Optical device and method for image correction

Abstract

The invention relates to an optical device comprising an illumination beam path (1) for guiding an illumination beam (BS), wherein the illumination beam (BS) is directed into a sample chamber (3) to illuminate a sample (4) optionally located therein; and / or a detection beam path (7) for guiding a detection beam (DS) coming from the sample chamber (3); and a detection unit (12) arranged in the detection beam path (7) for acquiring image data from a number of image locations. According to the invention, the device is characterized in that an optical means (8) is arranged in the illumination beam path (1) and / or in the detection beam path (7), the transmittance of which for the illumination radiation (BS) or for the detection radiation (DS) can be varied over the cross-section of the respective beam path (1, 7), wherein the varying transmittance of the optical means (8) is adapted to the respective beam path (1, 7) in such a way that a uniform distribution of the amount of light over the cross-section of the beam path (1, 7) is achieved. The invention also relates to a method for image correction using the device.

Description

[0001] The invention relates to a method and a device for correcting image data.

[0002] In optical imaging techniques, such as microscopy and photography, aberrations such as a drop in light intensity toward the edge of the image (peripheral light falloff, vignetting) or uneven light output across the image area occur independently of the sample. These can at least be reduced with various technical measures. However, all additional image enhancement measures also involve additional costs and technical effort.

[0003] The term shading (vignetting) refers to an effect that occurs independently of the specific sample being imaged, in which areas in the image, be it a captured image (camera image) and / or an image in an eyepiece, are darker than others. The light output achieved with a detector, for example, is not uniform across the sensor surface of the detector. Shading often becomes increasingly apparent towards the edges of an image (so-called marginal light falloff). The same effect can occur when an object (sample) is scanned point-wise or line-wise. In this case, the individual pieces of information (pixels) captured can have different brightness levels across the object field and / or along the scan line, depending on the system.

[0004] These variations in light output are caused, for example, by the optical conditions of an illumination and / or detection beam path and the optical elements contained therein in interaction with physical laws.

[0005] The lower intensities of the light to be detected (detection radiation) in the shaded areas also mean that the signal-to-noise ratio (SNR), i.e. the information content of the obtained image areas, is lower there than in the other areas.

[0006] Current practice is to perform a digital correction of the captured and stored image data after image acquisition. This correction consists of a pixel-by-pixel multiplication with a pre-determined correction value, whereby each pixel can be assigned an individual correction value.

[0007] While this approach can significantly improve the resulting images for the viewer, it does have drawbacks. While multiplying the correction value changes the intensity value and thus the brightness perceived by the viewer, it does not increase the SNR. Thus, the information content of the pixels and image areas corrected in this way remains at the original level.

[0008] However, a consistently high SNR across the image is essential for many subsequent analyses. Such analyses include, for example, the resolution and precise localization of individual fluorescent proteins or the determination of sample topographies.

[0009] A further disadvantage is that the correction must be applied to all images. Image stacks (z-stacks), time series, and the acquisition and simultaneous display of different image planes (“tiles”), in particular, require enormous computing power. This computing power may also mean that it is not possible to provide a corrected display in real time for fast acquisitions.

[0010] For samples sensitive to the bleaching effect of illuminating radiation, significant noise can occur, particularly in the center of the image, due to the uneven light distribution. Such uneven bleaching is particularly disadvantageous in time series and multiple exposures.

[0011] The invention is based on the object of proposing a way to reduce the disadvantages known from the prior art. In particular, a way to more evenly distribute the light output across the captured image is to be demonstrated.

[0012] The problem is solved by the subject matter of the independent claim and subordinate claims. Advantageous further developments are the subject matter of the dependent claims.

[0013] The problem is solved with an optical device comprising an illumination beam path for guiding illumination radiation and / or a detection beam path for guiding detection radiation coming from the sample chamber. In the case of an illumination beam path, the illumination radiation is directed into a sample chamber to illuminate a sample located there. A detection unit for acquiring image data from a number of image locations is also arranged in the detection beam path.

[0014] According to the invention, the device is characterized in that an optical means is arranged in the illumination beam path and / or the detection beam path, the transmittance of which varies for the illumination radiation or for the detection radiation across the cross-section of the respective beam path. The varying transmittance of the optical means is matched to the respective beam path in such a way that a uniform distribution of the amount of light across the cross-section is achieved. The uniform distribution is preferably achieved in a detection plane or image plane, in which the detection unit is advantageously arranged.

