Method for generating an overview image using a high-aperture lens

Abstract

Method for generating an overview image using a high-aperture lens (4) with the steps: - Providing an optical detection beam path (2) with the objective (4), wherein the detection beam path (2) in a first operating state has a first numerical aperture (NA1) and a first depth of field (ST1) which lies substantially in a focal plane of the detection beam path (2), and in the first operating state an image of a detection radiation with the first depth of field (ST1) and with a lateral image field is or can be detected, wherein the detection radiation is effected by directing at least one light sheet (16) along an illumination axis (15) into a sample volume (8) to be imaged located in the sample space; - transferring the detection beam path (2) into a second operating state by increasing the first depth of field (ST1) to a second depth of field (ST2), wherein the high-aperture lens (4) remains in the detection beam path (2), - adjusting a thickness of the light sheet (16) in the direction of a detection axis (3) of the lens (4) to the second depth of field (ST2) using an adjustable illumination device, so that the thickness of the light sheet (16) corresponds at least to the second depth of field (ST2); - in the second operating state, detecting a detection radiation, wherein the detection radiation coming from a sample space is collected by means of the objective (4), guided along the detection beam path (2) and detected as an image with a lateral image field by means of at least one detector (7) sensitive to the detection radiation, wherein the lateral image fields are the same in the first and second operating states.

Description

[0001] The invention relates to a method for generating an overview image using a high-aperture lens.

[0002] Microscopy techniques are typically used to examine small sections of larger objects. To identify a suitable section for the examinations and observations to be performed, so-called overview images are often created.

[0003] On conventional (wide-field) microscopes and laser scanning microscopes (LSM), the overview image is usually captured with an objective whose magnification is lower than that of the objective used for the actual examination. A lower magnification enlarges the field of view, allowing large areas of a sample to be imaged very quickly. However, reducing the magnification usually also reduces the numerical aperture (NA) and thus the lateral and axial resolution of the detection system. However, high resolution can be dispensed with for an overview image.

[0004] Based on these overview images, a user can define a suitable section of the sample (region of interest, ROI) for examination. This could be, for example, a specific cell in a cell cluster or biological tissue, which can then be observed and imaged using a high-magnification, high-resolution lens.

[0005] If a microscope system cannot switch between objectives of different magnifications, or only does so without significant disadvantages, the user is dependent on the existing objective. Such systems without the option of easily switching between different objectives often lack an interchangeable unit for cost reasons, or particularly demanding tolerances must be maintained when adjusting the respective objective.

[0006] If a microscope is set to use an existing objective with a particularly high magnification, overview images can only be acquired very slowly because small sections of the sample are captured with a high data density.

[0007] Light sheet microscopes are an example of microscope systems where changing objectives is at least difficult and where the objective typically used offers high magnification and a shallow depth of field. In these microscopes, a thin light sheet is directed into the sample volume to be imaged, and an image of the illuminated sample plane is captured using an objective of a detection beam path based on the detected detection radiation. The light sheet can be generated, for example, as a static light sheet using a cylindrical lens in the illumination beam path or by means of a light beam moving (scanned) in the light sheet plane (dynamic light sheet). The depth range from which detection radiation can be captured is further restricted by the thin light sheet, in addition to the limited depth of field of the objective.

[0008] Light sheet microscopes are typically used for three-dimensional images of biological samples. For example, creating a three-dimensional overview image using light sheets requires a large number of individual images, which is time-consuming and requires significant computing power.

[0009] The invention is based on the object of proposing a method by means of which two- and three-dimensional overview images of a sample volume can be created quickly and without changing the objective of the detection beam path.

[0010] The object is achieved by a method according to claim 1. Advantageous further developments are the subject of the dependent claims.

[0011] The method according to the invention serves to generate and optionally provide an overview image using a high-aperture objective. A high-aperture objective has, for example, a numerical aperture of NA = 0.5; 0.6; 0.75 and more, for example, 1.0. The objectives can be designed as immersion objectives. A suitable immersion medium is, for example, water.

[0012] The method comprises the following steps, which are carried out in the specified order, whereby intermediate steps not specified here are possible in further embodiments of the method.

