LIGHTING MODULE FOR ANGLE-SELECTIVE LIGHTING

Description

TECHNICAL AREA

[0001] Various embodiments of the invention relate to a laser scanning microscope and a microscope. In various embodiments, an illumination module for a corresponding optical device comprises a carrier with multiple light sources and optionally at least one light-transmitting area. BACKGROUND

[0002] US patent 2008 / 062424 A1 discloses a compact ring light that mimics the performance of a much larger ring light. It utilizes a ring light source and a conical or cylindrical reflector, causing the light to first cross the optical axis and then be reflected back towards the inspection area. This light is particularly useful for inspecting electronic semiconductor devices.

[0003] US Patent 2007 / 211460 A1 describes a multi-colored LED light source for transmitted light illumination that is compatible with most microscopes. The light source provides spatially and angularly uniform monochromatic illumination in red, green, or blue, or combinations of these colors. Wavelength changes are very rapid, which is desirable for automated sample scanning. The light source is inexpensive and compact; it can be housed in the condenser compartment of a typical microscope, eliminating the need for the standard white light source, condenser, and light filtering system. Various methods for shaping the light for illuminating microscopic samples are also provided. Furthermore, a method for acquiring full-color images using the existing light source and a monochrome camera is also provided.

[0004] EP 1 698 927 A1 discloses a confocal laser scanning microscope with an additional illumination source (halogen lamp) for transmitted light imaging of the sample. A mechanical tilting mirror can be used to switch between transmitted light illumination and transmitted light detection.

[0005] The operating instructions (Carl Zeiss LSM 700 Operating Manual, XP093155520 dated 1.9.2011) describe a halogen lamp.

[0006] Techniques for illuminating a sample from different lighting directions (angle-selective illumination or structured illumination) are known from DE 10 2014 112 242 A1. By combining the images obtained for the different lighting directions, a result image can be determined which, for example, exhibits phase contrast. Therefore, phase contrast imaging is possible using angle-selective illumination and subsequent digital post-processing.

[0007] It can sometimes be difficult to combine suitable illumination modules for angle-selective illumination with conventional optical devices, such as a laser scanning microscope (LSM) or a light microscope with a lens. This can be the case because such conventional optical devices often have limited installation space. SUMMARY

[0008] Therefore, there is a need for improved implementations of lighting modules for angle-selective illumination. In particular, there is a need for suitable lighting modules that can be flexibly integrated into the beam path of an optical device.

[0009] This problem is solved by the features of a laser scanning microscope as defined in the independent claim. The dependent claims define embodiments.

[0010] For example, an LSM comprises a first illumination module with a laser light source. The LSM also comprises a second illumination module with a support and with multiple light sources arranged on the support.

[0011] For such an LSM, aspects of angle-selective illumination can be combined with laser scanning imaging.

[0012] For example, the LSM can be configured for fluorescence imaging. For this purpose, the LSM includes a sample holder. The sample holder secures a sample object, such as a biological sample or a cell culture, in the beam path. The sample object can be, for example, a phase object that exhibits a significant phase change with respect to transmitted light. The laser light can excite fluorescence processes in the biological sample. The detector can then be configured to detect the corresponding fluorescence signal from the sample object.

[0013] For example, the detector can be designed as a photomultiplier (PMT). The detector can be arranged in either a transmission or reflection geometry, meaning it can be positioned behind (transmission beam path) or in front of (reflection beam path) the first illumination module of the sample holder. If the detector is arranged in the transmission beam path, a bright-field transmission image can be generated, for example.

[0014] For example, it is possible that the detector's detection spectrum differs from the laser light source's emission spectrum. This can occur because fluorescence processes can cause a wavelength shift between absorbed and emitted light. However, it is also possible that the detector's detection spectrum overlaps, at least partially, with the laser light source's emission spectrum.

[0015] It is generally possible for the LSM to include more than one detector for detecting the fluorescence signal. For example, a first detector could be located in the transmission beam path and a second detector in the reflection beam path. The second detector could also be supplemented by an Airy unit. Furthermore, the second detector could be positioned behind a scanning optic of the LSM relative to the sample holder.

[0016] For example, the support can be arranged in a beam path defined by the laser light source. For instance, the support could have an extent perpendicular to the beam path. The support could therefore extend in a plane perpendicular to the beam path. Alternatively, the support could extend in a plane that forms an angle between 0° and 90° with a central ray of the beam path. The support could thus be tilted relative to the beam path. It is also possible that the support could have a certain extent (thickness) parallel to the beam path.

[0017] In exemplary implementations, it is possible for the support to have at least one translucent area. This at least one translucent area can, for example, be located within an outer circumference of the support. In various examples, the at least one translucent area can be positioned within the support, i.e., spaced away from outer edges or a circumference of the support. Thus, an outer circumference of the support can enclose the at least one translucent area.

[0018] The at least one translucent region can, for example, allow the transmission of a significant proportion of light in a specific spectral range perpendicular to the substrate, i.e., along its thickness. In particular, the translucent region can, for example, have a transmittance greater than 20%, preferably >80%, and more preferably >90%. It is generally not necessary for the translucent region to have a correspondingly high transmittance over a particularly large spectral range. For example, it would be possible for the translucent region to have a significant transmittance only in a specific spectral range and a comparatively low transmittance in other spectral ranges.

[0019] For example, the beam path can pass at least partially through the at least one transparent area. It would therefore be possible for the second illumination module to be arranged in the beam path defined by the laser light source. For example, the second illumination module could be arranged concentrically with a central beam of the beam path.

[0020] By incorporating a second lighting module with a translucent area, angle-selective lighting techniques can be flexibly integrated into the LSM design. In particular, it may be possible to position the second lighting module within the beam path. This is because the translucent area ensures that the beam path is not altered, or not significantly altered, by the second lighting module. Various techniques can be used to create the translucent area within the support. The different examples described here for creating the translucent area can also be combined in various scenarios.

[0021] One example involves implementing the translucent area as a hole. This hole could, for instance, be a through-hole. In other words, it might be possible to implement the translucent area as a recess in the substrate material.

[0022] A hole that is arranged off-center on the support has a center point that is a certain distance from the center of the support.

[0023] According to the claimed invention, the center of the support is defined by the point on the support that is determined by a central ray of the beam path. The hole can have lateral dimensions of a plane defined by a surface of the support, which correlate with a beam width of the beam path in the region of the second illumination module. For example, the lateral dimensions of the hole can be more than 50% of the beam width of the beam path, preferably more than 90%, and particularly preferably more than 120%.