[0015] The core of the invention is to specifically reduce the amount of light, if necessary, in areas of an image to be captured in order to ultimately achieve a more even distribution of light intensities in the captured image data. Particular reference is made to an uneven distribution of light intensities caused by physical effects and technical conditions within the respective beam paths and not by a particular illuminated and / or imaged sample. As a result, the light intensity is more evenly distributed, particularly in the image plane, than would be the case without the optical means. The invention can be applied in the illumination beam path and / or the detection beam path of optical devices that operate according to the principle of wide-field image capture or point-by-point or line-by-line illumination or capture using a scanning device and the deflection of an illumination beam.The invention can therefore also be used for so-called light sheet microscopy.

[0016] The light intensities across the cross-section of the relevant beam path are adjusted by deliberately reducing the transmissivity, i.e. the permeability to radiation of a predetermined wavelength or at least a predetermined wavelength range. The term transmissivity here refers to a difference in intensities that exist between two positions in the beam path, depending on the wavelength and location. Rotations and reflections in the beam path are also permitted. In contrast to the solutions according to the prior art, the invention therefore specifically attenuates or suppresses light in order to achieve a uniform light distribution in favor of an improved SNR. A redistribution of light, however, is not the subject of the invention, even if this may occur to a minor extent.In contrast to the prior art, the invention deliberately accepts a reduced light output in order to achieve an improved SNR.

[0017] Detection radiation is understood to be electromagnetic radiation (light) that is emitted as a result of reflection and / or as a result of excitation, for example of fluorescence radiation.

[0018] A uniform distribution of the light intensity is achieved, for example, when the intensity values ​​across the cross-section of the beam path preferably fluctuate by a maximum of 2% around a common (arithmetic) mean or lie within a 99% confidence interval.

[0019] In a preferred embodiment of the device according to the invention, the optical means is designed in the form of a controllable optical array. Such an array can be, for example, an LCD array (liquid crystal display), an LCoS array (liquid crystal on silicon), or a DMS array (deformable micromirrors).

[0020] The filter effect to be adjusted can be selected based on simulations, available databases, and / or the result of individual measurements. For example, a specific device can be exposed to illumination radiation and / or detection radiation. The distribution of light intensities in the image plane is recorded and evaluated using the detector unit.

[0021] If a detector unit with a plurality of detector elements is used, for example a camera with a CCD, CMOS, sCMOS chip or a SPAD array, the respective recorded intensities for each detector element can be evaluated.

[0022] If the device according to the invention is used with a scanning device by means of which the illumination radiation is guided over the sample in the form of a spot or a linear illumination (line), the detector unit can also have only a single detector element (for example, a photomultiplier tube, PMT) in the case of point scanning. In this case, an image is obtained by arranging the image data acquired for each sample location (image points, pixels). The same applies to line scanning, in which case detector units with a plurality of detector elements or an array of a plurality of individual detectors (PMT) are used.The necessary information on the origin of the radiation detected or its position in the image to be captured can be determined, for example, based on the respective orientation of the scanning device and assigned to the respective image data.

[0023] The design or control of the filter effect of the optical device can be adapted to different requirements. For example, the filter effect can be higher in the center of the optical device than in the outer region, and can decrease radially from the center outwards. In another embodiment, the filter effect can be higher in the outer region and decrease radially towards the center.

[0024] A course or gradient of the filter effect of the optical means can be designed such that an approximately rectangular filter area with a first filter effect is created on the optically effective surface of the optical means, which filter area is surrounded by an area with a second filter effect. In one embodiment, the first filter effect can be higher than the second filter effect. In a second embodiment, the first filter effect can be lower than the second filter effect. In further embodiments, the approximately rectangular area of ​​the first filter effect can take up the entire surface of the optical means in one direction and be flanked only on at least one side by an area with a second filter effect. The embodiments with a rectangular filter area are suitable, for example, for use in combination with a cylindrical lens or a scanning device for generating a light sheet (see below).

[0025] In further embodiments, the optical means can be designed such that it has a region of the same filter effect in the passage area of ​​the beam path. For example, such a region is present in the center of the optical means. The optical means can also have several such regions, which can be designed, for example, in the form of rectangles ("tiles"), circular rings and / or sectors. Such a region is large in relation to the diameter or the clear width of the beam path and is, for example, one tenth, one eighth, one fifth, one quarter, one third or one half of the diameter or the clear width. The optical means can have a number of such regions that have different filter effects. The different filter effects do not have to be realized in the sense of one or more gradients, but can be designed and arranged as required.