[0013] An optical detection beam path is provided with the objective lens. The detection beam path can, for example, be present in a microscope and provided with it. The detection beam path can have two operating states, which can be set manually or automatically. In a first operating state, the detection beam path has a first numerical aperture and a first depth of field in a focal plane of the detection beam path. With the detection beam path in the first operating state, an image of a detection radiation is acquired with the first depth of field and with a lateral image field. Optionally, multiple images with the first depth of field can also be acquired. The detection radiation is generated by directing at least one light sheet along an illumination axis into a sample volume to be imaged located in the sample chamber.

[0014] In order to be able to adapt the light sheet, in particular its extension (thickness) in the direction of a detection axis of the objective, to the second depth of field, an adjustable illumination device is preferably used to generate the light sheet or light sheets.

[0015] In the first operating state, the thickness of the light sheet(s) may differ from the depth of field of the detection lens. In particular, the depth of field of the detection lens may be smaller than the thickness(es) of the light sheet(s). In the second operating state, however, the depth of field of the detection lens and the light sheet thickness(es) are preferably equal.

[0016] In a further process step, the detection beam path is converted to a second operating state by increasing the first depth of field to a second depth of field. The high-aperture lens remains in the detection beam path.

[0017] Once the second operating state is established, detection radiation is captured, originating from a sample space, in particular from a sample volume to be examined. The detection radiation is collected by the objective lens, guided along the detection beam path, and captured in an image plane as an image with a lateral image field by at least one detector sensitive to the detection radiation. The lateral image fields in the first and second operating states are largely identical.

[0018] The depth of field indicates the distance range in front of the lens from which outgoing detection radiation, imaged in an image plane of the detection beam path, produces a detailed, sharp image. A second depth of field, which is increased compared to a first depth of field, is therefore characterized by a larger absolute distance range. The mean positions of the distance ranges of the first and second depth of field can be the same.

[0019] The term numerical aperture in this description refers to the detection beam path unless explicitly stated otherwise.

[0020] The depth of field can be determined, in particular calculated, in a known manner. The wave-optical depth of field is used. The calculation can be carried out using the general formula d = λ / (2* n*sin(u)) 2). Where λ is the wavelength, n the refractive index and u the aperture angle. To approximate the half-width in the direction of the Z-axis, for example, 1.67* λ*n / NA 2 be used.

[0021] The second depth of field is increased by at least a factor of two compared to the first depth of field, but preferably by a factor in the range of two to ten, for example, in the range of four to nine. Increasing the depth of field can be achieved using various measures.

[0022] One possibility is to reduce the numerical aperture of the detection beam path. To do this, a diaphragm, particularly a pinhole or slit diaphragm, can be inserted into a pupil plane of the detection beam path, the effect of which reduces the numerical aperture of the detection beam path.

[0023] The aperture can be pivoted into or pushed into the detection beam path. In further embodiments, a controllable aperture element present in the detection beam path can also be used. For example, an LCD element (liquid crystal display) can be present or inserted in the detection beam path. As required, the aperture element is controlled by a control unit, and the desired opening width and shape of the opening can be set. A aperture whose opening width is mechanically adjustable can also be arranged in the detection beam path. The adjustable apertures can be used both in the first operating state and when generating the second operating state. It is also possible for an adjustable aperture to be pivoted into or pushed out of the detection beam path or to be pushed in or out.

[0024] Advantageously, the numerical aperture of the first operating mode is reduced by a factor of 0.5 or less, for example, 0.3. This achieves a sufficiently large increase in the second depth of field by approximately four or nine times. For example, lowering the numerical aperture from NA = 1.0 to NA = 0.3 (= factor 0.3) and adjusting the detection step sizes allows the overview image to be created approximately ten times faster, as explained in more detail below.

[0025] Another possibility for increasing the second depth of field is the application of the principle of extended depth of focus (EDoF). Various measures are known for this purpose, which can be used in the method according to the invention.

[0026] For example, a phase mask can be inserted into the detection beam path, as described in King et al. (King, SV et al. (2015), Spatial light modulator phase mask implementation of wavefront encoded 3D computational-optical microscopy; Applied Optics 54: 8587 - 8595). By inserting a cubic phase mask into the pupil of the detection beam path, a PSF is generated that has the shape of an Airy beam. This PSF has a greatly increased axial extension and thus enables imaging with a significantly increased depth of field.

[0027] In a further embodiment of the method, it is possible for an axicon and optionally a pair of convex optical lenses to be arranged in the detection beam path (US 9,494,785 B2, DE 10 2014 102 215 A1).

