MICROSCOPE AND METHOD FOR CAPTURING A MICROSCOPIC IMAGE AND USE OF A PLANAR REFLECTOR

Abstract

The invention relates to EPI lighting which allows transmitted light-bright field- or transmitted light-dark field-imaging or phase contrast imaging of a microscopic sample. For this purpose, a flat reflector is used which is located opposite the observer side and which brings about a deflection of the illumination beam of light. The flat reflector has a plane normal and an effective perpendicular which differs from the plane normal, or it is in the form of a retroreflector.

Claims

1. The use of at least one plate-shaped reflector for deflecting at least one illumination beam for illuminating at least one sample for recording at least one microscopic image of the sample from a first side using an image sensor, wherein the plate-shaped reflector has a plate normal and a substitute perpendicular, deviating from the plate normal, in respect of the illumination beam and the reflector is arranged on a second side which is opposite the first side with respect to the sample.

2. A method for recording a microscopic image of at least one region of at least one sample arranged in a sample plane from a first side, comprising generating at least one beam with the aid of at least one light source, guiding the beam through the sample plane to a plate-shaped reflector, the reflector being a retroreflector, deflecting the beam by the reflector, illuminating the sample with the deflected beam, recording the microscopic image using an image sensor.

3. The method or use as claimed in claim 1, wherein the sample is arranged in a horizontal sample plane and/or in that the microscopic image is recorded from below in relation to the force of gravity.

4. The method or use as claimed in claim 1, wherein the reflector is embodied in one piece as a plate or a film and/or in that the reflector is embodied as a layer on a carrier plate or a carrier film.

5. The method or use as claimed in claim 1, wherein there is a focal plane which is imaged on the image sensor in focus and in the focal plane there is a field of view which is captured by the image sensor and the illumination beam has an intersection with the focal plane before the deflection and the intersection contains the field of view.

6. The method or use as claimed in claim 1, wherein the beam is guided through the sample plane at a point lying outside a field of view.

7. The method or use as claimed in claim 1, wherein the deflected beam effects a transmitted light bright field illumination or a transmitted light dark field illumination.

8. The method or use as claimed in claim 1, wherein the deflected beam has a central ray which is inclined to an optical axis.

9. The method or use as claimed in claim 1, wherein the light source is an LED.

10. The method or use as claimed in claim 1, wherein a plurality of microscopic images of a plurality of samples and/or of one sample at a plurality of locations are recorded and in that a microscope camera, which comprises the image sensor and a camera lens, is moved, from the recording of one image to the recording of a next image, with respect to the samples or the sample in each case and the reflector is fixedly arranged with respect to the samples or the sample and the light source is fixedly arranged with respect to the microscope camera.

11. The method or use as claimed in claim 1, wherein the reflector is embodied as a periodic relief structure and at least two reflection surfaces are present in each period.

12. The method or use as claimed in claim 1, wherein the reflector is embodied as a microprism array and/or a microlens array.

13. The method or use as claimed in claim 1, wherein the reflector is embodied as a retroreflector embodied as a full cube microprism array or as a pyramidal triple microprism array or comprising encapsulated micro glass beads.

14. The method or use as claimed in claim 1, wherein the beam of the illumination incident on the sample is split in the sample and/or by refraction at a sample back side into at least one first beam and at least one second beam the second beam impinging on the reflector at a different angle of incidence to the first beam

15. The method or use as claimed in claim 1, wherein the beam of the illumination is guided through the microscope objective onto the sample.

16. The method or use as claimed in claim 1, wherein the reflector deflects an incident light ray of the beam by means of at least two successive individual reflections.

17. The method or use as claimed in claim 1, wherein the microscopic image is a phase contrast recording or a superposition of a transmitted light bright field image or a transmitted light dark field image with a phase contrast image.

18. The use as claimed in claim 1, wherein the reflector is embodied as a Fresnel prism, the Fresnel prism comprising several reflection surfaces with reflection surface normals and the reflection surface normals being inclined with respect to the plate normal.

19. A microscope for recording at least one transmitted light bright field image or transmitted light dark field image of at least one sample in at least one field of view, comprising a beam path comprising at least one illumination beam path and at least one imaging beam path, at least one light source for generating at least one beam, a plate-shaped reflector for deflecting the beam, the deflected beam being provided for illuminating the sample and the plate-shaped reflector having a plate normal and a substitute perpendicular, deviating from the plate normal, in respect of the illumination beam, at least one microscope objective for the imaging beam path, at least one image sensor.