[0026] In an advantageous development of the device according to the invention, a drive is provided by which the position of the optical means can be adjusted by a motor. In this way, the optical means can be adjusted and positioned both transversely to the beam path in an xy plane and, optionally, in a z direction along the beam path. Such an adjustment option can alternatively be implemented manually or with a manual drive; however, adjustment by a motor drive advantageously allows for the stored positions to be saved and efficiently readjusted.

[0027] In order to achieve an effective influence on the distribution of the light intensity, the optical means is advantageously arranged at the location of an intermediate image of the beam path.

[0028] To implement the invention, the respective beam paths and the at least one optical means are coordinated with one another. For this purpose, the distribution of the light intensity across the cross-section of the beam path can be recorded and evaluated using a detector unit located in a detection beam path. A neutral sample placed in the sample space, which in itself does not cause uneven intensity distributions, can be used as a reference. Such a neutral sample can also be used if the distribution of the light intensities in the interaction of an illumination beam path and a detection beam path is to be determined. Based on the result of the evaluation, it can be decided which areas of the cross-section of the beam path(s) are shadowed and how severe the shadowing is in each case.

[0029] If it is possible to position the optical means in the form of an optically active array by means of a motor-controlled or manual drive in the xy plane and / or in the z direction, this positioning option can also be used to adjust the distribution of the light intensities and to fine-tune it.

[0030] It is of course also possible that at least one optical means for uniform distribution of the light intensity is arranged in both an illumination beam path and a detection beam path.

[0031] The object of the invention is also achieved by a method for image correction in which a device according to the invention is used.

[0032] During the process, illumination radiation is directed into a sample chamber along an illumination beam path, and / or detection radiation coming from the sample chamber is guided along a detection beam path. The detection radiation is detected, and image data is acquired.

[0033] According to the invention, the method is characterized in that an optical means arranged in the illumination beam path and / or in the detection beam path is selected in advance or controlled during the method by means of control commands in such a way that its transmissivity (transparency) for the illumination radiation or for the detection radiation is varied or is varied across the cross-section of the respective beam path. The variation is carried out in such a way that, in particular in a detection plane of the detection beam path, a uniform distribution of the amount of light is effected across the cross-section of the beam path. The method can also be used for only one illumination beam path in order to achieve uniform illumination of a sample located in the sample chamber.

[0034] The method according to the invention achieves a uniform distribution of light intensities across the cross-section of the relevant beam path. This reduces technically induced effects that would otherwise lead to an uneven distribution. Deviating distributions of light intensities or light quantities caused by an imaged sample are not the subject of the method. The method therefore represents a correction method by means of which, when imaging a sample, the image data can be corrected for each image location. This correction can be complete or incomplete. Complete correction means that after correction using the method, a high and uniform SNR and uniform illumination are present across the entire image, whereby the SNR may only fluctuate across the image within predefined narrow limits.The fluctuation range is, for example, less than 10%, in particular less than 5%, preferably at most 2.5% of the arithmetic mean of the SNR values.

[0035] If a method design with an incomplete correction is selected, the image data are corrected accordingly by the effect of the optical means, but this does not necessarily achieve the above-mentioned narrow limits of the permissible fluctuation range of the SNR across the image.

[0036] In one embodiment of the method, an incomplete correction can be combined with a correction of the acquired and subsequently stored image data known from the prior art. This allows a sufficient improvement in the SNR to be achieved, and the qualitatively improved image data can optionally be post-processed using known routines. In another embodiment of the method, the incompletely corrected data in this sense is used without further correction. In this case, computing power and storage space can be saved.

[0037] The method according to the invention can be carried out uniformly, regardless of the currently used lens and / or illumination mode. In further embodiments, the optical means to be used and / or their control can be determined depending on the currently used lens and / or illumination mode, or can be determined in advance and stored for retrieval.

[0038] The optical device according to the invention can advantageously be used within the scope of the method according to the invention or independently thereof, wherein the controllable optical array is controlled in such a way that a diaphragm, in particular a rectangular diaphragm, a ring diaphragm, or a round / pinhole diaphragm, is created. Such uses are advantageous, for example, when the illumination radiation has a specific cross-sectional shape.

[0039] For example, a so-called cylindrical lens can be present in the illumination beam path, the effect of which focuses the illumination radiation in only one direction, so that a so-called static light sheet is or can be generated in the sample space.