[0028] The focal length can also be increased by inserting a ring diaphragm into a pupil plane of the detection beam path.

[0029] Furthermore, it is possible to incorporate an additional refractive optical element into the detection beam path to axially extend the point spread function (PSF), as is known from WO 2017 / 075275 A1. Spherical aberrations are specifically generated by means of a refractive optical element inserted between the objective lens and the sample to be imaged, resulting in an extended PSF in the direction of the objective lens's detection axis.

[0030] The detection radiation can be reflected illumination radiation or spectral components of the illumination radiation. The sample to be imaged, or components thereof, can also be excited to emit detection radiation. In particular, suitable molecules can be excited by excitation radiation as illumination radiation to emit fluorescent radiation as detection radiation.

[0031] Another possible approach to increasing the depth of field involves simultaneously detecting radiation from multiple parallel object planes. For this purpose, multiple light sheets can be generated and used in the different object planes, i.e., planes within the sample volume.

[0032] For example, it is possible to illuminate a sample volume to be imaged with a first light sheet and at least one further light sheet arranged parallel to the first light sheet along an illumination axis, wherein the light sheets are arranged one behind the other along a detection axis of the objective perpendicular to the illumination direction and overlap each other at most proportionally (US 2012 / 0224034 A1).

[0033] Alternatively, a sample volume to be imaged can be illuminated with a first light sheet and at least one additional light sheet arranged parallel to the first light sheet in one illumination direction and along one illumination axis each. The detection radiation generated by the respective light sheets is recorded along a detection axis. Each additional light sheet is generated shifted both in the detection direction, i.e., along the detection axis, and in the illumination direction relative to the first light sheet. The detection radiation of all light sheets is recorded simultaneously (WO 2016 / 189012 A1).

[0034] Without light sheets, detection radiation from different object planes can be captured with an increased depth of field by inserting a grating into a Fourier plane of the detection beam path. This grating splits detection radiation comprising multiple wavelengths into components according to their diffraction orders, and the components of the diffraction orders are simultaneously directed to different areas of an image plane. In the image plane, the components of the individual diffraction orders can be captured separately using a suitable detector, as is known from WO 2013 / 106731 A1.

[0035] In addition to a two-dimensional representation, the method according to the invention advantageously also enables the rapid creation of a three-dimensional overview image. For this purpose, after acquiring an image of the sample volume in the second operating mode with a first position of the light sheet, the light sheet is positioned relative to the sample volume. Despite the relative movement of the light sheet and the sample volume, the light sheet remains in the focal plane of the objective. The images of the thus offset light sheets can be saved and combined into a three-dimensional overview image using a computer.

[0036] To achieve high speed in the acquisition of the individual images and the creation of the overview image, the light sheets are offset by increments where there is no or only minimal overlap between the individual light sheets. For example, the individual light sheets overlap by no more than 50%, but preferably by no more than 40%, for example, 30%, 25%, 10%, or 0% of their thickness. This refers to the extent of the light sheets in the lateral image field.

[0037] The advantageous improvement in the detection speed achieved by the method according to the invention is illustrated by a numerical example: For a system with a 10x objective and a numerical aperture of the detection beam path NA = 1.0 (with water immersion), the half-width FWHM is x,y (Full Width at Half Maximum) approx. 250 nm and the half width in the z-direction FWHM z(≈depth of field) about 1000 nm. With a typical procedure, an image must be taken approximately every 300 nm (step size 300 nm).

[0038] When generating an overview image, it is common to accept limitations regarding the lateral resolution. For example, for an overview image with a 10x objective and NA = 0.3, the lateral half-width FWHM is x,y approx. 900 nm and in z-direction FWHM z Even 10-14 µm, depending on the immersion medium used. A typical sampling procedure requires an image to be acquired every 3-4 µm (step size 3-4 µm).

[0039] If the detection beam path has a numerical aperture NA = 0.3, approximately 10x fewer images need to be acquired than when using a detection beam path with an NA = 1.0.

[0040] To utilize the increased second depth of field with a reduced numerical aperture, a light sheet with a thickness that is at least equal to the second depth of field is required, and is preferably equal to the second depth of field. By reducing the numerical aperture of the detection objective from, for example, NA = 1.0 to NA = 0.3 using a pupil diaphragm and increasing the light sheet thickness, an increase in depth of field of approximately a factor of 10 can be achieved while accepting a reduced axial and lateral resolution. Accordingly, three-dimensional overview images can be acquired and provided ten times faster. Even higher factors are possible for even lower resolution requirements. The lateral field of view is retained.