[0024] By implementing the translucent area as a hole, the transmittance of the light-transmitting area of ​​the carrier can be made particularly high. Furthermore, the carrier can be manufactured easily, for example by drilling or milling.

[0025] In some examples, the hole can remain unobstructed when the second illumination module is placed in the LSM; that is, no other optically active elements can be located within the hole. However, in other examples, optical elements such as a lens, a mirror, a beam splitter, a grating, a filter, etc., could be located at least partially within the hole. For instance, in some examples, the detector could be located at least partially within the hole. The detector could therefore extend at least partially within the hole. For example, a sensitive area or sensor surface of the detector could have a surface oriented parallel to the surface of the support. Similarly, the hole could have lateral dimensions parallel to the surface of the support that correlate with the lateral dimensions of the detector's sensitive area.

[0026] Using such techniques, it is possible to ensure a particularly space-saving implementation. In particular, it may be possible to place the second illumination module in close contact with the detector, thus ensuring a high degree of integration.

[0027] Such an implementation of a combined second illumination module / detector can be particularly desirable with respect to a PMT in transmission geometry, i.e., for a scenario in which the detector and the second illumination module are positioned behind the sample holder relative to the first illumination module. In this way, bright-field fluorescence imaging, for example, can be performed using the detector.

[0028] In other examples, the second illumination module is not located in the beam path, but rather in front of the sample holder relative to the first illumination module. For example, the second illumination module could be positioned between a scan optic of the LSM and a lens of the LSM, either directly in the beam path or reflected in it. The scan optic and the specimen are positioned in front of the sample holder relative to the first illumination module. In this way, efficient illumination by the second illumination module can be achieved because the scan optic does not need to be traversed.

[0029] For example, the second illumination module could be positioned in an area between the lens and the scan optics where the beam path length is comparatively short – for instance, compared to other areas of the beam path between the scan optics and the lens. For example, the second illumination module could be positioned in the area between the lens and the scan optics near or at an intermediate image plane.

[0030] It would also be possible to arrange the second illumination module between the lens and the scan optics, reflected into the beam path. For this purpose, a partially reflective mirror could be used as the coupling element, forming a specific angle with the beam path. Alternatively or additionally, a dichroic filter could be used for coupling, for example, if different wavelength ranges are being utilized.

[0031] The detector can have a detection spectrum that differs, at least partially, from the emission spectrum of the light sources in the second illumination module. This allows, for example, fluorescence imaging to be performed in such a way that illumination of the sample by the multiple light sources of the second illumination module does not excite fluorescence processes, or only to a minimal extent. This ensures that the fluorescence imaging is not distorted, or not significantly distorted, by illumination using the light sources of the second illumination module. Consequently, it may be possible to perform phase-contrast imaging alongside fluorescence imaging without significantly reducing the accuracy of the fluorescence imaging.

[0032] The detectors of the LSM described above can be used to perform phase contrast imaging in various examples. In other words, it may be possible in different scenarios to use the detector(s) for imaging with illumination from the first illumination module as well as for imaging with illumination from the second illumination module. For example, a PMT can be used for phase contrast imaging with illumination from the second illumination module and based on angle-selective illumination techniques. In such an example, it may be unnecessary to provide a dedicated detector for angle-selective illumination techniques; this, in turn, reduces the required installation space.

[0033] In other examples, the LSM may include an additional detector. This additional detector can be configured for angle-selective imaging techniques. For example, it may have a comparatively high resolution, such as that of a PMT. Specifically, it may be configured for imaging when the sample is illuminated by the second illumination module. If the additional detector does not need to detect fluorescence signals, its sensitivity may be comparatively low, for example, compared to a PMT. In such cases, the additional detector may have a detection spectrum that at least partially overlaps with the emission spectrum of the light sources in the second illumination module. For instance, the additional detector could be a CCD sensor.Alternatively or additionally, the second detector could also include a CMOS sensor. This second detector could, for example, be operated as a camera. Including this second detector allows for optimized image acquisition with regard to phase contrast imaging.

[0034] In various examples, the additional detector can be positioned in a wide range of locations. In particular, its positioning can vary depending on the positioning of the second illumination module. Fundamentally, the additional detector could be operated in either transmission or reflection geometry relative to the second illumination module and the sample holder. For instance, the additional detector could be positioned between the scan optics and the objective lens. Alternatively, it could be located in the region of an intermediate image plane. For example, a beam splitter could be provided for the additional detector, thus creating a corresponding beam path.

[0035] The various light sources of a lighting module can be controlled separately. This means that the different light sources can be operated, for example, by separate control signals if needed. In the examples described herein, different light sources can be used to implement angle-selective lighting. For example, the light sources can be selected from the following groups: organic light-emitting diodes (OLEDs); solid-state LEDs (SLEDs); LEDs; halogen light sources; and laser diodes. It is possible, for example, that organic light-emitting diodes and halogen light sources have a larger lateral dimension parallel to a surface of the substrate than LEDs. By providing at least one translucent area, flexibility can be achieved with regard to the light sources arranged outside this area.

[0036] For example, the light sources could be arranged in a circular or ring shape.

[0037] For example, the light sources can be arranged in a matrix structure, i.e., with a one-dimensional or two-dimensional periodicity within a plane defined by a surface of the substrate. The matrix structure can correspond to a grid structure of the arrangement. In this case, the matrix structure can, for example, define a square, rectangular, or polygonal unit cell of the corresponding grid structure. By using the matrix structure, particularly flexible angle-selective illumination can be employed. In this way, particularly informative phase-contrast imaging can be implemented.

[0038] For example, it would be possible for the light sources to be arranged on a dome-shaped surface of the support (lighting dome).

[0039] In various examples, the distance between adjacent light sources can vary for angle-selective illumination. For instance, the light sources could be arranged on a substrate surface with a geometric fill factor of no more than 90%, preferably no more than 50%, and most preferably no more than 30%. In this way, it is possible for very little of the incident light to be absorbed or reflected by the light sources.

