阅读说明：本技术 用于操纵显微镜中的光路的方法和装置、用于摄取显微镜中的图像堆栈的方法 (Method and device for manipulating a beam path in a microscope, method for capturing a stack of images in a microscope ) 是由 弗罗里安·法尔巴赫 于 2019-03-18 设计创作，主要内容包括：本发明涉及一种用于操纵在显微镜(1)中特别是在光片显微镜(5)中的至少一个光路(8)的方法、一种用于在显微镜(1)中特别是在光片显微镜(5)中对图像堆栈(224)摄像的方法、一种用于操纵在显微镜(1)中特别是在光片显微镜(5)中的至少一个光路(8)的装置和一种非易失性的计算机可读的存储介质(163)。现有技术的方案具有如下缺点：不能利用任何浸渍介质(23)观察样本(21),并且该方法附加地受限于数值孔径(NA)较小的光学设备(9)。根据本发明的方法通过如下方法步骤改善了现有技术的方案：-求取布置在样本空间(17)中的样本(21)的和/或布置在样本空间(17)中的光学介质(35)的折射系数(n)；和-根据所求得的折射系数(n)来调节至少一个显微镜参数(2),用于操纵所述光路(8)。(The invention relates to a method for actuating at least one beam path (8) in a microscope (1), in particular in a light sheet microscope (5), to a method for imaging an image stack (224) in a microscope (1), in particular in a light sheet microscope (5), to a device for actuating at least one beam path (8) in a microscope (1), in particular in a light sheet microscope (5), and to a non-volatile computer-readable storage medium (163). The prior art solutions have the following drawbacks: the specimen (21) cannot be observed with any immersion medium (23), and the method is additionally limited to optical devices (9) with a small Numerical Aperture (NA). The method according to the invention improves the prior art solutions by the following method steps: -determining a refractive index (n) of a sample (21) arranged in the sample space (17) and/or of an optical medium (35) arranged in the sample space (17); and-adjusting at least one microscope parameter (2) for manipulating the beam path (8) as a function of the determined refractive index (n).)

1. Method for manipulating at least one beam path (8) in a microscope (1), in particular in a light sheet microscope (5), comprising the following method steps:

-determining a refractive index (n) of a sample (21) arranged in the sample space (17) and/or of an optical medium (35) arranged in the sample space (17); and

-adjusting at least one microscope parameter (2) for manipulating the light path (8) as a function of the determined refractive index (n).

2. Method according to claim 1, wherein the determination of the refractive index (n) of the sample (21) arranged in the sample space (17) and/or of the optical medium (35) arranged in the sample space (17) comprises a method step of reading in a corresponding value of the refractive index (n) by a user.

3. Method according to claim 1 or 2, wherein the focal position is adjusted in accordance with the ascertained refractive index (n) in at least one of the following method steps:

-changing the effective focal length of at least one optical device (9); or

-moving at least one optical device (9) along its respective optical axis (53).

4. A process according to any one of claims 1 to 3, wherein the process comprises the process steps of:

-moving the sample (21), wherein, upon said movement, an interface (235) between an immersion medium (23) in which the sample (21) is immersed and a medium (39), such as air, located before the illumination objective (7) is moved; and

-adjusting at least one microscope parameter (2) for manipulating the light path (8) as a function of the determined refractive index (n).

5. The method as claimed in claim 4, wherein in the method the position of the focal spot (19) is kept at a constant position within the image field (237).

6. The method according to claim 4 or 5, wherein the method step of manipulating the detection beam path (190) is carried out for a focal position (19) of the detection beam path if an optical medium (35) having a refractive index (n) different from the sample medium (27) and/or the immersion medium (23) is present between the detection optics (41) and the sample (21).

7. The method according to any of claims 1 to 6, wherein spherical imaging errors are corrected by manipulating the optical path (8) according to the determined refractive index (n) by varying the optical path length (176) of the optical path (8) according to the distance (r) from the optical axis (53).

8. The method as recited in claim 7, wherein changing the optical path length (176) includes moving at least one mirror segment (178, 180) and/or moving an interface of a deformable transmissive medium.

9. A method according to claim 7 or 8, wherein r is substantially according to a functional relationship²And r⁴Is superposed-r²And r⁴Are determined to change the optical path length (176), where r is equal to the distance from the optical axis (53).

10. The method according to any of claims 1 to 9, wherein the method steers the light path (8) according to wavelength by the following method steps:

(A) a method according to any of claims 1 to 5, manipulating the optical path (8) of the light (11) of the first wavelength; and

(B) at least one other wavelength is tuned.

11. The method according to claim 10, wherein the method step of adjusting at least one further wavelength comprises, according to the method of any one of claims 1 to 9, sequentially manipulating the optical path (8) of the light (11) of the further wavelength accordingly.

12. The method of any one of claims 1 to 11, wherein the method is repeated at set time intervals.

13. Method for imaging an image stack (224) in a microscope (1), in particular in a light sheet microscope (5), wherein the method comprises the following method steps:

-implementing a method for manipulating at least one light path according to any one of claims 1 to 12; and

-detecting a change in position of the sample (21) relative to the optical device (9), and/or detecting a change in the excitation wavelength (239) before adjusting the at least one microscope parameter (2).

14. Method for imaging an image stack (224) in a microscope (1), in particular in a light sheet microscope (5), wherein the method comprises the following method steps:

(8a) moving the focal plane (19) of the first optical device (9b) by a preset scanning distance (229);

(8b) according to one embodiment of the invention, the method for manipulating the beam path (8) in the microscope (1) according to one of claims 1 to 12, at least one beam path (8) of the first optical device (9b) is manipulated for correcting an imaging error of the first optical device (9 b); and

(8c) the focal plane (19) of the second optical device (9c) is moved or follows a following path (233) which is dependent on the refractive index (n) determined in method step (8 b).

15. The method of claim 13 or 14, comprising capturing and/or storing images (225) for producing an image stack (224).

16. The method according to claim 14 or 15, wherein method step (8c) comprises, according to one design of the method for steering the optical path (8) according to one of claims 1 to 12 of the present invention, steering at least one optical path (8) of the second optical device (9c) for correcting an imaging error of the second optical device (9 c).

17. The method of any one of claims 14 to 16, comprising:

-performing method steps (8a), (8b) and (8c) once; and

-repeating the method steps (8a), (8b) 'and (8c) n times, wherein the method step (8b)' only has the method step of adjusting at least one microscope parameter (2) as a function of the determined refractive index (n) in order to steer the beam path (2).

18. A method according to any of claims 13-17, further comprising the method step of detecting a trigger signal (241) for starting to perform a further method step.