[0041] The design options of the method according to the invention presented so far primarily concern applications in which the detection axis is directed essentially perpendicularly to the sample volume to be imaged and, for example, strikes a currently generated light sheet perpendicularly.

[0042] The proposed method is also applicable in situations where the detection axis is not perpendicular to the sample or a light sheet. In this case, significant advantages are achieved even for 2D overview images.

[0043] One example is the creation of an overview image of a sample or a sample volume in a standard sample carrier, such as Petri dishes, multi-well plates, or flat sample carriers made of glass or plastic, using an inverted light sheet microscope. The samples usually lie flat on these sample carriers and are relatively shallow compared to their spread on the sample carrier. Examples of this are biological cells, which have a sample thickness H of approximately 10–30 µm and a lateral extension up to the millimeter range. Other organisms, such as zebrafish or worms, also usually lie flat on the sample carrier.

[0044] For this reason, the user wants to obtain an overview image that allows them to orient themselves parallel to the sample carrier and survey the adjacent cell structures, cell organelles, the cells themselves, or sections of the organism, and select a region (ROI) for high-resolution image acquisition. Such a lateral overview image should be able to be acquired quickly.

[0045] For this purpose, a light sheet can be directed into the sample volume in the second operating mode. The sample rests on a surface of the sample carrier that serves as a support surface. The sample volume is therefore limited in one direction by the support surface.

[0046] The illumination axis forms an angle α of less than 90° with the support surface, thus the support surface and the sample volume are illuminated obliquely with the light sheet. The detection axis is perpendicular to the light sheet and thus also oblique to the support surface.

[0047] To achieve the second depth of field, for example, the numerical aperture of the detection beam path is reduced. At the same time, the extension of the light sheet in the direction of the detection axis is increased so that it coincides at least with the second depth of field. A number of images can be acquired, with the light sheet being displaced relative to the sample volume for each image acquisition, which advantageously corresponds at most to the second depth of field.

[0048] To obtain a 2D overview image, the 3D information must then be transformed into a plane that is as easy to handle as possible for the user. In the case of an inverted light sheet microscope, this is usually a plane parallel to the support surface of the sample carrier. This transformation can, for example, be a projection. Alternatively, a 3D overview can be displayed, as described above for the orthogonal case.

[0049] Ideally, projection occurs in the direction of the lens's detection axis, allowing the lens to achieve its highest possible resolution. The image data is mathematically projected onto the plane of the support surface with only minimal degradation due to the applied geometry factor.

[0050] The method according to the invention can also be used for systems with a lens changing device. This allows lens changes to be avoided, for example, to circumvent positioning tolerances or to simplify the workflow. The disadvantage of a constant image field is accepted.

[0051] The invention is explained in more detail below using exemplary embodiments and figures. They show: Fig. 1 a schematic representation of a first embodiment of a microscope with a detection beam path and a pivotable optical element; Fig. 2 a schematic representation of a second embodiment of a microscope with a detection beam path and a pivotable optical element; Fig. 3 a schematic representation of a third embodiment of a microscope with a detection beam path and a pivotable optical element; Fig. 4 a schematic representation of a microscope with a detection beam path and a closable optical element; Fig. 5 a schematic representation of a microscope with an additional refractive element in the detection beam path between the objective and the sample volume; Fig. 6 a schematic representation of a microscope with a scanner; Fig. 7 a schematic representation of a microscope with a detection beam path and an illumination beam path for the simultaneous generation of at least two light sheets along the detection axis and for the detection radiation of both light sheets; Fig. 8 shows a schematic representation of a first embodiment of the method according to the invention; Fig. 9 a schematic representation of a second embodiment of the method according to the invention and Fig. 10 a schematic representation of the implementation of the second embodiment of the method according to the invention.

[0052] In the Fig. Figures 1 to 7 schematically illustrate exemplary embodiments of microscopes 1 that incorporate modifications for implementing the method according to the invention. The relevant beam paths and technical components are highlighted, and additional technical components are omitted for clarity. The same reference numerals denote the same technical elements unless expressly stated otherwise.