[0040] For example, the substrate could be made of a translucent material, such as a solid, at least in the translucent area. For example, the substrate could be made of glass, at least in the translucent area. For example, the substrate could be made of transparent plastic film, at least in the translucent area. For example, the different light sources could be arranged on different sections of the plastic film. Particularly in conjunction with a relatively low geometric fill factor, with which the light sources are arranged on the substrate's surface, the use of a translucent material can ensure that the translucent area is formed between adjacent light sources and is comparatively large.In this way, it can be achieved that very little light from the beam path incident on the surface of the carrier is reflected or absorbed.

[0041] For example, the light sources could be arranged rotationally symmetrically with respect to a centrally located axis perpendicular to a surface of the substrate. Different light sources could, for instance, be positioned at different distances from this axis. In this way, it might be possible to implement the translucent area in a central region of the substrate. In particular, it might be possible to dimension the translucent area relatively large. Furthermore, such a rotationally symmetrical arrangement of light sources could enable the particularly efficient implementation of angle-selective illumination techniques.

[0042] In one example, the light sources could be positioned outside the translucent area on the substrate. In such a case, the translucent area could be formed by an element other than the light sources, for example, as described above, by a hole, a translucent solid material, etc.

[0043] In another example, it would also be possible for the light sources to at least partially form the at least one translucent area. For instance, the light sources could be implemented as organic light-emitting diodes (OLEDs), which are translucent. This would make it possible, on the one hand, to implement a particularly large translucent area, while simultaneously allowing for a particularly flexible arrangement of the light sources.

[0044] Such microscopes or laser scanning microscopes as described above can be used for angle-selective illumination. This makes it possible to generate a phase-contrast image of an object (result image).

[0045] In angle-selective illumination according to the claimed invention, the light sources are controlled separately and / or sequentially to generate light. This allows the illumination of an object from multiple directions. Illuminating the object from a specific direction involves controlling at least one light source of the illumination module. By controlling the light sources separately and / or sequentially, the object's image can be separated for the different illumination directions in corresponding measurement images. With sequential illumination of the object, the different measurement images can also be acquired sequentially, thus separating the illumination directions over time.Alternatively or additionally, the lighting directions can be separated by using different spectral ranges of light for the different lighting directions. Alternatively or additionally, the lighting directions can also be separated by using different polarizations of light for different lighting directions. In such cases, corresponding measurement images can also be acquired at least partially in parallel; the separation can be achieved by using appropriate color filters and / or polarization filters. Single-shot measurements may be possible.

[0046] According to the invention, a corresponding laser scanning microscope comprises a computing unit configured to control multiple light sources for the separate and / or sequential illumination of an object from multiple illumination directions. The computing unit can further be configured to combine multiple measurement images associated with the illumination directions to obtain a result image exhibiting phase contrast.

[0047] According to the invention, the lighting module comprises a carrier. The carrier may have a translucent area. The lighting module also comprises several light sources arranged on the carrier.

[0048] According to another example of the invention, the lighting module comprises a carrier, several light sources arranged on the carrier, and at least one detector mounted on the carrier. For example, an emission spectrum of the several light sources can be at least partially overlapping with a detection spectrum of the at least one detector. For example, it would be possible for the at least one detector to be mounted centrally or decentrally on the carrier.

[0049] According to the invention, the at least one detector is located at a distance from the center of the carrier, wherein at least one light source of the module is arranged between the center of the carrier and the at least one detector. BRIEF DESCRIPTION OF THE FIGURES

[0050] The properties, features and advantages of this invention described above, as well as the manner in which they are achieved, will become clearer and more easily understood in connection with the following description of the exemplary embodiments, which are explained in more detail in conjunction with the drawings. FIG. 1 is a schematic top view of a lighting module for angle-selective lighting and with a carrier that has a light-transmitting area according to various embodiments. FIG. 2 is a schematic side view of the lighting module for angle-selective lighting according to FIG. 1 . FIG. 3 is a schematic side view of the lighting module for angle-selective lighting according to FIG. 1 , where in the example the FIG. 3 a detector is partially located inside the hole. FIG. 4 schematically illustrates an LSM according to various embodiments, where in the example the FIG. 4 The lighting module is arranged for angle-selective illumination and with the carrier in the area between a scan optic of the LSM and a lens of the LSM in the beam path of a laser light source of the LSM. FIG. 5 schematically illustrates an LSM according to various embodiments, where in the example the FIG. 5 The illumination module for angle-selective illumination is arranged with the carrier in relation to the laser light source of the LSM behind a sample holder of the LSM in a transmission beam path, wherein in the example the FIG. 5 Furthermore, the detector is partially formed in the hole located centrally on the carrier. FIG. 6A schematically illustrates an LSM according to various embodiments not covered by the claimed invention, wherein in the example the FIG. 6A The lighting module is arranged for angle-selective illumination and is reflected into the beam path of the laser light source in the area between a scan optic of the LSM and a lens of the LSM. FIG. 6B schematically illustrates an LSM according to various embodiments, where in the example the FIG. 6B A further detector is arranged between the scan optics and the lens of the LSM, wherein the further detector is set up for angle-selective illumination of a sample object when illuminated by the illumination module. FIG. 7 schematically illustrates a conventional LSM that includes an illumination module with a halogen light source for phase contrast imaging. FIG. 8A Figure 1 schematically illustrates a microscope which does not fall under the claimed invention, but whose features can contribute to a better understanding of the invention, with a camera according to various embodiments, wherein the illumination module for angle-selective illumination is arranged in a transmission beam path associated with the camera. FIG. 8B Figure 1 schematically illustrates a microscope which does not fall under the claimed invention, but whose features can contribute to a better understanding of the invention, with a camera according to various embodiments, wherein the illumination module for angle-selective illumination is arranged in a transmission beam path associated with the camera. FIG. 8C Figure 1 schematically illustrates a microscope which does not fall under the claimed invention, but whose features can contribute to a better understanding of the invention, with a camera according to various embodiments, wherein the illumination module for angle-selective illumination is arranged in a transmission beam path associated with the camera. FIG. 9A Figure 1 schematically illustrates a microscope which does not fall under the claimed invention, but whose features can contribute to a better understanding of the invention, according to various embodiments, wherein the illumination module for angle-selective illumination is arranged in a beam path associated with the camera. FIG. 9B Figure 1 schematically illustrates a microscope which does not fall under the claimed invention, but whose features can contribute to a better understanding of the invention, according to various embodiments, wherein the illumination module for angle-selective illumination is arranged in a beam path associated with the camera and an imaging optic is arranged in the area of ​​a hole arranged centrally on the support. FIG. 9C Figure 1 schematically illustrates a microscope which does not fall under the claimed invention, but whose features can contribute to a better understanding of the invention, according to various embodiments, wherein the illumination module for angle-selective illumination is arranged in a beam path associated with the camera and the camera is coupled to the illumination module. FIG. 10A Figure 1 schematically illustrates a microscope which does not fall under the claimed invention, but whose features can contribute to a better understanding of the invention, according to various embodiments, wherein the illumination module for angle-selective illumination is arranged in a detection beam path. FIG. 10B schematically illustrates the lighting module for angle-selective lighting and with the carrier and the multiple light sources according to various embodiments, wherein the light-transmitting area is designed as a hole arranged decentrally on the carrier. FIG. 11 Figure 1 schematically illustrates the lighting module for angle-selective lighting according to various embodiments, wherein the carrier of the lighting module is made of translucent material and the light sources of the lighting module are arranged on the surface of the carrier with a low geometric fill factor. FIG. 12 The figure schematically illustrates the lighting module for angle-selective lighting, wherein the light-transmitting area of ​​the lighting module is designed as a hole arranged centrally on the carrier, which is not covered by the claimed invention. FIG. 13 The figure schematically illustrates the lighting module for angle-selective illumination, wherein the multiple light sources of the lighting module are implemented by organic light-emitting diodes, and wherein the lighting module has a central hole, which is not part of the invention. FIG. 14 The figure schematically illustrates the lighting module for angle-selective lighting, wherein the multiple light sources are implemented by halogen light sources, and wherein the lighting module has a central hole, which is not part of the invention. FIG. 15 The figure schematically illustrates the lighting module for angle-selective lighting according to various embodiments, wherein the light-transmitting area and the multiple light sources are implemented by organic light-emitting diodes, wherein the lighting module has a central hole, which is not part of the invention. FIG. 16 is a schematic top view of a lighting module for angle-selective illumination and with a carrier on which, according to various embodiments, a detector is mounted, wherein, in contrast to the claimed invention, the carrier has no light-transmitting area and the detector is arranged centrally on the carrier. FIG. 17 is a schematic side view of the lighting module for angle-selective lighting according to FIG. 16 . FIG. 18 schematically illustrates an optical device according to various embodiments with a detector and a computing unit. FIG. 19 is a flowchart of a process according to different embodiments. DETAILED DESCRIPTION OF EXECUTION FORMS