20. A microscope for recording at least one image of at least one sample in at least one field of view, comprising a beam path comprising at least one illumination beam path and at least one imaging beam path, at least one light source for generating at least one illumination beam, a plate-shaped reflector for deflecting the illumination beam, the deflected illumination beam being provided for illuminating the sample, and the reflector being embodied as a retroreflector, at least one microscope objective for the imaging beam path, at least one image sensor, wherein the illumination beam is guided through the microscope objective before being deflected at the reflector.

21. The microscope as claimed in claim 19, wherein at least one second light source is present in addition to the first light source and a second illumination beam is able to be generated using the second light source and the second light source is operable independently of the first light source, and the plate-shaped reflector is moreover provided for deflecting the second beam the deflected second beam being provided for illuminating the sample.

22. The microscope as claimed in claim 19, wherein there is a focal plane which can be imaged on the image sensor in focus, and the illumination beam has an intersection with the focal plane in the beam path before the deflection at the reflector and the intersection contains the field of view.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0083] The present invention will become more fully understood from the detailed description given hereinbelow and the accompanying drawings which are given by way of illustration only, and thus, are not limitive of the present invention, and wherein:

[0084] FIG. 1 shows a first exemplary embodiment.

[0085] FIG. 2 shows a second exemplary embodiment.

[0086] FIG. 3 shows a third exemplary embodiment.

[0087] FIG. 4 shows a fourth exemplary embodiment.

[0088] FIG. 5 shows a fifth and a sixth exemplary embodiment.

[0089] FIG. 6 shows a seventh exemplary embodiment.

[0090] FIG. 7 shows a first embodiment of the illumination beam path in a sectional plane.

[0091] FIG. 8 shows a second embodiment of the illumination beam path in a sectional plane.

[0092] FIG. 9 shows a light source.

[0093] FIG. 10 shows an eighth exemplary embodiment.

[0094] FIG. 11 shows a ninth exemplary embodiment.

[0095] FIG. 12 shows a tenth exemplary embodiment.

[0096] FIG. 13 shows a beam deflection at an element of a microprism array.

[0097] FIG. 14 shows a section from a reflector.

DETAILED DESCRIPTION

[0098] FIG. 1 shows a first exemplary embodiment. An apparatus for recording at least one microscopic image 1, which apparatus can also be referred to as a microscope, is shown. The optical axis 2 of the microscope objective lies in the z-direction. A sample 7 with transparent and/or semitransparent objects 9 is situated on a sample carrier 10. The microscope comprises a microscope objective 20 with an optical axis 2. The light source 17 is arranged slightly in front of the pupil plane 22. The pupil plane is the xy-plane in which the stop 21, which can also be referred to as the pupil, is situated. This arrangement of the light source causes a slight divergence of the beam 3. Alternatively, the light source can be arranged in the pupil plane in order to generate a parallel beam (not shown).

[0099] The plane 16 is the focal plane, in which objects are imaged on the image sensor 25 in focus. The focal plane simultaneously is the sample plane 8 in which the sample is arranged.

[0100] The beam 3 is guided through the sample plane 8 to a plate-shaped reflector 11. The plate-shaped reflector 11 has a plate normal 12 and a substitute perpendicular 14, deviating from the plate normal, in respect of the illumination beam 3. The reflector 11 is embodied as a Fresnel prism. The Fresnel prism comprises a plurality of reflection surfaces 13 with reflection surface normals 14. The reflection surface normals are inclined with respect to the plate normal 12. The reflection surface normals each are the incidence perpendicular of an incident ray. The incidence perpendicular corresponds to the substitute perpendicular of the reflector. The reflector has a periodic structure with a period 29 in the x-direction. Each period 29 comprises a reflection surface. The steep flanks between the reflection surfaces 13, however, are not intended for reflection.

[0101] The deflection of the beam 3 by the reflector 11 is also shown. The deflected beam 4 has a central ray 5. The sample is illuminated with the deflected beam 4.

[0102] In addition, a camera 23 is shown, which comprises a camera lens 24 and an image sensor 25. One or more microscopic images of the sample can be recorded with an image sensor 25. In order to clarify the imaging beam path, light rays 6 from the object are shown here.

[0103] The illumination shown in FIG. 1 is a bright field transmitted light illumination. The direction of gravity here is the z-direction. The sample is therefore illuminated from below and observed from below.

[0104] FIG. 2 shows a second exemplary embodiment. Here, in contrast to the first exemplary embodiment, the reflector is embodied as something approximating a retroreflector in relation to the beam 3. It is characteristic that the incident light rays are almost reflected back into themselves. The other reference signs correspond to those of the first exemplary embodiment. The illumination shown in FIG. 2 is a combined reflected light and transmitted light dark field illumination.