[0053] In a first embodiment ( Fig. 1), a detection beam path 2 is present in the microscope 1, in which an objective 4 for capturing detection radiation, at least one optical lens 5, for example a tube lens, and a detector 7 are arranged along a detection axis 3, and in an image plane 6. The detection axis 3 is directed into a sample space (not shown in detail), in which a sample to be imaged with a sample volume 8 is placed.

[0054] Furthermore, a control unit 9 and drive 10 are provided, by means of which an optical element 11 can be introduced into and removed from a pupil plane 12 of the detection beam path 2 in a controlled manner (symbolized by the double arrow). The control unit 9 is connected to the detector 7, the drive 10, and an illumination device in a manner suitable for data exchange. The control unit 9 is also connected to a display or screen 24 on which the acquired image data of the operating states, an overview image, and / or raw image data can be displayed.

[0055] Furthermore, an illumination beam path 13 of the illumination device is provided, of which only one illumination lens 14 is shown, with which an illumination radiation is shaped into a light sheet 16 and directed along an illumination axis 15 into the sample volume 8. The plane of the light sheet 16 determines a sample plane 17 currently to be imaged. The light sheet 16 illuminates the sample plane 17 and generates detection radiation through reflections and / or through excitation of light emissions, in particular fluorescent radiation. This detection radiation can be partially collected by the lens 4, which functions as a detection lens, and guided along the detection axis 3 to the detector 7 in the detection beam path 2, where it can be captured or imaged as an image of the detection radiation.

[0056] The Fig. Figure 1 shows the detection beam path 2 in a first operating state. The optical element 11 is a diaphragm that is pivoted out of the detection beam path 2. In this first operating state, the detection beam path 2 has a first numerical aperture NA1 (NA1 = 1.0). Due to this first numerical aperture NA1, the image is captured with a first depth of field ST1 and a lateral image field. The light sheet 16 lies in the area of ​​the first depth of field ST1.

[0057] To effect a second operating state of the detection beam path 2, the control unit 9 generates a control command and outputs it to the drive 10, which in turn inserts the optical element 11 into the detection beam path 2 (shown with a dashed solid line). Due to the action of the optical element 11, the detection beam path 2 now has a second numerical aperture NA2, which is smaller (NA2 = 0.3) than the first numerical aperture NA1. This reduced second numerical aperture NA2 causes an increase in the depth of field, so that the detection beam path 2 now has a second depth of field ST2. The extent of the lateral image field is the same as that in the first operating state. In further embodiments of the invention, the first or second operating state can also be manually adjustable additionally or alternatively.

[0058] At the same time, a light sheet 16 is generated in the illumination beam path 15, the extent of which in the direction of the detection axis 3 - also referred to as thickness - is at least as large as the second depth of field ST2 and which lies in the area of ​​the second depth of field ST2.

[0059] It is now possible to move the light sheet 16 relative to the sample volume 8 with step sizes (see Fig. 8 to 10) to positions where sample planes 17 that barely overlap or are adjacent to one another are illuminated and images of the sample planes 17 can be acquired. The sample volume 18 can be scanned more quickly with the light sheets 17 of increased thickness than with a light sheet 16 whose thickness is designed for high-resolution image acquisition.

[0060] In further embodiments of the microscope and further embodiments of the method, the optical element 11 can also be a slit diaphragm or a rectangular diaphragm instead of a pinhole diaphragm.

[0061] If a change in the depth of field is to be carried out using the EDoF principle, the optical element 11 can be designed in the form of a ring diaphragm in the pupil plane 12. To implement the EDoF principle, the optical element 11 can also comprise several elements and, for example, be formed by an axicon and a convex lens arranged downstream of the axicon, as shown in Fig. 1 is symbolically shown in the image inset. When applying the EDoF principle, the numerical aperture of detection beam path 2 is not changed or only optionally changed.

[0062] In a further embodiment, the optical element 11 can be a Spatial Light Modulator (SLM), as shown in Fig. 2 is shown in simplified form (see also King, SV et al. (2015), Spatial light modulator phase mask implementation of wavefront encoded 3D computational-optical microscopy; Applied Optics 54: 8587 - 8595). The optical element comprises a mirror M1, a spatial light modulator SLM (spatial light modulator), and a mirror M2. Mirror M1 reflects the light onto the spatial light modulator SLM and from there via mirror M2 back onto the detection axis 3. The numerical aperture is maintained.