19. The method according to any of claims 1 to 18, wherein the determination of the refractive index (n) of the sample (21) and/or of an optical medium (35) arranged in the sample space (17) comprises the method steps of:

(i) Focusing measuring light (65) into the sample space (17) by means of an optical device (9), wherein the measuring light (65) is transmitted through the optical medium (35) and a further optical medium (9) on a sample side (15) of the optical device (9);

(ii) detecting measuring light (65b) reflected by the reflection component (49) and transmitted through the other optical device or said optical device, with a detection device (57) or with a position-resolved detector (58);

(iii) determining a working distance (79) between the optical device (9) and the reflective component (49) based on the measurement light (65) detected by the detector (63), wherein for the working distance (79) a focal point (19) of the measurement light (65) is located on the reflective component (49);

(iv) varying at least one of the following parameters:

(iv.1) a distance between the optical device and the sample medium (117);

(iv.2) a distance between the reflector and the further optical medium (113);

(iv.3) the divergence of the measurement light (65),

wherein the change results in a determined distance change (121) of the focal position (123) of the measurement light (65);

(v) (iv) determining a further working distance (127) according to method steps (i) to (iii);

(vi) determining a working distance change (129) between the working distance (79) and the further working distance (127); and

(vii) The refractive index (n) is determined on the basis of the distance change (121) and the working distance change (129).

20. The method according to one of claims 1 to 19, wherein the determination of the refractive index (n) of the sample (21) and/or of the optical medium (35) arranged in the sample space (17) further comprises the method steps of:

(I) -injecting measurement light (65) into the sample space (17) obliquely with respect to the optical axis (53) by means of an optical device (9);

(II) causing incident light (11) to be reflected on a reflective member (49) provided at a first location (207) in the sample space (17);

(III) imaging the reflected light (65b) onto a position-resolved detector (58);

(IV) analyzing the signal detected by the detector (58) on the position-resolved detector (58) for the magnitude and/or offset of the reflected light (65 b);

(V) moving the reflective component (49) to a second position (208) along the optical axis (53) and performing method steps (III) and (IV); and

(VI) for a first position (207) and a second position (208) of the reflective member (49), the refractive index (n) is found based on a magnitude and/or a shift of the reflected light (65 b).

21. The method according to any of claims 1 to 20, further comprising reading out calibration data (188), wherein at least one microscope parameter (2) is adjusted depending on the ascertained refractive index (n) and/or depending on the read-out calibration data (188).

22. A non-transitory computer-readable storage medium (163) comprising a program for implementing the method according to any one of claims 1 to 21.

23. An apparatus for manipulating at least one light path (8) in a microscope (1), in particular in a light sheet microscope (5), comprising:

-a refractive index finding module (56) for finding the refractive index (n) of the sample (21) and/or for finding the refractive index (n) of an optical medium (35) arranged in the sample space (17); and

-at least one optical path manipulator (170) for adjusting at least one microscope parameter (2) based on the determined refractive index (n) in order to manipulate at least one optical path (8).

24. The apparatus of claim 23, wherein the optical path manipulator (170) comprises at least one component of the group consisting of:

-an optical component having an electrically adjustable focal length (182);

-an actuator module (87);

-a corrective ring (184);

-a correction plate module for introducing a correction plate into the light path;

-an optical device (9) equipped with a corrective ring (184);

-a deformable mirror (172);

a hollow part filled with a transparent liquid medium, with at least one transparent entrance face and/or exit face, wherein the at least one transparent entrance face and/or exit face is deformable.

25. The device according to claim 23 or 24, characterized by a timer module which outputs a start signal at set time intervals for starting the measurement of the refractive index (n) and/or the manipulation of the light path (8).

Technical Field

The invention relates to a method for actuating at least one beam path in a microscope, in particular in a light-sheet microscope, a method for recording an image stack in a microscope, in particular in a light-sheet microscope, a device for actuating at least one beam path in a microscope, in particular in a light-sheet microscope, as a function of the refractive index, and a non-volatile computer-readable storage medium.

Background

In the prior art, so-called decontamination methods are known which allow the sample to be held in an immersion medium, wherein the immersion medium can be adapted in terms of its refractive index to the sample to be examined or can homogenize the refractive index of the sample. The latter method does not lead to the refractive index of the sample also being adapted to the refractive index of the medium. A number (more than 20) of such impregnation media are known, which may be based on ethanol or sugar. With this method, live samples cannot be examined because these have undergone a large chemical change, for example by removal of fat.

On the other hand, there have been efforts to implant living cells and tissues into a medium having a refractive index equal to that of the object to be examined. In addition to their different refractive indices, the respective solvents are used in different mixing ratios in the various immersion media. This results in large changes, in particular uncertainties, in the refractive index of the immersion medium which has just been used.

The refractive index of the immersion medium is primarily responsible for the occurrence of aberrations, in particular defocus and spherical aberration. These refractive index related effects can degrade the photomicrograph.

If a so-called light sheet microscope is used for examining the specimen in the immersion medium, the refractive index which is not taken into account can lead to the focal plane of the detection optics being displaced over a distance by a difference in refraction due to the refractive index of the immersion medium and/or of the specimen, with the result that the focal plane can no longer overlap with the light sheet produced by the illumination objective (i.e. a two-dimensional illumination plane). That is, defocus in a light sheet microscope can cause significant quality loss of the captured image.

For this reason, iterative and image-based algorithms or methods based on these algorithms are known from the prior art, which modify at least one microscope parameter on the basis of the image quality, so that aberrations which may occur can be compensated for in an approximate manner.

In the prior art purification method, it is preventively preferable to use an objective lens with a small Numerical Aperture (NA), because the above-mentioned aberrations are negligible for these small NA objective lenses. The aberrations are also negligible in confocal microscopy methods, which use, for example, a single objective lens, so that both the illumination beam path and the detection beam path are subjected to aberrations in the same "direction", i.e., with the same sign. It is however desirable that also objectives with large NA can be used, in order to achieve a higher resolution with these objectives and to detect the light from the sample more efficiently.

Disclosure of Invention

The object of the invention is thus to be able to observe a sample in any immersion medium and to use an objective lens with a large NA in the observation.

The above object is achieved by a method according to the invention for actuating at least one beam path, comprising the following method steps:

-determining a refractive index of a sample arranged in the sample space and/or of an optical medium arranged in the sample space; and

adjusting at least one microscope parameter for manipulating the beam path as a function of the determined refractive index.

The object mentioned at the outset is achieved by a method for imaging an image stack in a microscope, comprising the following method steps: determining a refractive index of a sample arranged in the sample space and/or of an optical medium arranged in the sample space; -detecting a change in the position of the sample relative to the optical device, and/or detecting a change in the excitation wavelength before adjusting at least one microscope parameter; and adjusting at least one microscope parameter for manipulating the beam path as a function of the determined refractive index.

The object mentioned at the outset is achieved by an apparatus according to the invention in that the apparatus comprises: a refractive index finding module for finding the refractive index of the sample and/or finding the refractive index of an optical medium arranged in the sample space; and at least one optical path manipulator for adjusting at least one microscope parameter based on the determined refractive index in order to manipulate at least one optical path.

The object is further achieved in that the non-volatile computer-readable storage medium mentioned in the opening paragraph contains a program for implementing the method according to the invention.