[0105] FIG. 3 shows a third exemplary embodiment. In contrast to the above exemplary embodiments, the illumination beam 3 is guided past the objective 20 on the outside in this case. The light source 17 here comprises a dedicated collimation apparatus (not shown) for generating a parallel beam. On the basis of this exemplary embodiment, the intersection 26 of the beam 3, i.e., of the beam before the deflection, with the focal plane 16, which also lies in the sample plane 8 in this case, is also shown. The field of view 27 is also shown. The intersection 26 lies outside the field of view 27 in this case. This is a transmitted light dark field illumination. The reflector is embodied in such a way that a rising flank 30 and a falling flank 31 with respect to the z-direction are present in each period. Only the longer rising flanks are used as reflection surfaces 13. The other reference signs correspond to those of the previous exemplary embodiments.

[0106] FIG. 4 shows a fourth exemplary embodiment. This is a transmitted light bright field illumination, the illumination beam 3 being guided past the objective 20 on the outside. The other reference signs correspond to those of the previous exemplary embodiments.

[0107] FIG. 5 shows a fifth and a sixth exemplary embodiment. In the fifth exemplary embodiment, a first light source 17.a is provided for generating the first beam 3.a. The reflector 11 has V-grooves, a flank 30 rising with respect to the z-direction and a falling flank 31 of the same length being present in each period 29. Both flanks are used as reflection surfaces 13. The shown rays of the beam 3.a are deflected by a first reflection 15.a at a reflection surface and a subsequent second reflection 15.b at another reflection surface. As a result, the substitute perpendicular 14 is dependent on the direction of the incident ray. The first substitute perpendicular 14.a here designates the substitute perpendicular of the central ray of the incident beam 3.a. The interaction of the two individual reflections creates the first deflected beam 4.a, with which the sample is illuminated under an oblique incidence of light. The roof angle 32 is selected here to be less than 90°. The other reference signs correspond to those of the previous exemplary embodiments. In a development of the sixth exemplary embodiment, three reflections (not shown) are provided for deflecting the beam. For this purpose, the reflector can be embodied as a microprism array, for example as a full cube or pyramidal triple microprism array.

[0108] In the sixth exemplary embodiment, a second light source 17.b is additionally provided, which can be operated independently of the first light source 17.a. A first image is recorded with the first light source switched on. Then the first light source is switched off and the second light source is switched on. Since the substitute perpendicular depends on the direction of incidence of the light, the reflector 11 now has a second substitute perpendicular 14.b and a second incident beam 3.b is deflected into a second deflected beam 4.b and illuminates the sample from a different direction than the first deflected beam 4.a. A second image of the sample is then recorded under this illumination. A difference image can be calculated from these two images, in which the contrasts of the observed objects can be improved.

[0109] FIG. 6 shows a seventh exemplary embodiment. A plurality of microscopic images of a plurality of samples 7a-c are recorded here. For this purpose, use is made of a scanner unit 33, which carries a microscope camera 23, the objective 20 and the light source 17. This scanner unit is arranged so as to be displaceable 34 in an xy-plane below the samples. The microscope camera comprises the image sensor 25 and a camera lens 24. The light source 17 is fixedly arranged with respect to the microscope camera.

[0110] A displacement 34 of the scanner unit 33 with respect to the samples 7 is provided in each case from the recording of one image to the recording of the next image. The reflector 11 is fixedly arranged with respect to the samples.

[0111] FIG. 7 shows a first embodiment of the illumination beam path in a sectional plane. The sectional plane is the focal plane 16 in this case. The intersection 26 of the incident beam with the focal plane, the field of view 27 and the sample area 28 illuminated by the deflected beam are shown here. It is evident that this is a combined incident light and transmitted light illumination. Such an illumination is shown in the second exemplary embodiment in FIG. 2.

[0112] FIG. 8 shows a second embodiment of the illumination beam path in a sectional plane. The sectional plane is the focal plane 16 in this case. The intersection 26 of the incident beam with the focal plane located outside of the field of view, the field of view 27 and the sample area 28 illuminated by the deflected beam are shown here. It is evident that this is a transmitted light illumination.

[0113] FIG. 9 shows a light source. The light source is an LED 17. A diffuser 18 and light source stops 19 are situated in front of the light-emitting surface. A homogeneous directional distribution of the illumination light over a limited area can thus be achieved.