[0063] Instead of using a reflective SLM, such as an LCOS (liquid crystal on silicon) SLM, a transmissive SLM can be used. This is placed in the beam path and controlled accordingly. Mirrors M1 and M2 can be omitted.

[0064] In a further possible embodiment of the microscope 1 and an embodiment of the method, the optical element 11 is realized by an arrangement of several elements, by the effect of which different diffraction orders of the collected detection radiation are separated and focused on different areas of the detector 7 ( Fig. 3). After passing through an intermediate image plane 19, the detection radiation reaches the pupil plane 12, which is also a Fourier plane. A multifocal grating 20 is arranged in the pupil plane 12, the effect of which separates the different diffraction orders of the detection radiation. Each diffraction order corresponds to a different sample plane 17. In this embodiment of the method, the light sheet 16 contains several sample planes 17.

[0065] Downstream of the multifocal grating 20 are a chromatic correction grating 21, a prism 22, and a lens 52. The chromatic correction grating 21 and the prism 22 correct the chromatic dispersion of the detection radiation caused by the multifocal grating 20. The lens 52 focuses the diffraction orders onto the various areas of the detector 7.

[0066] In a fourth embodiment, the optical element 11 remains in the pupil plane 12 ( Fig. 4). The optical element 11 can be controlled by the control unit 9 and the drive 10 and can, for example, be a diaphragm with an adjustable hole diameter, an adjustable rectangular cutout, or an adjustable slit width. A diaphragm based on a liquid crystal element can also be used as the adjustable optical element 11. The resulting diaphragm opening can be adjusted depending on its control.

[0067] With the above-mentioned embodiments, it is possible to switch between the first depth of field ST1 and the second depth of field ST2 or a further depth of field by changing the numerical aperture of the detection beam path 2.

[0068] The EDoF principle can be implemented in a further embodiment of the microscope 1 and the method for generating the second depth of field ST2 by arranging an optical element 11 in the form of a refractive element, for example a body made of a material transparent to the detection radiation, between the objective 4 and the sample volume 8 ( Fig. 5). The refractive element can, for example, have a refractive index in a range of n = 1.0 to 2.0 and a thickness in the direction of the detection axis between 5 and 100 mm (from WO 2017 / 075275 A1). In one possible embodiment of the invention, a thin glass plate is used as the refractive element, the refractive index of which is, for example, n = 1.5 and the thickness of which is, for example, d = 0.15 to 1 mm. Fig. 3 shows the second operating state, in which the optical element 11 is pivoted into the detection beam path 2. The optical effect of the optical element 11 designed and arranged in this way results in an extension of the point image blurring function in the direction of the detection axis 3. The thickness of the light sheet 16 is, in turn, adapted to the first or second operating state and the respective depths of field ST1 or ST2.

[0069] Instead of using two light sheets 16.1, 16.2 or a thick light sheet 16 to illuminate the depth of field, a thin light sheet 16 can also be used, which is scanned along the optical axis 3 of the detection beam path 2 during the detection period of the detector 7, for example the exposure time of a camera. For the controlled generation of the scanning movement of the illumination radiation, a scanner 18 is arranged in the illumination beam path 3. The scanner 18 can be controlled by means of the control unit 9. Since the detector 7 is integrated over the detection period, in the case of a camera over the exposure time, an effect is achieved with this embodiment that corresponds to the use of a thick light sheet. In the Fig. 6, such a scanning movement along the optical axis 3 is symbolized by a double arrow in the illumination lens 14.

[0070] The second depth of field ST2 can also be achieved in further embodiments of the method by simultaneously directing at least two light sheets 16, for example a first light sheet 16.1 and a second light sheet 16.2, into the sample volume 8. In the Fig. In the simplified embodiment shown in Figure 7, each of the light sheets 16 is aligned on one side of the sample plane 17 and parallel to it. The light sheets 16 adjoin one another in the sample plane 17 (shown with emphasized thick light sheets 16.1, 16.2 for clarity).

[0071] In a further possible embodiment, at least two light sheets 16 are arranged offset from one another along the detection axis 3 and perpendicular to it (not shown).

[0072] The principle of the method according to the invention is based on the Fig. 8 to 10. In a first embodiment of the procedure according to the Fig. 8, the detection axis 3 is directed perpendicular to the sample volume 8.