The method according to the invention, the device according to the invention and the non-volatile computer-readable storage medium according to the invention for carrying out the method thus have the following advantages: they enable rapid, sample-preserving, deterministic and non-iterative manipulation of at least one optical path of the microscope based on the refractive index of the sample and/or immersion medium.

The method according to the invention, the device according to the invention and the non-volatile computer-readable storage medium according to the invention can each be further improved by special designs. The individual features of the inventive design can be combined and/or omitted here as desired, without depending on the technical effect achieved with the omitted features.

The method according to the invention can be used for the nearly arbitrary manipulation of the at least one optical path, but in particular also for compensating at least one imaging error introduced by the sample and/or the optical medium.

The above-mentioned method according to the invention, device according to the invention and storage medium according to the invention have the following advantages compared to prior art solutions: the refractive index of the sample and/or of the immersion medium surrounding the sample can be measured automatically and can be determined quantitatively on the basis of the measured refractive index, i.e. decisively, not on the basis of an iterative method, in particular without the need for a picture of the image. For this purpose, the sample does not need to be arranged in the sample space, so that the method according to the invention or the device according to the invention can protect the sample. Furthermore, the method according to the invention is contactless.

An immersion medium refers to a medium which can be located in the sample space, in particular surrounding the sample in the sample space. The immersion medium can likewise be located in the region between the specimen and the front lens of the respective objective.

The beam path to be manipulated, in particular of a light sheet microscope, may be, for example, an illumination beam path and/or a detection beam path. The two beam paths for illumination and detection are preferably manipulated, in particular the aberrations occurring can be compensated for in the respective beam path.

Optical media refer to the following materials: which is transparent to the wavelength or wavelengths employed in the microscope and has optical properties specific to that wavelength such as refractive index and dispersion. The optical medium may particularly and without limitation include the above mentioned ethanol or sugar based immersion medium, but may also include water, glycerol and air. The method according to the invention or the device according to the invention can therefore also be used without immersion medium (objective lens located in air).

The determination of the refractive index of the sample arranged in the sample space and/or of the optical medium arranged in the sample space comprises method steps in which the respective value is entered by the user, whereby a simple design of the method according to the invention can be achieved. In this embodiment, the user can select the refractive index from a selection list of the specified immersion medium, for example, but can also enter the refractive index specified by the user. The adaptation to precisely this immersion medium can thus be accelerated when repeatedly measuring with the immersion medium that has been used in advance and is therefore known.

In another design, a method for imaging an image stack may include the steps of: (8a) moving a focal plane of a first optical device by a preset scanning distance; (8b) according to one embodiment of the method for manipulating an optical path in a microscope, at least one optical path of a first optical device is manipulated for correcting an imaging error of the first optical device; and (8c) moving or following the focal plane of the second optical device by a following path which is dependent on the refractive index determined in method step (8 b).

In an advantageous embodiment of the method, the method may comprise capturing and/or storing images for producing an image stack. In particular, n images may be captured and/or stored, which form an image stack. Preferably, all images of the image stack are recorded with the imaging errors corrected, i.e. after the corresponding beam path has been steered.

In a further embodiment, according to one embodiment of the method according to the invention for manipulating an optical path in a microscope, in method step (8c), a step of manipulating at least one optical path of the second optical device is additionally carried out for correcting an imaging error of the second optical device, as a result of which the aforementioned method for imaging an image stack can be improved. Both the illumination beam path and the detection beam path can thus be adapted to the unknown medium, in particular the refractive index of the medium, in the microscope.

In a further possible embodiment of the method according to the invention for imaging an image stack, method steps (8a), (8b) and (8c) can be repeated in a modified manner. It is thus possible that in method step (8b) of manipulating at least one optical path of the first optical device, in a second execution of method steps (8a) - (8c), the refractive index is not re-found. The modified method step (8b)' may therefore only have a method step for adjusting at least one microscope parameter for manipulating the beam path as a function of the determined refractive index. In this case, the refractive index is determined in method step (8b) when it is first performed. This has the following advantages: in the case of imaging an image stack, the refractive index determination is carried out only for the first image to be imaged, while the other images of the image stack do not have to be determined again for the refractive index (which has already been determined). This may speed up the capturing of the image stack.

According to method step (8c), preferably n image acquisitions are performed, wherein n denotes the total number of images of the image stack.

In particular, after the execution of the method steps (8a), (8b) and (8c), the method steps (8a), (8b)' and (8c) are repeated (n times) at arbitrary frequencies, depending on the desired number of images of the image stack. Here, it is not necessary to repeatedly find the refractive index before each image capturing.

In a further embodiment of the method for imaging an image stack, however, method steps (8a), (8b) and (8c) can be carried out in each case at any frequency, depending on the number of images to be imaged which has been determined in advance, wherein the image acquisition can be carried out in each case after method step (8 c). Such a design of the method according to the invention may preferably be applied in samples having a large (or detectable) refractive index gradient. Possible imaging errors can thus be corrected for each imaged plane.

If the refractive index variation in the space containing the image stack is negligible, a single refractive index determination in method step (8b) may speed up the method, since the refractive index determination does not have to be repeated for each subsequent image acquisition.

In a further embodiment of the method according to the invention for imaging a stack of images, the refractive index can be detected as soon as the user has loaded the sample into the microscope before the first detection of the image. Different triggers, for example computer-initiated triggers, may be considered here, once a new project is created by the user. The measurement can also be triggered manually. This may particularly preferably be done before the user views the preview images that are required to be oriented in the sample and measured based on the preview images (i.e. camera the image stack). That is, the correction is performed during the orientation phase, i.e. already before the actual measurement (imaging of the image stack). The refractive index values determined in the orientation phase can be used in a method for imaging an image stack, so that a separate determination of the refractive index is no longer necessary in the design of the method.

The method thus comprises method steps of determining and storing the refractive index, wherein the refractive index may be determined and/or stored for different excitation and/or excitation wavelengths. These stored values of the refraction index, advantageously in combination with the calibration data stored for the optical device, have the following advantages when measured:

the focal point of the illumination optics remains unchanged, for example when the position of the imaging plane relative to the sample is changed, or when the excitation wavelength is changed;

-if there is a sudden change in the refractive index in the probe beam path, imaging the image stack with the spatial distance between the images captured corrected;

-compensating for residual errors when using an optical device with a corrective ring; and

-correcting the imaging errors mentioned previously.

Such a design of a method for imaging an image stack may thus comprise the following method steps:

b1 measuring and/or storing the refractive index;

b2 detecting changes in the position of the sample relative to the optical device (e.g. illumination and/or detection optics) and/or detecting changes in the excitation wavelength, reading out the measured values stored in method step (b1) and/or previously stored calibration data; and

b3 adjusting at least one microscope parameter for manipulating the beam path according to the determined refractive index.

Method step (b2) may preferably be performed simultaneously with one of the aforementioned changes, but may also be performed after the change has been made.