[0114] FIG. 10 shows an eighth exemplary embodiment. Here, the reflector 11 is embodied as a retroreflector. When the sample 7 is transilluminated, the beam 3 of the illumination is split into a plurality of beams. This can arise from differences in the refractive index in the sample and/or the refraction at a curved sample back side 35. A first 3.a, a second 3.b and a third beam 3.c are illustrated. These are incident on the retroreflector 11 from different directions. Each beam is reflected back against the direction of incidence at the retroreflector; specifically, the first beam 3.a is reflected back into the first deflected beam 4.a, the second beam 3.b is reflected back into the second beam 4.b and the third beam 3.c is reflected back into the third deflected beam 4.c. Substitute perpendiculars can be assigned to the individual beams here, the direction of which corresponds to the respective emergent ray. The first substitute perpendicular 14.a corresponds to the first deflected beam 4.a, the second substitute perpendicular 14.b corresponds to the second deflected beam 4.b and the third substitute perpendicular 14.c corresponds to the third deflected beam 4.c. The description of the beam path by means of the substitute perpendiculars is redundant in the case of a retroreflector, since the direction of the deflected ray counter to the incident ray is already clearly described by the function of the retroreflector. The beams are deflected by a plurality of reflections; a first reflection 15.a and a second reflection 15.b are given by way of example. In the advantageous embodiment of the reflector as a microprism array, three reflections are provided for deflecting each ray. This can result in a beam offset between the incident and emergent rays. The maximum beam offset is smaller, the smaller the selected period 29, i.e., the structure size of the retroreflector. The extent of the prisms in the case of a prism array or the diameters of glass spheres in the case of embedded glass spheres can be considered as the structure size here. In order to advantageously minimize the beam offset, the use of microprism arrays or the smallest possible embedded glass spheres for the retroreflector can be advantageous. This is because the deflected beams 4.a, 4.b, 4.c are to impinge on the sample back side as close as possible to the beams 3.a, 3.b and 3.c, respectively. Then they can be diffracted in the opposite way to the latter. In this way, transmitted light illumination can be achieved which is parallel or divergent in the same way as the incident beam of the illumination 3. The illustration shows an axially parallel bright field illumination, i.e., the beam of the illumination 3 runs parallel to the optical axis 2. In a development of the exemplary embodiment, not shown, oblique bright field illumination can be provided. In the case of the latter, the beam of the illumination 3 runs at an angle to the optical axis 2. In this exemplary embodiment, the illumination beam is reflected in with a partially transmissive mirror 38.

[0115] The illustration also shows an optional configuration for recording a Hoffman modulation contrast image. This optional configuration comprises a modulator 37. The latter comprises three segments of different optical attenuation, which are indicated by dashed lines of different widths. This modulator is normally provided for the observation beam path (not shown). The illumination light is also passed through the modulator in this case. The optional configuration also includes a slit stop (slotted stop) 19. Said stop can be fixed or rotatable and/or displaceable. This stop is partially covered by a polarizer, which is shown in dashed lines. In addition, a further polarizer 39 can optionally be provided, which acts on the entire illumination beam used. The latter can be rotatable.

[0116] FIG. 11 shows a ninth exemplary embodiment. In contrast to the aforementioned exemplary embodiment, oblique dark field illumination is provided in this case. A retroreflector 11 is also used in this example. In a development of this exemplary embodiment, provision is made of a second light source (not shown), which can be operated independently of the first light source 17. A respective partial image can then be recorded with one light source switched-on in each case and the microscopic image can be created as a difference image of the two partial images.

[0117] FIG. 12 shows a tenth exemplary embodiment. A phase plate 36, which is embodied as a phase ring, is provided in this case. A first illumination beam path 3, which emanates from a first point light source 17.a, is shown. In addition, further point light sources can be specified, for example a second point light source 17.b in the sectional image shown. In this exemplary embodiment, a ring-shaped illumination is provided, which can be viewed as a plurality of light sources arranged in a ring. The individual light sources 17.a, 17.b can be fed by a single light source 17. As a result, the ring-shaped illumination can be coherent in order to achieve a phase contrast recording as a microscopic image. In this exemplary embodiment, the illumination beam is reflected in with a partially transmissive mirror 38.

[0118] In an alternative development of this exemplary embodiment, the phase plate is omitted and one or more bright field recordings of the sample are recorded with oblique illumination.

[0119] FIG. 13 shows a beam deflection at an element of a microprism array. Each beam is deflected by a first 15.a, a second 15.b and a third reflection 15.c at one of the surfaces of the microprism 40 in each case.

[0120] FIG. 14 shows a section from a reflector. The reflector is a retroreflector, which is embodied as a full cube microprism array in this exemplary embodiment. The microprism array includes many tri-faceted microprisms 40. Such retroreflectors can be used in the above exemplary embodiments.

[0121] The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the invention, and all such modifications as would be obvious to one skilled in the art are to be included within the scope of the following claims.