[0051] The present invention is now explained in more detail with reference to preferred embodiments and the drawings. In the figures, identical reference numerals denote identical or similar elements. The figures are schematic representations of various embodiments of the invention. Elements depicted in the figures are not necessarily shown to scale. Rather, the various elements shown in the figures are represented in such a way that their function and general purpose are understandable to a person skilled in the art.

[0052] The following describes techniques related to an illumination module that can be used for angle-selective illumination of a sample object. The illumination module comprises several light sources arranged at intervals from each other, thus enabling illumination of the sample object from multiple directions. Measurement images corresponding to the individual illumination directions can then be combined. This allows for the creation of a final image with phase contrast through digital post-processing.

[0053] Several examples illustrate the particularly flexible combination of such an illumination module for angle-selective illumination with optical devices of varying designs. Various examples describe how such an illumination module can be combined with a light microscope (LSM). Further examples describe how such an illumination module can be combined with a reflected-light microscope or a transmitted-light microscope.

[0054] For example, various examples describe how such an illumination module can be combined with a laser light source of an LSM. The illumination module could be structurally connected or coupled to a PMT arranged in a transmission geometry, which is configured to detect a fluorescence signal from a corresponding sample object. For this purpose, the illumination module can, for example, have a transparent area through which light can pass to the detector. For instance, the illumination module could be implemented as a transparent area by means of a carrier with a centrally arranged recess / hole, which is not covered by the claimed invention. In this case, it may be possible to arrange the PMT at least partially within the hole. It is also possible, for example, for the emission spectrum of the light sources of the illumination module to differ from the detection spectrum of the PMT.The detection spectrum of the PMT can be tuned to the wavelength of a laser light source and / or to the wavelength of a fluorescence signal; the emission spectrum of the light sources of the illumination module can, for example, be in the infrared spectral range.

[0055] In other examples, the lighting module could have a perforated support, with no further optical elements arranged in the hole(s). In other examples, different optical elements could be arranged in the area of ​​the at least one hole, such as a lens, a Bertrand lens with angle-selective shading, a grating, etc.

[0056] In various examples, it may be possible to combine the illumination module with an optical microscope that is not covered by the claimed invention, but whose features can be combined with the invention. The illumination module can, for example, be arranged in a detection beam path or a further beam path of the illumination module, the latter being at least partially different from the detection beam path. In various examples, it is possible to combine such an illumination module with an overview camera. The overview camera can, for example, be configured to capture an overview image of a sample holder of the microscope. A corresponding beam path associated with the camera can therefore have a comparatively lower magnification.

[0057] The microscope can be, for example, a conventional reflected-light microscope or a transmitted-light microscope. Such techniques can be used particularly in connection with wide-field microscopy, in which an overview image of a sample object is created and fine positioning takes place based on this overview image.

[0058] The individual design and arrangement of the light sources of such a lighting module for angle-selective illumination can vary in different examples. For instance, an LED array could be used as the corresponding matrix structure. In other examples, light sources with a comparatively large lateral extent could also be used, such as halogen light sources or organic light-emitting diodes (OLEDs). These could, for example, be arranged in different quadrants on the substrate with respect to a centrally located hole, which is not part of the claimed invention and forms the translucent area. It is also optionally possible for the OLEDs themselves to be translucent and thus implement the translucent area.

[0059] In various examples, it is unnecessary for the lighting module to have a translucent area. For example, in such examples, it would be possible for the lighting module to have a continuous, non-translucent support. For example, at least one detector could be applied to the support, for instance, by gluing it on. The detector could be positioned centrally or, according to the claimed invention, decentrally on the support.