[0073] The sample rests on a support surface 23.1 of a sample carrier 23. The sample volume 8 is limited in one direction by the support surface 23.1 with regard to its possible extent. For further explanation, a Cartesian coordinate system is used whose x-axis and y-axis are orthogonal to each other and each parallel to the support surface 23.1, while the detection axis 3 runs in the direction of the z-axis. The sample volume 8 has a height H. The light sheet 16 is shown at a first position P1, which it occupies, for example, during the first operating state. The light sheet 16 is generated parallel to the support surface 23.1 in an xy plane, so that in the Fig. 8 to 10 only lateral cross-sections of the light sheet 16 are visible. The detection beam path 2 has a first numerical aperture NA1 = 1.0. The corresponding point spread function PSF 1,0 is in Fig. 8 is schematically shown as an oval next to the sample volume 8 with respect to its lateral (xy plane) and axial (z-axis direction) extent and indicates the first depth of field ST1. In order to completely scan the sample volume 8 in the direction of the detection axis 3 with the first numerical aperture NA1, a first step size SW1 would be required, with which the light sheet 16 could be moved in the axial direction relative to the sample volume 8, for example, into a second position P2 1,0 would have to be postponed.

[0074] If, however, the second operating state is established and the detection beam path 2 is operated with a second numerical aperture NA2 = 0.3, the result is a point spread function PSF 0,3which is considerably longer in the axial direction than the point spread function PSF 1,0 and indicates the second depth of field ST2.

[0075] To completely scan the sample volume 8 in the direction of the detection axis 3, a second step size SW2 that is larger than the first step size SW1 would be possible. Reducing the required number of image acquisitions and evaluations enables rapid provision of an overview image of the sample volume 8.

[0076] The first step size SW1 and the second step size SW2 each represent maximum step sizes that allow a continuous scanning of the sample volume 8 using the light sheet 16. Smaller step sizes can also be selected, in which case more images must be acquired for a complete scan.

[0077] In the Fig. 9 again shows a sample with a sample volume 8 on a support surface 23.1 of a sample carrier 23. The detection axis 3, along which the detection radiation is collected and guided, is directed obliquely onto the sample volume 8. The detection axis 3 and the support surface 23.1 enclose an angle α, which in the exemplary embodiment is α = 60°. As already Fig. As described in Figure 8, the first depth of field ST1, assuming a first numerical aperture of NA1 = 1.0, is smaller than the second depth of field ST2, assuming a second numerical aperture of NA2 = 0.3, in a second operating state. To completely scan the sample volume 8, the light sheet 16 must again be shifted with the step sizes SW1 and SW2, respectively.

[0078] In order to be able to display an overview image in an xy plane parallel to the support surface 23.1 to a user of the microscope 1 and the user of the method according to the invention, the acquired image data are converted into this xy plane and can then be displayed on a display or screen 24. Alternatively, a three-dimensional display is possible.

[0079] In the Fig. 10 shows the light sheet 16 at the first position P1. The detection beam path 2 is in the second operating state and has a second depth of field ST2, a numerical aperture NA = 0.3 and a corresponding point spread function PSF. 0,3 After detection of the detection radiation at the first position P1, the light sheet 16 is moved parallel to the support surface 23.1 by the second step size SW2 to the second position P2 0,3 shifted and detection radiation is recorded again. The point spread functions PSF0,3 at both positions P1 and P2 0,3 are directly adjacent to each other, so that despite the large second step size SW2, a gapless scanning of the sample volume 8 is possible. For illustration, the sections of the sample volume 8 are delimited by broken solid lines, which are located at the positions P1 and P2 0,3 be recorded.

[0080] The displacement of the light sheet 16 between the individual positions P1 and P2 0,3 along the detection axis 3 and parallel to the support surface 23,1 leads to “slices” of image data arranged next to one another, which can be projected into the xy plane or displayed three-dimensionally with little computational effort. Reference symbol 1 microscope 2 Detection beam path 3 detection axis 4 Lens 5 optical lens 6 Image plane 7 Detector 8 sample volumes 9 Control unit 10 Drive 11 optical element 12 pupil plane / Fourier plane 13 Illumination beam path 14 Illumination lens 15 Lighting axis 16 light sheets 16.1 first light sheet 16.2 second light sheet 17 Sample level 18 scanners 19 Intermediate image plane 20 multifocal grid 21 chromatic correction grid 22 Prism 23 sample carriers 23.1 Support surface 24 screen 52 optical lens BS beam splitter H Sample height M Mirror P1 first position of the light sheet P2 second position of the light sheet PSF point spread function SLM spatial light modulator ST1 first depth of field ST2 second depth of field SW step size α angle (between detection axis 3 and support surface 23.1)