In a further embodiment of the method according to the invention, the method can adjust the focal position as a function of the ascertained refractive index in at least one of the following method steps: -changing the effective focal length of at least one objective lens; or-moving at least one objective along its respective optical axis.

It is thus possible to move the sample by means of the method according to the invention, wherein during said movement the interface between the immersion medium in which the sample is immersed and the medium, for example air, located in front of the illumination optics or the illumination objective is also moved. In a second step, the illumination beam path can be manipulated with respect to its focal position and the imaging deviations that occur in some cases can be corrected. The position of the focal spot can in this case be kept particularly preferably at a constant position within the image field. By changing the position of the interface, the corrected imaging error can change, resulting in a need for re-correction using this method.

If there are also media with different refractive indices between the detection optics and the sample (for example, if the sample is arranged in a cuvette between the illumination optics and the detection optics), the method can be adapted to the further interface in such a way that the detection beam path can additionally be manipulated with respect to its focal point, wherein this manipulation of the detection beam path can also include correcting imaging errors of the detection optics.

This design has the following advantages: manipulation of the focal position may be used to adapt the microscope to the refractive index of the sample itself and/or to the immersion medium surrounding the sample. In particular aberrations caused by unknown refractive indices can be compensated. In this way, the focal position in the light sheet microscope is particularly advantageously adjusted, since in the light sheet microscope it is advantageously ensured that the focal plane of the detection objective and the illumination plane of the illumination objective can overlap, so that a clear image of the two-dimensional region illuminated by the light sheet can be obtained.

The effective focal length and the focal position of the at least one objective are thus possible microscope parameters. At least one of these parameters can be set in the respective device by means of the beam path manipulator.

In a further embodiment of the method according to the invention, the spherical imaging error can be corrected by changing the optical path length of the optical path as a function of the distance from the optical axis by manipulating the optical path as a function of the determined refractive index.

Imaging aberrations of the sphere occur especially for non-paraxial light of the beam and increase with distance from the optical axis. Thus, with this design it is possible to use a volume of about 1cm ³And sample sizes above, almost completely illuminate the optical system and use its aperture.

In a further embodiment of the method according to the invention, this spherical imaging deviation can also be compensated only partially or even overcompensated.

In a special design of the aforementioned method according to the invention, the changing of the optical path distance may comprise moving at least one mirror segment and/or moving an interface of the deformable transmissive medium.

In particular, in a further special design, the functional relation r can be taken into account²And r⁴Is superposed-r²And r⁴Are determined-to change the optical path distance, where r is equal to the distance from the optical axis.

In other words, in the method according to the invention, the optical path length can be increased or decreased as a function of the distance from the optical axis (i.e. as a function of r) (that is to say the sum of the respective geometric partial distances is multiplied by the respective refractive index occurring along the respective partial distance).

The change in the optical path length can be expressed by Δ x, and in particular by the mathematical expression Δ x ═ a × r²+B*r⁴To describe. Here, r is the distance from the optical axis in the pupil of the optical system under consideration, and a or B is a freely selectable antecedent factor which allows weighting of squared or quadratically (doubly squared) portions. The optical path distance variation described by this mathematical expression can thus be used to compensate for spherical aberration occurring in media and/or samples of unknown refractive index. The third-order spherical aberration (when the aberration is described by means of Zernicke-polynomials) is described by means of a squared or a fourth-power component. Said aberrations can be compensated with the method according to the invention or the device according to the invention of this design.

In a corresponding design of the device according to the invention, the optical path manipulator may comprise at least one component of the following group: (a) an optical component having an electrically adjustable focal length; (b) an actuator module for moving the at least one optical device; (c) a correction ring; (d) a correction plate module for introducing a correction plate into the optical path; (e) an objective lens equipped with a corrective ring; (f) a deformable mirror; and (g) a hollow member filled with a transparent liquid medium, with at least one transparent entrance and/or exit surface, wherein the at least one transparent entrance and/or exit surface is deformable.

The components (a), (f) and (g) may be designed to vary the effective focal length of at least one objective lens. Component (b) may be designed to move at least one objective lens along its respective optical axis.

The components (e), (d) and (e) can be designed to apply spherical aberration to the optical path, that is to say, in particular, to correct an optical path which already has spherical aberration, with the opposite spherical aberration.

Components (f) and (g) may be used to both vary the optical path length as a function of distance from the optical axis and (additionally or alternatively) vary the effective focal length of the respective objective lens.

One special design of component (a) may be an Electrically Tunable Lens (ETL), and component (d) may in particular introduce into the optical path a correction plate for correcting spherical aberration and/or defocus.

The components (a), (b), (f) and (g) are variably adjustable, thus allowing flexibility in the application of the device according to the invention or of the method according to the invention. In particular, the component (g) can be used to deform the entrance surface and/or the exit surface appropriately, so that their radial thickness profile can be equal to the functional relation r²And r⁴A superposition of r²And r⁴The weights of (a) are determined well, respectively.

Since the components (a) to (g) described above differ both in the dynamic range (i.e. the range of optical path lengths that can be varied) and in their speed, different optical path manipulators can advantageously be combined with one another. In a possible embodiment, the basic imaging errors can thus be corrected slowly by means of the correction ring (c). In particular, a certain refractive index n can be corrected by means of the correction ring, wherein this slow correction takes place over a large dynamic range. A slow correction can be combined, for example, with a component (g) which, although not having the same dynamic range as the correction ring, can correct the residual error much faster. This may be advantageous, for example, when the correction ring does not take into account the dispersion of all the materials used and thus has residual errors that can be compensated for by the second component.

In an advantageous embodiment of the method according to the invention for imaging an image stack, the components described above can be advantageously applied to at least one, preferably all, method steps of the method for imaging an image stack. For example, in method step (8a), the focal plane of the detection optics can be moved by means of a deformable mirror, and the same deformable mirror is also used to manipulate the beam path of the detection optics in method step (8 b). Thus, the imaging error of the detection optics can be corrected. The following of the focal plane of the second optical device, in this case the illumination objective, can take place with a further deformable mirror. Alternatively, a tilting mirror may be used instead of another deformable mirror.

The method according to the invention can be further developed in that the beam path is manipulated as a function of the wavelength by the following method steps: (A) according to one of the aforementioned designs of the method of the invention, the optical path of the light of the first wavelength is manipulated; and (B) according to one of the aforementioned designs of the method of the invention, at least one other wavelength is adjusted and the optical path of the light of the other wavelength is correspondingly, sequentially manipulated.

In a variant of the method, it is possible in method step (B) to adjust only at least one further wavelength, so that for this further wavelength the beam path of the light of this further wavelength is also based on the refractive index measured for the first wavelength in method step (a).