[0060] Various effects can be achieved using such techniques. For example, it may be possible to combine different imaging techniques. Conventional analog imaging techniques—such as analog reflected-light microscopy, analog transmitted-light microscopy, or fluorescence imaging—can be combined with techniques based on digital post-processing. Furthermore, it is possible to combine fluorescence imaging techniques with non-fluorescence imaging techniques. Digital techniques related to angle-selective illumination can, for example, enable fully or at least semi-automatic generation of suitable contrast—as described in connection with DE 10 2014 112 242 A1. Such techniques can therefore be performed even without in-depth expert knowledge.Particularly in the context of fluorescence imaging, using suitable emission spectra for the illumination module's light sources can prevent biological sample objects from being consumed by angle-selective illumination techniques, thus avoiding any degradation of the fluorescence imaging. Furthermore, it is possible to implement optical devices that combine different imaging techniques, as described above, while using a common objective lens. This saves space, reduces costs, and decreases complexity. For example, angle-selective illumination techniques can be used to generate phase-contrast images, potentially eliminating the need for dedicated differential phase-contrast (DIC) or Zernike optics.

[0061] FIG. 1 illustrates an exemplary lighting module 100. FIG. 1 This is a top view of the lighting module 100. The lighting module 100 comprises a support 101. For example, the support 101 can be designed as a solid plate. The support 101 can be made of transparent, i.e., translucent material such as glass or plastic, or it can be made of opaque material. For example, the support 101 could be made of metal, such as steel or aluminum. The support can be designed as a plate.

[0062] On carrier 101, in this example, the FIG. 1 A total of four light sources 111 are arranged offset from a geometric center corresponding to an axis 221. The light sources 111 are arranged in different directions with respect to the axis 221. In particular, the light sources 111 are arranged in different quadrants with respect to the axis 221. For example, the light sources 111 can be implemented by light-emitting diodes (LEDs). Other types of light sources 111 can also be used. FIG. 1 It is evident that the light sources 111 are arranged at different positions with respect to the geometric center of the support 101. This allows for the implementation of angle-selective illumination, for example, in a scenario where the central axis 221 runs along a central ray of a beam path of an optical device that accommodates the illumination module 100. In particular, the light sources 111 are arranged rotationally symmetrically with respect to the central axis 221. This can be helpful when angle-selective illumination is to be implemented for significantly different directions, for example, to generate a particularly strong phase contrast.

[0063] A translucent area 112 is arranged in the region of the geometric center of the support 101. In one example, the translucent area 112 can be implemented using a translucent solid material; examples of translucent materials would be, for example, glass, plastic, plastic film, etc. The translucent material can, for example, be embedded in and firmly bonded to the surrounding material of the support 101. For example, the translucent area 112 could be implemented as a glass plate embedded in the metallic support. In another example, the translucent area 112 can be implemented as a recess or hole.

[0064] While with regard to the example of the FIG. 1 While a single, continuous translucent area 112 is shown, it may be possible in other examples to provide more than one translucent area. For example, several separate holes could be provided. Appropriate techniques, as described above, can be used in relation to FIG. 1 explained and applied.

[0065] The translucent area allows the lighting module 100 to be combined with an optical device with particular flexibility. For example, the lighting module 100 can be positioned within the beam path of the optical device; light can then pass through the light-translucent area 112 along the beam path of the lighting module 100. In this way, the lighting module 100 can be flexibly integrated into the optical device.

[0066] FIG. 2 This is a side-section view of the lighting module 100. FIG. 1 along the dashed-double-dotted line AA' in FIG. 1 In FIG. 2 Surfaces 110A and 110B of the support 101 are shown. FIG. 2 In particular, a top surface 110A and a bottom surface 110B of the carrier 110 are shown. The top surface 110A and the bottom surface 110B are planar; in other examples, they could also have a non-planar shape.

[0067] Out of FIG. 2 It is evident that, for example, light incident from above or below along the central axis 221 can pass through the illumination module 100 in the region of the transparent area 112 without significant reflection or absorption. Therefore, it is possible to flexibly combine the illumination module 100 with various optical devices, such as an LSM or a conventional reflected or transmitted light microscope. In particular, it may be possible to position the illumination module 100 in the beam path of an optical device.

[0068] In FIG. 2 The lighting directions 91 associated with the light sources 111 are also shown.

[0069] FIG. 3 This illustrates aspects relating to the arrangement of a detector 230 in the transparent area, which is configured as a hole centrally located on the support 110. For an example in which the transparent area 112 is configured as a hole, it is possible to arrange other optically active elements, such as a detector 230, at least partially within the hole. Such a scenario is shown in FIG. 3 shown in the example of the FIG. 3 A PMT, which can be configured, for example, to detect a fluorescence signal from a biological sample, is arranged in the area of ​​the hole that implements the transparent region 112. Specifically, a sensitive area 231 of the detector 230 is spaced apart from the top surface 110A; this means that the detector 230 projects beyond the top surface 110A along the axis 221. This allows light incident from above to be detected by the detector 230 without significant losses due to the illumination module 100.

[0070] FIG. 4 illustrates aspects relating to an LSM 200. The LSM 200 comprises a first illumination module 211, which includes a laser light source. The laser light source defines a first beam path 261 (in FIG. 4 (shown in dashed lines). A collimator optic 212 is provided. A scanning optic 213 is arranged in the beam path 261. An objective 214 is arranged near a sample holder 215. The scanning optic 213 and the objective 214 are arranged in front of the sample holder 215 and directly in the beam path 261 with respect to the first illumination module 211. The sample holder 215 is configured to fix a sample object in the beam path 261. The LSM 200 also includes a detector 230, e.g., a PMT. The detector 230 is suitable for detecting a fluorescence signal from the sample object.

[0071] In the example of the FIG. 4 The detector 230 is arranged behind the sample holder 215 with respect to the illumination module 211, i.e., in the transmitted beam path 221. In other examples, the detector 230 could also be arranged in front of the sample holder 215 with respect to the illumination module 211. For this purpose, an output coupling with respect to the beam path 261 could be provided, for example in the form of a partially reflective mirror as a beam splitter (in FIG. 4 (not shown).

[0072] FIG. 4 This also illustrates aspects relating to the lighting module 100 for angle-selective illumination. For example, the lighting module 100, which was previously described in relation to the FIGs. 1 - 3 was discussed, will be used. FIG. 4 Figure 1 illustrates an exemplary arrangement of the lighting module 100 in the beam path 261. In the example of the FIG. 4 The illumination module 100 is arranged directly in the beam path 261 between the scan optics 213 and the lens 214. It is evident that the light-transmitting area 112 has a lateral dimension perpendicular to the beam path 261 that is greater than the beam distance of the beam path 261. Therefore, it is possible that the light along the beam path 261 passes at least partially or mostly through the illumination module 100 or the light-transmitting area.

