20, 20′, . . . if possible in order to allow good optical penetration. The specimens 1, 1′, 1″, 1′″ are each brought into focus and detected at an appropriate time interval one after the other on the stage 3. In the process, the optimal position of each specimen 1, 1′, 1″, 1′″ has been defined once at the beginning of the measurement and can now be repeatedly and automatically called up and taken up. The stage 3 can be moved in all three spatial directions by means of a movement arrangement 5. The specimens 1, 1′, 1″, 1′″ also remain in their place and therefore in the medium when a new position is taken up.

(44) Using the arrangement according to an embodiment of the invention, large specimens can easily be positioned on the stage 3 and do not have to be infused in an agarose cylinder as described in the prior art, as a result of which said specimens become inaccessible for later manipulation. On the contrary, a small stand of agarose is sufficient here, for example 2% agarose with a low melting point (low melting agarose).

(45) In FIG. 4a) a positioned fish egg is shown schematically in this manner as the specimen 1, while FIG. 4b) shows a fish embryo, which was positioned with a drop of agarose, as the specimen 1.

(46) FIG. 4c) is a side view of a zebra fish embryo as the specimen 1 in a chamber or a chamber that is easy to penetrate optically as a slide 2 and exhibits recesses at the growth points of the organism. FIG. 4d is a view from above of the zebra fish embryo as the specimen 1 in the chamber acting as the slide 2. If fish embryos are used, said embryos must be anaesthetised.

(47) C. High-Resolution Light Sheet Microscopy Using Lenses of Identical Or Different Numerical Aperture and Magnification

(48) The construction for high-resolution light sheet microscopy of small specimens, for example cells and sub-cellular structures, and of large specimens, including fish embryos and plants, for example, using lenses of identical or different numerical aperture and magnification follows the schematic views of FIGS. 5 and 6.

(49) According to FIG. 5, the specimen is illuminated by an illuminating lens 10 having a high numerical aperture (NA>0.8 μm) in order to generate as thin a light sheet as possible (d<1 μm). The light sheet is generated at a high emission angle and detected by the detection lens 20. Provided the geometry and free working distance to the illuminating lens 10 allow, a structurally identical illuminating lens 10′ can be added, as shown in FIG. 5, for double-sided illumination and thus double-sided detection can be achieved. In FIG. 5 the use of immersion lenses is shown by way of example.

(50) In order to move the specimen through the light sheet, the at least one illuminating lens 10, 10′ and the stage 3 remain fixed, while the angle of the optical axes 11, 11′ of the at least one illuminating lens 10, 10′ in relation to the stage 3 is changed by means of a device 15, 15′ provided for this purpose. The detection lens 20 is repositioned synchronously to the movement of the light sheet for optimal detection.

(51) The preparation for large specimens is as shown in FIG. 4. A two or three-dimensional in vivo cell culture or fixed cells and sub-cellular structures, for example a cytoskeleton, are, as shown in FIG. 6, prepared for high-resolution light sheet microscopy.

(52) In this case, FIG. 6a) shows the preparation of a two-dimensional in vitro cell culture on a layer of agarose, while the preparation of a three-dimensional cell culture in a medium that is appropriate therefore, for example in agarose, collagen, a hydrogel or perfluoroethylene propylene copolymer film is shown in FIG. 6b).

(53) D. Light Sheet Microscopy in Combination With Light Microscopy

(54) All of the constructions described under items A. to C. can be combined with known inverse light microscopes. Using the selected arrangement it is possible to take over the stage 3, including, for example, a piezo stage, as well as the detection lens 20 directly with slight modifications.

(55) As can be seen from FIG. 7, the use of a nosepiece 40, which has a series of detection lenses 20, 20′, 20″, 20′″, 20″″, 20′″″ for capturing the detection beam, is also possible.

(56) E. Light Sheet Microscopy in Combination With Epifluorescence and/or High-Resolution Microscopy

(57) All of the constructions and combinations described thus far can be combined with epifluorescence and/or with known devices for high-resolution microscopy. In an embodiment, epifluorescence is incorporated by the second illuminating lens 10′.

(58) In an alternative embodiment, the second illuminating lens 10′ serves to use so-called photoactivation localization microscopy (PALM), a method known from high-resolution microscopy, and to combine it with light sheet microscopy.

(59) While the invention has been illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered illustrative or exemplary and not restrictive. It will be understood that changes and modifications may be made by those of ordinary skill within the scope of the following claims. In particular, the present invention covers further embodiments with any combination of features from different embodiments described above and below. Additionally, statements made herein characterizing the invention refer to an embodiment of the invention and not necessarily all embodiments.

(60) The terms used in the claims should be construed to have the broadest reasonable interpretation consistent with the foregoing description. For example, the use of the article “a” or “the” in introducing an element should not be interpreted as being exclusive of a plurality of elements. Likewise, the recitation of “or” should be interpreted as being inclusive, such that the recitation of “A or B” is not exclusive of “A and B,” unless it is clear from the context or the foregoing description that only one of A and B is intended. Further, the recitation of “at least one of A, B and C” should be interpreted as one or more of a group of elements consisting of A, B and C, and should not be interpreted as requiring at least one of each of the listed elements A, B and C, regardless of whether A, B and C are related as categories or otherwise. Moreover, the recitation of “A, B and/or C” or “at least one of A, B or C” should be interpreted as including any singular entity from the listed elements, e.g., A, any subset from the listed elements, e.g., A and B, or the entire list of elements A, B and C.

LIST OF REFERENCE NUMERALS

(61) α, α′ Angle between the optical axis 11, 11′ of an illuminating lens 10, 10′ relative to the optical axis 21, 21′ of a detection lens 20, 20′ β, β′ Angle of entry into an illuminating lens 10, 10′ 1, 1′ . . . Specimen (sample) 2 Slide (mount) 3 Stage (sample stage) 4, 5 Movement arrangements 10, 10′ Illuminating lenses 11, 11′ Associated optical axes 12, 12′ Emission cones 15, 15′ Device for changing the angle β,β′ 20, 20′ . . . Detection lenses 21, 21′ Associated optical axes 22, 22′ Entry cones 31,31′ Light Sources (excitation lasers) 32, 32′ Focusing arrangements 33, 33′ Specimen illumination region (light sheet) 35, 35′ Detection beam 36, 36′ Detection device (detector, camera) 37 Deflection mirror (dichromatic or normal) 38 Device for oblique plane microscopy (OPM) according to Cutrale et al., see above) 39 Reverse beam splitter 40 Nosepiece