It is also conceivable that a further embodiment of the method according to the invention comprises the following method steps: (a') finding a refractive index of a sample arranged in the sample space and/or an optical medium arranged in the sample space for light of the first wavelength; (B') adjusting at least one other wavelength and, for light of this second wavelength, correspondingly, sequentially finding the refractive index of the sample arranged in the sample space and/or of the optical medium arranged in the sample space; (C') according to one of the previously described designs of the method of the invention, the light paths of the light of the first wavelength and of the at least one further wavelength are manipulated.

This wavelength dependent correction can be performed using common wavelength independent correction components such as deformable mirrors or ETLs. If such a corrective component is used, the correction is generally not capable of simultaneously calibrating different color aberrations for multiple wavelengths, as opposed to using a corrective ring. In this case, the imaging can be carried out sequentially for different wavelengths, wherein significantly faster components such as deformable mirrors or ETLs can reduce the possible time loss of sequential imaging due to their switching times, which are, for example, significantly less than 10 ms.

It should be mentioned here that, for example, correction rings which allow adaptation to media with different refractive indices, it is also possible to correct only a certain dispersion for the different refractive indices, i.e. for water and a wavelength of light of about 500nm (green light), the dispersion n is 1.33, for glycerol, the dispersion n is 1.42, but not for substances with different dispersions. The number of substances with different refractive indices, whose dispersion can be corrected for the respective refractive index, is also limited, and for example, different dispersions of media cannot be corrected individually for n 1.33, n 1.37, n 1.41, n 1.45, etc.

In this method, in particular for wavelengths determined beforehand (by the user), the refractive index can be determined and, after the measurement, a wavelength-dependent correction or manipulation takes place in at least one optical path, for example the illumination and detection optical paths.

The method according to the invention can be further improved in that provision is made for the method to be repeated at set time intervals.

This can be advantageous in particular for the following samples and/or immersion media: which takes into account the change in refractive index over time, for example due to evaporation. With this design it is also possible to detect changes in the immersion medium of and/or around the specimen when observing a living specimen and to correct them accordingly.

In a corresponding embodiment of the device, a timer module can be provided which outputs a start signal at set time intervals for starting the measurement and/or the manipulation of the at least one beam path.

In a further embodiment of the method according to the invention, the determination of the refractive index of the sample and/or of the optical medium arranged in the sample space comprises the following method steps:

(i) focusing the measurement light into the sample space by means of an optical device, wherein the measurement light is transmitted through the optical medium and a further optical medium on a sample side of the optical device;

(ii) detecting, with a detection device, measurement light that is reflected by the reflection member and transmitted through another optical device or the optical device;

(iii) determining a working distance between the optical device and the reflective component based on the measuring light detected by the detector, wherein the focal point of the measuring light is located on the reflector for the working distance;

(iv) varying at least one of the following parameters:

the distance between the optical device and the sample medium;

the distance between the reflector and the further optical medium;

the divergence of the measurement light, wherein the change results in a determined distance change of the measurement light focus position;

(v) (iv) determining a further working distance according to method steps (i) to (iii);

(vi) calculating the working distance change between the working distance and the other working distance; and

(vii) the refractive index is found based on the distance change and the working distance change.

This design, in particular the design of the method steps for finding the refractive index of the sample, is advantageous because it allows the refractive index to be measured without the sample or scattering medium in the sample space.

According to the method of the invention, in order to determine the refractive index of the sample and/or of the optical medium arranged in the sample space, the following method steps can alternatively or additionally be included:

the method comprises the steps of enabling measuring light to enter a sample space obliquely relative to an optical axis by means of an optical device;

reflecting the incident light on a reflective member provided at a first location in the sample space;

imaging the reflected light onto a position-resolved detector;

analyzing the signals detected by the detector for the size and/or offset of the reflected light on a position-resolved detector;

moving the reflective member to a second position along the optical axis and performing method steps (iii) and (iv); and

And calculating the refractive index based on the magnitude and/or offset of the reflected light for the first and second positions of the reflective member.

If the above-described method is carried out only up to method step (d), the size of the focal spot on the position-resolved detector can already give an indication about the distance of the reflection component from the effective focal plane of the optical device. The distance is related to the refractive index of the medium and/or of the sample and also to the path traveled by the light in the medium and/or the sample.

If a medium, for example air, is present between the optical device and the reflecting member, the objective lens being coordinated with the refractive index of the medium, then the focal point lies on the optical axis when the reflecting member is located in the focal plane. Such measurements may be taken into account for calibration. Depending on the movement of the reflecting component, the focal spot is moved laterally on the position-resolved detector, which allows the refractive index to be determined.

A further advantageous embodiment of the method according to the invention may also comprise reading out calibration data, wherein at least one microscope parameter may be adjusted as a function of the ascertained refractive index and/or as a function of the read-out calibration data.

A corresponding design of the device according to the invention may thus comprise a memory module in which calibration data of at least one optical apparatus may be stored, wherein these calibration data may be recalled by the control unit.

If calibration data are stored in the method according to the invention or in the device according to the invention, it is known how large the spherical error (or defocus) of the optical device is in relation to the refractive index and the wavelength. The refractive index or spherical aberration stored as a corrective value may also be stored in accordance with the settings of the corrective element used, e.g. a corrective ring. It is thus possible to take the corrections of these parts into account when compensating. In addition to the data that can be obtained by means of the method according to the invention, however, these component-specific values can also be provided "on the factory side" therewith, since these values relate to the properties of the optical device that remain the same (apart from the wavelength-and/or refractive index-dependent dependence stored in the calibration data). These calibration data can in principle be provided for all optical components used in the optical system, i.e. for example in the microscope, i.e. stored in the memory unit.

The control unit of the device according to the invention can also be designed to automatically or manually measure the refractive index of the sample and/or of the immersion medium and to operate at least one beam path in the microscope as a function of the determined refractive index, so that the beam path is adapted to the determined refractive index. Particularly preferably, the optical path is manipulated to compensate for occurring aberrations such as defocus or spherical aberration. The control unit can thus be designed to determine the refractive index and to control the at least one beam path manipulator.

It is also possible that a personal computer which reads into the non-volatile storage medium controls the method steps of the method according to the invention and, for example, calculates the refractive index. Since microscopes of the prior art increasingly have computer-based control and/or analysis sections, the non-volatile computer-readable storage medium according to the invention is particularly advantageous since it allows retrofitting existing microscopes. Furthermore, in general, microscopes of the prior art already have a special design of possible optical path manipulators, such as actuators and correction rings, which can be used when carrying out the method according to the invention.

Drawings

The subject matter of the invention will also be described in detail with the aid of exemplary figures. Examples of advantageous designs of the invention are shown in the figures, in which the technical features of the respective designs can be combined with one another and/or omitted as desired, if not dependent on the technical effect achieved with the respective omitted technical features. For clarity, identical features and features of identical function are denoted by the same reference numerals.