[0073] The lighting module 100 defines another beam path 222 (dotted-dashed line in FIG. 4 The beam path 222 can also be used to illuminate the sample object (in FIG. 4 (not shown) on the sample holder 215. For example, detector 230 or another detector (in FIG. 4 (not shown) are used.

[0074] FIG. 5 illustrates aspects relating to an LSM 200. The LSM 200 according to the example of the FIG. 5 This essentially corresponds to the LSM 200 as shown in the example of the FIG. 4 However, in the example of the FIG. 5 The lighting module 100 is arranged at a different location with respect to the beam path 261. In the example of the FIG. 5 The illumination module 100 is arranged in the transmission beam path 261, that is, on the opposite side of the sample holder 215 with respect to the illumination module 211. In the example of the FIG. 5 The detector 230 is partially located in the hole implementing the light-transmitting area 112.

[0075] Despite the spatial proximity between the illumination module 100 and the detector 230, the interaction between these elements 100 and 230 can be comparatively weak. This can be achieved, for example, by ensuring that the detection spectrum of the detector 230 differs from the emission spectrum of the light sources 111.

[0076] While in FIG. 5 Where the detector 230 is arranged in the hole of the light-transmitting area 112, in other examples the detector 230 and the illumination module 100 could be arranged separately from each other in the transmission beam path, e.g. according to an arrangement as in FIG. 4 discussed.

[0077] The preceding examples illustrated scenarios in which the support 110 of the lighting module 100 has a translucent area 112, implemented, for example, by a hole. In other examples, however, it is possible that the support 110 does not have a corresponding translucent area 112. Such a scenario is described in the example of... FIG. 6A illustrated.

[0078] Fig. 6A illustrates aspects relating to an LSM 200, which is not covered by the claimed invention. The LSM 200 essentially corresponds to the aspects described above. FIGs. 4 and 5 LSMs 200 were discussed. In the scenario of FIG. 6A The illumination module 100 is arranged in a mirrored position between the scan optics 213 and the lens 214. A partially reflective mirror 243 is provided for this purpose, which combines the beam path 222 associated with the illumination module 100 with the beam path 261. A light-transmitting area 112 is not provided.

[0079] FIG. 6B illustrates aspects relating to an LSM 200. In particular, illustrates FIG. 6B Aspects relating to another detector 241 (in FIG. 6B For clarity, detector 230 is not shown; it could be arranged in the transmission or reflection beam path. The additional detector 241 can be used to detect signals when the sample object passes through the additional illumination module 100 (in FIG. 6B (Not shown for the sake of simplicity) is illuminated. In the example of the FIG. 6B The detector 241 is arranged in a mirrored position between the scan optics 213 and the lens 214. For this purpose, a further optic 242 and a coupling unit 243 are provided. The arrangement of the detector 241 in FIG. 6B is merely exemplary; in other implementations, the detector 241 could, for example, be located behind the scan optics 213 with respect to the sample holder 215, or it could be located in the transmission beam path 261, i.e., behind the sample holder 215 with respect to the illumination module 211.

[0080] Detector 241 could be, for example, a CCD or CMOS sensor. Detector 241 could, for example, be part of a camera. Detector 241 could, for example, generate an overview image. However, detector 241 could also generate a highly magnified image.

[0081] FIG. 7 illustrates aspects relating to a conventional LSM 200A according to reference implementations, which, in addition to the illumination module 211 with the laser light source, also includes another illumination module 261A, which, in combination with a suitable lens (in FIG. 7 (not shown) can be used for phase contrast imaging. In such a conventional LSM 200A, it may be necessary to combine the lens 214 with another lens (in FIG. 7 (not shown) to replace. This increases the required installation space and necessitates complex mechanics. Furthermore, it may not be possible, or only possible to a limited extent, to simultaneously implement lighting via both the lighting module 211 and the lighting module 261A. For example, depending on the selected lighting, the mirror 261B may need to be tilted.

[0082] FIG. 8A Illustrates aspects relating to an optical microscope 300, for example a reflected light microscope or a transmitted light microscope, which in its entirety does not fall under the claimed invention. The microscope 300 comprises an eyepiece 315 and an objective 314. The eyepiece 315 defines a detection beam path 321 (in FIG. 8A (shown with the dashed line).

[0083] While in the example of the Fig. 8A Although an eyepiece 315 is shown, the detection beam path 321 could also be combined with other units in other examples, for example with a CCD camera, etc. The same applies to all examples described herein. The microscope 300 also includes a sample holder 215, which is designed to fix a sample object in the beam path 321.

[0084] In the example of the Fig. 8A A camera 310 is arranged in the beam path 322 – which does not pass through the objective 314 – and is used to provide an overview image of the sample holder 215 or the sample object. The illumination module 100 is also arranged in the beam path 322. The central axis 221 is arranged concentrically with a central ray 221 of the beam path 322. The central ray corresponds to the optical axis of the microscope 300. The support 110 of the illumination module 100 is rigidly coupled to a camera body 311. The illumination module 100 can be configured according to the other examples described herein. In particular, the illumination module 100 has several light sources 111 (in Fig. 8A (not shown). The illumination module 110 is arranged such that light can pass along the beam path 322 through the light-transmitting area 112, for example, a centrally or decentrally arranged hole (not covered by the invention). The camera 310 is partially arranged in the hole. By providing the illumination module 110 in the beam path 322, a particularly space-saving implementation of angle-selective illumination can be achieved.

[0085] Fig. 8B and Fig. 8C illustrate aspects relating to an optical microscope 300, for example, a reflected light microscope or a transmitted light microscope. The optical microscope 300 according to the Figs. 8B , 8C This is essentially equivalent to the optical microscope 300 according to the Fig. 8A In particular, the camera 310 and the illumination module 100 for angle-selective illumination are arranged in the transmission beam path 322. However, in this example, the Fig. 8B The carrier 110 is not rigidly coupled to the camera body 311. In the example of the Fig. 8B An imaging optic 312 associated with the camera 310 is partially arranged in the light-transmitting area 112. In the example of the Fig. 8C The imaging optics 312 are again partially arranged in the light-transmitting area 112, but the carrier 110 is rigidly coupled to the camera body 311.