[0040] This light sheet should exhibit a uniform distribution of light intensity across its cross-section. This type of illuminating radiation is used, for example, in so-called single-plane illumination microscopy (SPIM).

[0041] The invention is explained in more detail below using exemplary embodiments and figures. They show: Fig. 1 shows a first embodiment of a device according to the invention with transmitted light illumination; Fig. 2 shows a second embodiment of a device according to the invention with illumination in transmitted light and optical means in both beam paths; Fig. 3 shows a third embodiment of a device according to the invention with illumination in the form of a light sheet; Fig. 4 shows a fourth embodiment of a device according to the invention with a scanned illumination and an optical means in the detection beam path; Fig. 5 shows a fifth embodiment of a device according to the invention with a scanned illumination and an optical means in the illumination beam path; Fig. 6 shows a first embodiment of an optical means with a symmetrical radial progression of the filter effect; Fig. 7 shows a second embodiment of an optical means with an asymmetric radial progression of the filter effect; Fig. 8 shows a third embodiment of an optical means with filter areas each having a different filter effect; Fig. 9 shows a fourth embodiment of an optical means with a region of a first filter effect and a second filter effect; and Fig. 10 shows a further embodiment of an optical means with a region of a first filter effect and a second filter effect.

[0042] The invention is explained using schematic illustrations. The same reference numerals denote the same technical elements unless expressly stated otherwise. It should be noted that the corrections explained below refer to the most uniform distribution of light intensities possible, which are determined by technical constraints and not by any sample 4 that may be present.

[0043] In a device according to the invention according to a first embodiment ( Fig. 1) an illumination beam path 1, illustrated by an optical axis, is provided, along which an illumination beam BS is or can be directed into a sample chamber 3 by means of an illumination objective 2. A sample 4 (shown with a broken solid line) can optionally be present in the sample chamber 3, which is arranged on a sample holder 5, for example in the form of a glass or a sample table (object plane OE).

[0044] Coming from the sample chamber 3, a detection radiation DS can be guided along a detection beam path 7 and detected in transmitted light and wide field by means of a detection lens 6. Due to the action of the detection lens 6, the detected detection radiation DS is directed into an intermediate image plane ZB in which an optical means 8 is arranged. In all exemplary embodiments, the optical means 8 is a controllable optically active array by means of which a uniform distribution of the light intensities of the detection radiation DS can be brought about across the cross-section of the detection beam path 7 used for image acquisition. The detection radiation DS, whose intensity distribution is thus influenced, is focused by means of an optical system, represented simply by an optical lens 11, into an image plane BE in which a detector unit 12 is arranged.This is used to capture the detection radiation DS as image data for later imaging.

[0045] The optical device 8 is optionally connected to a drive 9 (broken solid line) and can be moved either motor-driven or manually in each of the x, y, and z directions of a Cartesian coordinate system. In the motor-driven version, the drive 9 is connected to a control unit 10 in the form of a computer. This is configured to generate control commands by which the drive 9 can be controlled.

[0046] The control unit 10 is optionally connected to the detector unit 12 in order to generate the control commands taking into account acquired image data and / or parameters derived therefrom.

[0047] The optical device 8 is designed in the form of an active optical array. A data connection exists between the optical device 8 and the control unit 10 in order to control the optical device 8 and adjust its optical effects using control commands from the control unit 10.

[0048] The Fig. The second embodiment shown in Figure 2 has an optical means 8 in both the illumination beam path 1 and the detection beam path 7. These can also be controlled by the control unit 10.

[0049] With the optical means 8 in the illumination beam path 1, a uniform distribution of the light intensity of the illumination radiation BS can be achieved over the used cross-section of the illumination beam path 1, which in turn enables a more uniform illumination of a sample 4 present in the sample chamber 3. The effect of the optical means 8 in the detection beam path 7 can already be Fig. 1 described.

[0050] The invention can further be implemented in a so-called light sheet microscope ( Fig. 3). Illumination beam path 1 and detection beam path 7 are directed into the sample chamber 3 from a common side, so that the sample holder 5 does not need to be illuminated.

[0051] A light source 13 for generating and providing illumination radiation BS, particularly in the form of coherent radiation (laser radiation), is present in the illumination beam path 1. The illumination radiation BS is shaped or deflected in a controlled manner by a beam-shaping means 14 in order to generate a light sheet 16 in the sample chamber 3 in cooperation with the illumination objective 2 and, if appropriate, other optical elements (not shown) present in the illumination beam path 1. The illumination beam path 1 is directed into the sample chamber 3 at an angle greater than 0° and less than 90°.