FIG. 1 shows a prior art light sheet microscope;

FIG. 2 shows a device according to the invention;

Fig. 3 and 4 show a device according to the invention, in particular a first measuring method for determining the refractive index;

fig. 5 shows method steps for determining the refractive index according to a first measurement method;

FIG. 6 shows method steps for determining the refractive index according to a second measurement method;

FIGS. 7 and 8 show possible designs of the light path when determining the refractive index according to the second measurement method; and

fig. 9 and 10 illustrate a method for capturing an image stack.

Detailed Description

Fig. 1 shows a microscope 1, which is designed as a confocal microscope 3 or as a light sheet microscope 5. The microscope 1 comprises an optical device 9 designed as an illumination objective 7, which transmits illumination light 11 of an excitation wavelength 239 from an illumination side 13 of the illumination objective 7 along an optical path 8 to a sample side 15 of the illumination objective 7 and focuses the illumination light 11 in a sample space 17 shown with a dashed line. The optical path 8 of the illumination objective is an illumination optical path 8 a.

A focal spot 19 is formed inside a sample 21, wherein the sample 21 is located in a sample vessel 25 filled with an immersion liquid 23. The focal point 19 defines a focal plane, which is also designated by reference numeral 19.

The immersion liquid 23 can be understood as a sample medium 27 having a refractive index n. The refractive index n may also be synonymously referred to as the refractive index n.

The optical system 29, which comprises the optical device 9, the sample vessel 25 and the sample medium 27 contained therein, is influenced by the refractive index n of the sample medium 27 such that the spatial position 31 of the focal point 19 can be varied for different refractive indices n.

At the sample side 15 of the optical device 9, the illumination light 11 propagates through a free light space 33 shown with dashed lines.

In both the free light space 33 and the sample vessel 25, there is an optical medium 35, which in the example shown is air 37 in the free light space 33 and in the sample vessel 25 is the sample medium 27.

The air 37 in the free light space 33 is equivalent to having a refractive index n₁Of another optical medium 39. The sample medium 27 has a refractive index n₂The sample 21 having a refractive index n₃. All refractive indices n₁～n₃May be different from each other.

The microscope 1 shown in fig. 1 also comprises detection optics 41, which are known from the prior art and therefore are not described in detail.

In the adjusted state 43, the focal point 19 of the illumination light 11 is located exactly in the focal plane 45 of the detection optics 41 along the optical axis 53 and is centered in the image field along the illumination direction (parallel to the focal plane 45). Due to the change in the refractive index n, deviations from the adjusted state 43 occur, so that no clear imaging (not shown) can be achieved with the microscope 1.

In particular, the microscope 1 shown in fig. 1 can be used both as a confocal microscope 3 and as a light sheet microscope 5. For use as an optical sheet microscope 5 (which is illustrated in fig. 1), the microscope 1 has a reflection surface 47 of a reflection element 49 arranged in the sample space 17, wherein the reflection element 49 is arranged and fixed on a detection objective 51 of the detection optics 41. The reflecting member 49 thus forms a kind of reflector 55.

In fig. 1, the illumination light 11 is reflected on an inclined reflection surface 47, and in addition to this reflection surface, the reflection element 49 has a further reflection surface 47 which is oriented substantially perpendicularly to the optical axis 53 of the optical device 9 and of the detection objective 51. In the design of the microscope 1 shown in fig. 1, the optical axes 53 of the optical device 9 and of the detection objective 51 overlap, and in other designs can be arranged parallel to one another (see fig. 3 or 4).

Fig. 2 shows a schematic structure of a device 85 designed as a microscope 1 for manipulating at least one beam path 8.

Fig. 2 furthermore shows a PC 162 which serves to control the device 85 according to the invention designed as a microscope 1 and reads from a non-volatile computer-readable storage medium 163 and executes a program for carrying out the method according to the invention.

Instead of the PC 162, a microcomputer such as Arduino may also be used. Such a microcomputer (not shown) may be provided in addition to a PC for controlling the microscope, for example. A memory can be provided in the PC 162 or microcomputer, which memory contains calibration data of the optical device and/or of the correction element used and is supplied to the microscope for manipulation of the beam path.

In addition to the structure shown in fig. 1, the microscope also has a refractive index determination module 56, which is schematically illustrated in the form of a rectangle. The refractive index calculation module 56 may comprise a detection device 57, which in turn may comprise an aperture 59, for example in the form of a pinhole 61, and a detector 63.

In a second design of the refractive index determination module 56a (shown on the left in fig. 2), instead of the detection device 57, a position-resolved detector 58 can be used. In both cases, the measurement light 65b reflected back along the beam path 8 is focused by the lens 192 on the detector 63 or the spatially resolved detector 58.

It is to be noted that in fig. 3 and 4, a measurement light 65 is shown, which is referred to as an incident measurement light 65a, which is reflected at the reflection member 49 and reaches the refractive index calculation module 56 or 56a as the reflected measurement light 65 b.

The apparatus 85 of fig. 2 also has a plurality of optical path manipulators 170. These optical path manipulators are in particular deformable mirrors 172 which have (at least in some regions) variably adjustable bends 174 and, in particular, for the optical path 8 of the detection optics 41, it can be achieved that the optical path length 176 in the edge region 178 of the deformable mirror 172, i.e. the outer optical path length 176a, is reduced or increased in comparison to the optical path length 176 at the center 180 of the optical path 8, i.e. the central optical path length 176 b.

The device 85 of fig. 2 also includes an optical component 182, abbreviated ETL, with an electrically adjustable focal length. With the ETL182, it is possible to vary the effective focal length (not shown) of the detection optics 41 and thus to compensate for the refractive index n of the focal point 19 of the detection optics 41 and of the immersion liquid 23₂The associated offset (not shown).

The microscopic parameters 2, such as spherical aberration or effective focal length, can thus be adjusted by means of the optical path manipulator 170.

Both the detection optics 41 and the illumination objective 7 comprise a correction ring 184, which is only schematically illustrated as a rectangle in fig. 2.

The optical path manipulator 170 shown in fig. 2 may be arranged in different combinations in different designs of the device 85. That is, the deformable mirror 172, the ETL182, and the one or more corrective rings 184 are optional.

Although ETL 182 can substantially correct for the shift in focus 19, either deformable mirror 172 or corrective ring 184 can change optical path length 176, particularly as a function of distance r from optical axis 53.

Fig. 3 and 4 schematically show an apparatus 85 according to the invention, in particular a first measuring method for determining the refractive index n.

The device 85 comprises: an optical device 9 which can be translated 89 by means of the actuator module 87; a reflection element 49 which images the reflected measuring light 65b by means of the optical device 9 onto a measuring surface (not shown) of the detection device 57 or onto the position-resolved detector 58 (see fig. 2) when the reflection element 49 is at a working distance 79 from the optical device 9. In this design, the optical device 9 is identical to the further optical device 9 a.

The detection optics 41 shown have an image field 237 which is preferably substantially maintained, i.e. unchanged, even when the light path is manipulated.

By means of the at least one actuator module 87, the distance 93 between the reflector 55 and the focal point 19 of the measuring light 65 can be varied.