[0086] FIG. 9A illustrates aspects relating to an optical microscope 300, for example a reflected light microscope or a transmitted light microscope, which in its entirety does not fall under the claimed invention. The microscope of FIG. 9A This is essentially equivalent to the microscope 300. FIGs. 8A-8C . However, the beam path 322 associated with camera 310 (in FIG. 9A (shown with the dashed-dotted line) also through the objective 314. In particular, the beam path 322 is reflected into the detection beam path 321. For this purpose, the microscope 300 includes an output coupling unit 313, for example, a beam splitter such as a partially reflective mirror. The imaging optics 312 are arranged in the region of the beam path 322. The illumination module 110 is also arranged in the region of the beam path 322. In particular, the illumination module 110 is arranged such that light can pass along the beam path 322 through the transparent area 112. The central axis 221 is arranged concentrically with a central ray of the beam path 322. By providing the illumination module 110 in the beam path 322, a particularly space-saving implementation of angle-selective illumination can be achieved.

[0087] For example, it would be possible for camera 311 to capture an image of the sample object illuminated by angle-selective illumination using the illumination module 110. It would also be possible to provide another detector (in FIG. 9A (not shown); this could be arranged in the transmission beam path 322 or in the reflection beam path 322.

[0088] FIG. 9B illustrates aspects relating to a 300mm microscope. The 300mm microscope is an example of the... FIG. 9B This basically corresponds to the microscope 300 of the example. FIG. 9A In the example of the FIG. 9B The imaging optics 312 are partially arranged within the light-transmitting area 112 of the illumination module 100. The light-transmitting area 112 can be configured as a hole, as described above. For example, a lens, a grating, and / or a filter of the imaging optics 312 could be arranged within the hole. This allows for a particularly space-saving integration of the illumination module 100.

[0089] FIG. 9C illustrates aspects relating to a 300mm microscope. The 300mm microscope is an example of the... FIG. 9C This basically corresponds to the microscope 300 of the examples of FIGs. 8A-8C and 9A-9B In the example of the FIG. 9C The lighting module 100 is combined with the camera 311. This ensures a particularly space-saving integration of the lighting module 100. For example, a sensor area of ​​the camera 311 could be located adjacent to or within the light-transmitting area 112. The camera 311 can, for example, be permanently connected to the lighting module 110.

[0090] The microscope 300 of FIGs. 8A , 8B , 8C , 9A , 9B , 9C , 10The device could include an additional illumination module. This could be used to illuminate the sample when viewed through the eyepiece. The additional illumination source could, for example, consist of collimator optics and a light source. Alternatively, the illumination module 100 could be used to illuminate the sample when viewed through the eyepiece. For this purpose, several light sources 111 could be operated simultaneously to ensure uniform illumination of the sample.

[0091] Fig. 10A illustrates aspects relating to a 300mm microscope. The 300mm microscope is an example of the... Fig. 10A This basically corresponds to the microscope 300 of the examples of FIGs. 8A-8C and 9A-9C and therefore, in its entirety, does not fall under the claimed invention. In the example of the Fig. 10A The illumination module 100 is arranged directly in the detection beam path 321 and extends transversely to the detection beam path 321. This ensures particularly space-saving integration of the illumination module 100. A separate additional beam path 322 is not required.

[0092] FIG. 10B Figure 1 illustrates aspects relating to the illumination module 100 according to the invention. The illumination module 100 comprises a light-transmitting area 112, which is designed as an off-center hole. The hole 112 has a distance 119 from the central axis 221, which, when the illumination module 100 is arranged in an optical device 200, 300, can be coincident with a central ray of the respective beam path.

[0093] A lighting module 100 according to the example of the FIG. 10B This may be particularly desirable with regard to an arrangement of the illumination module 100 in the transmitted light beam path 322 of an optical microscope 300, i.e. with regard to the detection beam path 321 behind the detector, cf. FIGs. 8A-8C . Then direct light, generated by centrally arranged light sources 111 - i.e. between the central axis 221 and the hole 112 - can enter the lens 314.

[0094] In the example of the FIG. 10B The distance 119 is chosen to be so small that a projection 118 of the smallest aperture of the lens 314 onto the support 110 (in FIG. 10B (shown with the dashed line) includes light sources 111 and the hole 112 is arranged adjacently. This allows bright-field imaging to be achieved.

[0095] While the example of FIG. 10B In relation to the translucent area 112 implemented through a hole, similar techniques can also be used for differently implemented translucent areas in other examples.

[0096] FIG. 11 illustrates aspects relating to the lighting module 100. In FIG. 11 Figure 1 shows an example in which the support 111 is made of a translucent solid material. The spaces between the various light sources 111 then implement the translucent area 112.

[0097] The light sources 111 are in the example of the FIG. 11 arranged in a matrix structure with a hexagonal unit cell. Other arrangements of the light sources 111 would also be possible, e.g., a matrix structure in a rectangular unit cell, etc., or a disordered arrangement.

[0098] In the example of the FIG. 11 are the light sources 111 on the top side 110A of the carrier 110 (in the drawing plane of the FIG. 11 ) arranged with a comparatively low geometric fill factor of less than 30% (in FIG. 11 The geometric fill factor corresponds to the sum of the areas of all black areas relative to the total area of ​​the carrier 110). Due to the comparatively small geometric fill factor, it is possible for a significant amount of incident light to pass through the carrier 110 of the lighting module 100 in the area of ​​the spaces between the different light sources 111.

[0099] FIG. 12 illustrates aspects relating to the lighting module 100. In FIG. 12 An example is shown in which the carrier 110 is made of non-translucent material, for example aluminum or a plastic material. In the example of FIG. 12 The light-transmitting area 112 is formed by a centrally arranged hole, which is not covered by the claimed invention. For example, it may be possible that when the illumination module 100 is arranged in a microscope or LSM, the space within the light-transmitting area 112 / the hole remains free, or that other optically active elements, such as a detector, a lens, etc., are arranged within the hole.

[0100] While in FIG. 12 In an example not covered by the claimed invention, in which the translucent area 112 is arranged in the center of the support 110, in examples according to the invention the translucent area 112 is also arranged outside the center of the support 110. It is also not generally necessary for the translucent area 112 to be symmetrical with respect to a central axis.

[0101] FIG. 13 Illustrates aspects relating to the lighting module 100, wherein the lighting module 100 has a central hole not covered by the invention. In FIG. 13 The different quadrants of the support 110 are occupied by laterally extended light sources 111. For example, the light sources 111 in the example of the FIG. 13 This can be implemented using organic light-emitting diodes. With such a design of the lighting module 100, it is possible to illuminate the sample object from extended solid angles.