[0052] In one embodiment, the beam-shaping means 14 can be a cylindrical lens, the effect of which focuses a beam of illumination radiation BS in a direction transverse to the optical axis (illumination beam path 1). As a result, a light sheet 16 (static light sheet) is generated in the sample chamber 3 in an xz plane orthogonal to the illumination beam path 1. In this embodiment, the coordinate systems used (x, y, z or x', y', z') are each aligned such that the z-direction and the z'-direction run along the illumination beam path 1 and the detection beam path 7, respectively.

[0053] In a further embodiment, the means for beam shaping 14 can be a scanning device 15, by means of which an illumination beam can be quickly moved back and forth in the xz plane (scanned) and as a result a light sheet 16 (dynamic light sheet) can be generated in the sample space 3.

[0054] Detection radiation DS excited by the light sheet 16 is collected by the detection lens 6 and guided along the detection beam path 7 to the optical means 8, where it is imaged onto the detector unit 12 and detected. In the illustrated embodiment, an optical means 8 is arranged only in the detection beam path 7. In further possible embodiments, an optical means 8 can additionally or alternatively be arranged in the illumination beam path 1. The optical axis of the detection beam path 7 is directed at an angle of approximately 90° to the xz plane of the light sheet 16.

[0055] In the Fig. 4 and Fig. Figure 5 shows exemplary embodiments in which the illumination and detection radiation DS functions are performed with a common lens 2, 6 (the beam path is shown only schematically). Along a path between a beam splitter 17 and the sample chamber 3, the illumination beam path 1 and the detection beam path 7 coincide (designated 1, 7).

[0056] In the embodiment according to the Fig. 4, the illumination radiation BS emitted by the light source 13 is deflected in a controlled manner by means of a scanning device 15 and reaches the beam splitter 17, through whose wavelength-dependent effect the illumination radiation BS is reflected to the objective 2, 6. The scanning device 15 can be designed as a point scanner or as a line scanner. By means of the objective 2, 6, the deflected and shaped illumination radiation BS is focused into the sample chamber 3 and a sample 4 optionally located there. As a result of the deflections by the scanning device 15, the sample 4 is scanned with the focused illumination radiation BS. The respective scanned sample locations (in a point scan) or sample regions (e.g.in a line scan) are determined and stored by detecting information on the current deflection of the scanning device 15 and, if applicable, information on the focus position of the lens 2, 6, transmitting this information to the control unit 10 and storing it there in such a way that it can be assigned to the associated acquired image data.

[0057] The detection radiation DS detected by the lens 2, 6 reaches the color splitter 17 and, due to its different, generally longer, wavelength compared to the illumination radiation BS, is transmitted through it and directed onto the optical means 8 by means of an optical system shown as an example of an optical lens 11 and modified according to the set properties of the optical means 8, in particular corrected with regard to its distribution. The corrected detection radiation DS is detected in the form of image data by the detector unit 12. In further embodiments, a pinhole or slit aperture (pinhole) can additionally be located in a (further) intermediate image plane ZB in the detection beam path 7, for example, to create a confocal beam path. Such aperture effects can also be generated by means of an optical means 8 if this is designed as an optically active array (see also Fig. 9 and Fig. 10).

[0058] The Fig. Figure 5 shows an embodiment of the device according to the invention, in which the optical means 8 is arranged in the illumination beam path 1. This allows a more uniform distribution of the light intensities at different applied scanning angles and correspondingly different beam paths used within the illumination beam path 1.

[0059] Various possible designs of the optical means 8 are shown in the Fig. 6 to 10. The Fig. Figure 6 schematically shows a continuous filter effect (filter gradient) and a filter effect that decreases from the center to the edge. In other designs, rings with decreasing filter effect can also be realized, as already indicated in the drawing.

[0060] A radially asymmetric distribution of the filter effect distribution is in Fig. 7 is shown as an example. Here, too, the reduction in filter effectiveness can occur continuously or gradually.

[0061] The graduated filters according to the Fig. 6 and Fig. 7 can also have combinations of continuously and gradually changing filter effects in further designs. Furthermore, all designs can have filter effects that increase toward the edge.

[0062] Graduated filter according to the Fig. 6 and Fig. 7 can be implemented by means of a suitable and appropriately controlled optical array.