The schematic in fig. 3 shows the following: the focal point 19 of the measurement light 65 is spaced apart from the reflective member 49, and a distance 93 between the reflective member 49 and the focal point 19 of the measurement light 65 can be measured.

The device 85 further comprises an analysis unit 95, which is shown in detail only for the device 85 of fig. 4. The evaluation unit 95 is connected in data-transmitting manner, i.e. via a data line 97, to the detection device 57 or the position-resolving detector 58 (which is only schematically illustrated as a rectangle), to a working distance determination module 99 and to a refractive index module 101 for determining the refractive index n, wherein the refractive index module 101 is connected in data-transmitting manner to the actuator module 87 or actuator modules 87 and the working distance determination module 99, wherein the connection is centrally performed by a control unit 103. In other designs, the index module 101 is directly coupled to the actuator module 87.

The controller 103 may receive a trigger signal 241 which may be generated by a computer when creating a new item, in the case of loading a sample into a microscope, or manually by a user. The trigger signal 241 shown encoded can cause the method according to the invention to start.

The analysis unit 95 may be part of the refractive index calculation module 56.

The working distance determining module 99 is also connected to the actuator module 87 via the controller 103. The controller 103 may also be connected to at least one optical path manipulator 170 (see fig. 2) via at least one further manipulation output 186 in a data-transmitting manner. In the design shown, the controller is connected to an optical path manipulator 170 which is designed as an actuator module 87.

Either the working distance finding module 99 or the refractive index module 101 has a data output 105.

The evaluation unit 95 may also comprise a memory unit 107 in which, for example, a predefined function 109 or a measured value 111 is or may be stored. Furthermore, calibration data 188 can be stored in the memory unit 107, which calibration data detect possible imaging errors of the optical components used, such as the illumination objective 7 and/or the detection optics 41, for example, so that these imaging errors can be taken into account when the beam path 8 is manipulated according to the method or the device according to the invention. In particular for an objective with a correction means, for example, the color dependency that exists for different settings of the refractive index n can be stored. For an adjustable lens, for example, deviations from an ideal lens can be stored in relation to a set focal length.

The reflective member 49 of fig. 3 and 4 is at a distance 113 from an interface 235 between the optical medium 35 and another optical medium 39, in this case air 37. For simplicity, the wall 115 of the sample vessel 25 is considered infinitely thin and is not considered.

The optical device 9 is at a distance 117 from the sample medium 27, also here the wall 115 is not considered.

The state of fig. 4 can be substantially derived from the state of fig. 3 by increasing the distance 117 between the optical device 9 and the sample medium 27, whereupon the actuator module 87 follows the detection optics 41 of the optical device 9; alternatively, the distance 113 between the reflector 55 and the further optical medium 39 is reduced, after which the optical device 9 is moved away from the reflector 55 by the actuator module 87.

Situation a) is shown in fig. 3 by means of detail 119. In this section, it can be seen that a change in the distance 117 between the optical device 9 and the sample medium 27 leads to a defined, i.e. measurable, distance change 121 in the focal position 123 of the measuring light 65.

In fig. 3, the reflective member 49 is located at a working distance 79 of the optical device 9, while in fig. 4 at least one parameter 125 has been changed, including the distance 113 and the distance 117, in order to adjust a further working distance 127.

The working distance 79 of fig. 3 and the further working distance 127 of fig. 4 are transmitted from the working distance calculation module 99 in the form of a working distance value 131 (schematically represented by electrical signals) to the controller 103, wherein the controller calculates a working distance change 129 from the working distance value 131 of the working distance 79 and the working distance value 131 of the further working distance 127 by means of a calculation module (not shown), which is transmitted to the refractive index module 101 in the form of a working distance change value 133. Furthermore, the controller 103 determines the distance change 121 on the basis of the connection of the transmission data to the actuator module 87 and transmits the distance change to the refractive index module 101 in the form of the distance change value 135. The distance variation value 135 is shown schematically and purely exemplarily in fig. 4 in the form of a triangular pulse for the purpose of differentiation.

Based on the working distance change 133 and the distance change 135, the refractive index module 101 calculates the refractive index n, or a measurement proportional to the refractive index n, and provides it as a refractive index value 137 at the data output 105. The refractive index values 137 are schematically represented by sine waves for distinction.

A possible solution for finding the working distance 79 will be described with the aid of fig. 5. A parameter 145 (such as voltage or current) detected by the detection device 57 is shown, which parameter is shown both for the working distance 79 and for the further working distance 127 as a function of the distance change 121. Specifically, fig. 5 shows the respective predetermined functions 109 which are adapted to the measured values 111, wherein the predetermined functions 109 are represented by gaussian functions 147.

The gaussian 147 has only two parameters 125, namely a half-value width 149 and a center 151, where the center is located at the extremum 153 of the gaussian 147. For the gaussian function 147, the number N of parameters 125 is two. If other predetermined functions 109 are used, the number of required measurements 111 equals the number N of parameters 125 of the function 109 used.

A differently plotted gaussian function 147 allows the working distance 79 to be calculated as well as the further working distance 127 and the resulting working distance variation 129. From the working distance variation 129 and the distance variation 121, the refractive index module 101 (see fig. 4) can calculate the refractive index or refractive index n.

Fig. 6 shows a detail of a device 85 according to the invention, in particular a second measurement method for determining the refractive index n.

A reflective element 49 is shown which, as shown on the right, can be located directly on the detection optics 41, wherein the reflective element 49 is located in the immersion liquid 23, i.e. the optical medium 35.

Along the detection light path 190, the measurement light 65 is introduced via a lens 192 into the sample vessel 25, in which the optical medium 35 is located.

If the further optical medium 39 located between the lens 192 and the sample vessel 25 has the same refractive index n as the optical medium 35₁(i.e., n)₁＝n₂) A first immersion light path 194, shown by a short dashed line, results.

If the optical density of the other optical medium 39 is greater than the optical density of the optical medium 35 (i.e., n)₁>n₂) The probe optical path 190 is interrupted and a second immersion optical path 196, shown in solid lines, is obtained.

In both cases, the associated immersion beam path 194, 196 strikes the reflection element 49 and is reflected by it, so that the reflected measuring light 65b is guided along the corresponding measuring beam path 198 to the spatially resolved detector 58. This can likewise take place via a lens 192, which focuses the reflected measuring light 65 b.

If the first measurement beam path 200 resulting from the first immersion beam path 194 is observed and compared with the second measurement beam path 202 resulting from the second immersion beam path 196, it is clear that the second point of incidence 206 of the second measurement beam path 202 emerges laterally offset from the first point of incidence 204 of the first measurement beam path 200 on the position-resolved detector 58.

Furthermore, if the reflection element 49 is moved to a second position 208, which is indicated by a dashed line, a third measuring beam path 210 is generated, which impinges on the position-resolved detector 58 at a third impingement point 212. Analysis of the points of incidence 204, 206, 212 and in particular the variations between them provides the refractive index n of the optical medium 35 in relation to the distance variations 121 of the reflective member 49₂。

Furthermore, adjusting the autofocus may also be achieved using the method schematically shown in fig. 6. This is known from the prior art and will not be described in further detail here.