Claims

[1] Method for generating an overview image using a high-aperture lens (4) with the steps: - Providing an optical detection beam path (2) with the objective (4), wherein the detection beam path (2) in a first operating state has a first numerical aperture (NA1) and a first depth of field (ST1) which lies substantially in a focal plane of the detection beam path (2), and in the first operating state an image of a detection radiation with the first depth of field (ST1) and with a lateral image field is or can be detected, wherein the detection radiation is effected by directing at least one light sheet (16) along an illumination axis (15) into a sample volume (8) to be imaged located in the sample space; - transferring the detection beam path (2) into a second operating state by increasing the first depth of field (ST1) to a second depth of field (ST2), wherein the high-aperture lens (4) remains in the detection beam path (2), - adjusting a thickness of the light sheet (16) in the direction of a detection axis (3) of the lens (4) to the second depth of field (ST2) using an adjustable illumination device, so that the thickness of the light sheet (16) corresponds at least to the second depth of field (ST2); - in the second operating state, detecting a detection radiation, wherein the detection radiation coming from a sample space is collected by means of the objective (4), guided along the detection beam path (2) and detected as an image with a lateral image field by means of at least one detector (7) sensitive to the detection radiation, wherein the lateral image fields are the same in the first and second operating states. [2] Method according to claim 1, characterized by that in the second operating state the second depth of field (ST2), and thus the thickness of the light sheet (16), is increased by at least a factor of two compared to the first depth of field (ST1). [3] Method according to claim 1 or 2, characterized by that the second depth of field (ST2) is achieved by reducing the first numerical aperture (NA1) of the detection beam path (2) to a second numerical aperture (NA2). [4] Method according to claim 3, characterized by that an optical element (11) in the form of a pinhole, slit or rectangular diaphragm is introduced into a pupil plane (12) of the detection beam path (2), the effect of which brings about the second numerical aperture (NA2) of the detection beam path (2). [5] Method according to claim 1, characterized bythat the second depth of field (ST2) is achieved according to the principle of the increased focal length by introducing a phase mask, an axicon, a ring diaphragm in a pupil plane or an additional refractive optical element for the axial extension of the point image blurring function into the detection beam path (2) as an optical element (11). [6] Method according to claim 1, characterized by that the second depth of field (ST2) is achieved by simultaneously detecting detection radiation from several sample planes (17) lying parallel to one another. [7] Method according to claim 6, characterized by , that - a sample volume (8) to be imaged is illuminated with a first light sheet (16.1) and at least one further light sheet (16.2) arranged parallel to the first light sheet (16.1) along a respective illumination axis (15), wherein the light sheets (16.1, 16.2) are arranged one behind the other along a detection axis (3) perpendicular to the illumination axis (15) and overlap one another at most proportionally; or - a multifocal grating (20) is introduced into a pupil plane (12), a detection radiation comprising several wavelengths is split into components according to their diffraction orders, and the components of the diffraction orders are directed simultaneously onto different areas of an image plane (6); or - a sample volume (8) to be imaged is illuminated with a first light sheet (16.1) and at least one further light sheet (16.2) arranged parallel to the first light sheet (16.1) in an illumination direction and along a respective illumination axis (15), and detection radiation caused by the respective light sheets (16.1, 16.2) is detected along a detection axis (3), wherein the illumination axes (15) and the detection axis (3) each enclose an angle different from zero, and each further light sheet (16.2) is shifted in the detection direction, i.e. along the detection axis (3), as well as in the illumination direction to the first light sheet (16.1), and the detection radiation is detected simultaneously. [8] Method according to claim 7, characterized bythat after the acquisition of an image of the sample volume (8) in the second operating state with a first position of the light sheet (16), the light sheet (16) is displaced relative to the sample volume (8) and a further image is acquired. [9] Method according to claim 7, characterized by that the sample volume (8) is limited in one direction by a support surface (23.1) of a sample carrier (23) and after the acquisition of an image of the sample volume (8) with a first position of the light sheet (16), the light sheet (16) is displaced relative to the sample volume (8) and parallel to the support surface (23.1) and a further image is acquired.

Images