[0063] A design with clearly defined areas B1 to Bn each with a specific filter effect is available in the Fig. 8 is shown as an example. The ranges B1 to Bn can be controlled independently of each other, each with a specific filter effect.

[0064] Two special designs of the optical means 8 and its possible uses are described in the Fig. 9 and Fig. 10. In both embodiments, a first area B1 with a first filter effect and a second area B2 with a second filter effect are realized.

[0065] With an execution according to the Fig. 9, a pinhole can be created as an alternative to correcting the intensity distribution in a first application if the filter effect of the first region B1 is set to a maximum, but in any case significantly higher than the filter effect of the second region B2. The filter effect of the second region B2 can be minimal to allow unhindered passage of the respective radiation.

[0066] In a second alternative use, the filtering effect of the first region B1 can be less than the filtering effect of the second region B2. If the filtering effect of the first region B1 is minimal and the filtering effect of the second region B2 is as great as possible, a ring aperture can be created in this way.

[0067] In a further embodiment, the second region B2 can be formed in the form of a rectangle ( Fig. 10). In addition to a correction, a first alternative application can create a slit diaphragm by setting the filter effect of the first area B1 to maximum and the filter effect of the second area B2 to as low as possible. Such control can be used, for example, in conjunction with a cylindrical lens present in the illumination beam path 1 to block unwanted radiation components.

[0068] In a second alternative use, the filtering effect of the first area B1 is minimal and the filtering effect of the second area B2 is maximized. In this way, radiation components can be filtered out in a strip, for example, a centrally located one. Reference symbol 1 Illumination beam path 2 lighting lens 3 rehearsal room 4 Sample 5 Sample holder 6 Detection lens 7 Detection beam path 8 optical means 9 Drive 10 Control unit 11 optical lens 12 Detector unit 13 Light source 14 means of beam shaping 15 Scanning device 16 light sheets 17 beam splitters B1, B2,...Bn area of ​​a first filter effect, ..., area of ​​an n-th filter effect BE image plane BS illumination radiation DS detection radiation OE object level E.g. intermediate image plane

Claims

[1] Optical device comprising a - Illumination beam path (1) for guiding an illuminating radiation (BS), wherein the illuminating radiation (BS) is directed into a sample space (3) in order to illuminate a sample (4) optionally located therein; and / or - a detection beam path (7) for guiding a detection radiation (DS) coming from the sample chamber (3); and - a detection unit (12) arranged in the detection beam path (7) for capturing image data from a number of image locations; characterized by , that - an optical means (8) is arranged in the illumination beam path (1) and / or in the detection beam path (7), the transmittance of which optical means for the illumination radiation (BS) or for the detection radiation (DS) can be varied over the cross section of the relevant beam path (1, 7), wherein the varying transmittance of the optical means (8) is matched to the relevant beam path (1, 7) in such a way that a uniform distribution of the amount of light over the cross section of the beam path (1, 7) is effected. [2] Optical device according to claim 1, characterized by that the optical means (8) is a controllable optical array. [3] Optical device according to one of the preceding claims, characterized by that a drive (9) is present and the position of the optical means (8) is motor-adjustable by the action of the drive (9). [4] Optical device according to one of the preceding claims, characterized bythat the optical means (8) is arranged in an intermediate image (ZB) of the beam path (1, 7). [5] Optical device according to one of the preceding claims, characterized by that the optical means (8) has a region of the same filter effect in the passage region of the beam path (1, 7) [6] A method for image correction using the device according to any one of claims 1 to 5, wherein - an illumination radiation (BS) is directed along an illumination beam path (1) into a sample space (3); - a detection radiation (DS) coming from the sample chamber (3) is guided along a detection beam path (7); and - the detection radiation (DS) is detected and image data is acquired characterized by , that - an optical means (8) arranged in the illumination beam path (1) and / or in the detection beam path (7) is controlled by means of control commands during the method in such a way that its transmittance for the illumination radiation (BS) or for the detection radiation (DS) is varied over the cross section of the relevant beam path (1, 7), so that in an image plane (BE) of the detection beam path (7) a uniform distribution of the amount of light over the cross section of the beam path (1, 7) is effected. [7] Use of an optical device according to one of claims 1 to 5, characterized by that the optical means (8) is designed in the form of a controllable optical array and is controlled in such a way that an aperture, in particular a rectangular aperture, a ring aperture or a pinhole aperture, is effected.

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