Fig. 7 and 8 show possible designs of the beam path 8 shown in a simplified manner when determining the refractive index according to the second measurement method of fig. 6. For simplicity, the sample vessel is not shown and the reflection of the detection light path 190 is made at the location of the reflective member 49.

In addition to the structure shown in fig. 6, these figures also show a deflection mirror 214, which deflects the measurement light 65 towards the reflection member 49.

These detection beams 190 differ in that they are focused on a deflection mirror 214 in fig. 7. This results in the reflected measuring light 65b being collimated by the two lenses 192 onto the position-resolved detector 58.

In this configuration, a change in the refractive index n causes the wide spot 216 to change its position on the position-resolved detector 58 as a whole. Furthermore, if reflective member 49 is moved along optical axis 53, this causes the wide spot 216 to decrease or increase.

In the arrangement of the detection beam path 190 of fig. 8, however, it impinges collinearly on the deflection mirror 214. Imaging by means of the two lenses 192 results in an intermediate focus 217 on the reflection element 49 on the one hand and a focus 19 or a focused spot 218 on the position-resolved detector 58 on the other hand.

In addition to the different necessary analysis algorithms, these two configurations also allow for varying both the light intensity 220 in the sample space and the light intensity 222 on the detector. The arrangement of fig. 8 is thus preferred in the sample space, for example in the case of a small intensity of the measuring light 65 or in the case of a severe weakening thereof.

The correlation of fig. 7 is advantageous compared to the correlation of fig. 8 if the intensity of the measurement light 65 is in the boundary region of the dynamic range of the position-resolved detector 58.

The method for taking a stack of images will be briefly described with the aid of fig. 9 and 10.

An apparatus 85 is shown with which a three-dimensional photograph is to be produced from a sample 21 located in the sample space 17. This is done by imaging an image stack 224, which is schematically shown next to the sample vessel 25. The image stack 224 includes a plurality of individual images 225.

To capture such an image stack 224, the detection optics 41 may be moved in a movement direction 226, for example. In fig. 9, the focal point 19 of the detection optics 41 has been located outside the sample 21, i.e. the sample has been moved from a first position 207 (shown in dashed lines) to a second position 208.

Fig. 10 shows the movement path x of the detection optics 41 over time t, wherein a first slope 228 is obtained from the scanning distance 229 and the time t, which first slope ultimately represents the speed of movement of the detection optics 41 in the movement direction 226.

Since the focal point 19 of the illumination light 11 will have a lateral offset 230 when the illumination objective 7 is stationary, the illumination objective 7 must also be moved in the direction of movement 226. For both movements, for example, actuator modules 87 can be used separately.

However, since the path portion 165 in the optical medium and the path portion 167 in the other optical medium change relative to one another when the detection optics 41 are moved, the illumination objective 7 must be moved in the direction of movement 226 with a second slope 232 which is smaller than the first slope 228. At the same time t, the illumination objective 7 must be moved to followDistance 233. If the optical medium 35 and the further optical medium 39 have the same refractive index n₂Or n₁The first slope 228 is equal to the second slope 232. However once the refractive index n of optical medium 35 is reached₂Different from the refractive index n of the other optical medium 39₁The slopes 228, 232 will be different.

With regard to the method for imaging an image stack described with the aid of fig. 9 and 10, the detection optics 41 can be regarded as the first optical device 9b and the illumination objective 7 as the second optical device 9 c.

If in a further embodiment of the method the illumination objective 7 is first moved (wherein in this case the illumination objective 7 can be referred to as the first optical device 9b), the detection optics 41 are followed in a next method step. In this case, the detection optics 41 correspond to the second optical device 9 c.

In one embodiment of the method according to the invention, the refractive index of the specimen 21 and/or of the immersion medium 23 can thus be measured at each intermediate position during the imaging of the image stack 224, i.e. for each image of the image stack 224, while in another embodiment of the method according to the invention the refractive index of the specimen 21 and/or of the immersion medium 23 is measured once and the difference between the slopes 228 and 232 is calculated from this refractive index. The latter design of the method according to the invention is advantageous in particular in the case of the aforementioned purification method, in which the refractive index n of the immersion medium 23 is matched to the refractive index of the sample 21.

List of reference numerals

1 microscope

2 microscope parameters

3 confocal microscope

5 light sheet microscope

7 illumination objective

8 optical path

8a illumination light path

9 optical device

9a further optical device

9b first optical device

9c second optical device

11 illumination light

13 illumination side

15 sample side

17 sample space

19 Focus/focal plane

21 sample

23 impregnating liquid

25 sample vessel

27 sample Medium

29 optical system

31 spatial position

33 free light space

35 optical medium

37 air

39 another optical medium

41 detection optics

43 adjusted state

45 focal plane

47 reflective surface

49 reflective member

51 detection objective

53 optical axis

55 reflector

56 refractive index calculation module

Second design of 56a refractive index calculation Module

57 detection device

58 position resolving detector

59 aperture

61 pinhole

63 Detector

65 measuring light

65a of measuring light

65b reflected measuring light

79 working distance

85 device

87 actuator module

89 translate

93 distance between reflector and measuring light focus

95 analysis unit

97 data line

99 working distance solving module

101 refractive index module

103 controller

105 data output terminal

107 memory cell

109 a function predetermined in advance

111 measured value

113 distance between reflector and another optical medium

115 wall

117 distance between optical device and sample medium

119 part of

121 distance variation

123 focal position

125 parameter

127 another working distance

129 working distance variation

131 working distance value

133 working distance change value

135 distance change value

137 refractive index value

147 gauss function

149 half width

151 center

Extreme value of 153

162 PC

163 non-volatile computer-readable storage medium

165 parts of the path in the optical medium

167 a part of the path in another optical medium

170 optical path manipulator

172 deformable mirror

174 curved part

176 optical path length

Optical path length outside 176a

176b optical path length in the center

178 edge region

180 center

182 optical component with electrically adjustable focal length

184 correction ring

186 steering output

188 calibration data

190 probe optical path

192 lens

194 first immersion light path

196 second immersion light path

198 measurement light path

200 first measuring light path

202 second measuring light path

204 first point of incidence

206 second point of incidence

207 first position

208 second position

210 third measuring beam path

212 third point of incidence

214 deflection mirror

216 wide spot

217 intermediate focus

218 focused spot

220 light intensity in sample space

222 light intensity on the detector

224 image stack

225 image

226 direction of movement

228 first slope

229 scan distance

230 lateral offset

232 second slope

233 following course

235 interface

237 image field

239 excitation wavelength

241 trigger signal

Refractive index of n

n₁Refractive index of another optical medium

n₂Refractive index of sample medium

n₃Refractive index of sample

Distance r

time t

x path of movement