[0102] FIG. 14 Illustrates aspects relating to the lighting module 100, wherein the lighting module 100 has a central hole not covered by the invention. In FIG. 14 The various light sources 111 are designed as halogen light sources. These are arranged symmetrically with respect to a central axis.

[0103] FIG. 15 Illustrates aspects relating to the lighting module 100, wherein the lighting module 100 has a central hole not covered by the invention. In FIG. 15 The light-transmitting area 112 is implemented by light-transmitting organic light-emitting diodes 111. The support 110 can, for example, be made of non-transparent material.

[0104] From a comparison of FIGs. 10-15 It is evident that the design of the lighting module 100 can vary considerably in different examples. In particular, designs such as those relating to the FIGs. 10-15 They are shown and can be combined with each other.

[0105] FIG. 16 illustrates aspects relating to a lighting module 1000. The lighting module 1000 according to the example of the Fig. 16 This basically corresponds to lighting module 100 according to the example of the Fig. 1 . In this case, the carrier 101 of the lighting module 1000 - in contrast to the example of the Fig. 1 and not according to the invention - no light-transmitting area. In the example of the Fig. 16 A detector 1230 is mounted on the carrier 101. For example, the detector 1230 can be mounted on the top surface 110A of the carrier 101, cf. FIG. 17 Then the sensor surface 1231 is spaced away from the surface 110A. For example, the detector 1230 can be glued, screwed or otherwise attached to the carrier 101.

[0106] In the examples of FIGS. 16 and 17 The detector 1230 is arranged centrally on the support 101. This means, in particular, that the detector 1230 is arranged centrally with respect to the axis 221. In other examples according to the invention, the detector 1230 is arranged off-center on the support 101.

[0107] For example, it would be possible that different techniques are applied to detector 1230 that were previously used in relation to Fig. 10B as described. It would therefore be possible for the detector 1230 to have a distance from the center of the support 110, wherein, according to the claimed invention, at least one of the light sources 111 of the illumination module 100 is arranged between the center of the support 110 and the detector 1230. The distance between the detector 1230 and the center of the support 110 can, in turn, correspond to a projection 118 of the aperture of the lens 314 onto the support 110. This can promote bright-field illumination.

[0108] In the examples of FIGS. 16 and 17Module 1000 typically has a single detector 1230. However, in various examples, it would be possible for module 1000 to have more than one detector, for example, two or three detectors. These multiple detectors could be spaced apart from each other on the support 101. For example, the multiple detectors 1230 could be located on different sides of axis 221 or in different quadrants with respect to axis 221.

[0109] FIG. 18Figure 200, 300 is a schematic representation of the optical device 200, 300, which is configured for angle-selective illumination according to various examples described herein. The optical device 200, 300 comprises the detector 241, which detects light emitted from the light sources 111 of the illumination module 110. The optical device 200, 300 also comprises a processing unit 299. The processing unit 299 is configured to control the illumination module 100 or the light sources 111 for separate and / or sequential illumination of the sample object from multiple illumination directions. For example, different light sources 111 can be activated separately and / or sequentially. The processing unit 299 is further configured for each illumination direction to control the detector 241 to acquire a respective measurement image of the object. The processing unit 299 is also configured to combine the measurement images to obtain a result image.The resulting image exhibits phase contrast. The computing unit 299 can be configured to apply digital post-processing techniques disclosed in DE 10 2014 112 242 A1.

[0110] Fig. 19 This is a flowchart of a procedure according to various examples. For example, computing unit 299 could be set up to execute the procedure according to Fig. 19 to carry out.

[0111] First, in step 2001, a current illumination direction 91 is selected. Then, in step 2002, one or more light sources 111 are controlled so that the sample object is illuminated from the selected illumination direction. In step 2003, a corresponding measurement image is acquired, for example, using a suitable detector 241, such as a CCD detector or a CMOS detector, an overview camera, and / or a photomultiplier. The detector 241 can be controlled appropriately for this purpose. The measurement image is acquired while the sample object is illuminated from the current illumination direction.

[0112] Then, in step 2004, it is checked whether it is necessary to acquire another measurement image from a further illumination direction 91. If this is the case, steps 2001-2003 are repeated.

[0113] Subsequently, in step 2002, a result image is generated. This result image exhibits phase contrast for a depicted object (phase contrast image). The phase contrast image is determined by combining the measurement images acquired in the iterations of step 2003.

[0114] While the measurement images typically exhibit no or insignificant phase contrast, combining these images can generate a phase contrast image with a significant phase contrast component. In particular, compared to other conventional phase contrast imaging techniques, the method described here offers the advantage of a particularly simple implementation of phase contrast imaging, as well as a particularly simple, and therefore cost-effective and robust, design of the optical device.

[0115] For example, the lighting directions could form pairs or be arranged in pairs. It's possible that each lighting direction is assigned to only one pair. However, it's also possible that at least some of the lighting directions are assigned to multiple pairs. The measurement images of the lighting directions belonging to each pair can then be combined to obtain the respective result image.

[0116] Different criteria can apply to assigning two illumination directions 91 to a pair. For example, geometric criteria can apply to the illumination directions 91 of a pair, such as with respect to the optical axis; in this way, it may be possible, for example, to generate a particularly high phase contrast component in the phase contrast image. For example, the illumination directions of a pair could be symmetrically aligned with respect to the axis of the optical device along which an idealized light beam experiences no or only slight deflection (optical axis) and / or with respect to a plane that includes the optical axis. Alternatively or additionally, it would also be possible to consider the time of illumination and acquisition as an alternative or additional criterion for assigning two illumination directions 91 to a pair; for example,Those illumination directions 91 for which the respective measurement image is acquired immediately or in close succession can form a pair; in this way, for example, a certain robustness against motion artifacts could be achieved. In general, as an alternative or additional criterion for the membership of two illumination directions 91 in a pair, a subsequent evaluation for generating the phase contrast image can also be considered; for example, a single result image could always be generated for the two measurement images of a pair by combining these measurement images.

[0117] It is possible that the two illumination directions of a pair enclose correlating angles with the optical axis. Correlating angles can mean, for example, essentially the same angles or angles of essentially the same magnitude; "essentially" can be characterized, in particular, by technical limitations in accuracy, such as systematic or statistical errors in the acquisition of the measurement images by the optical device and / or structural limitations of an illumination device. If angles are implemented that are absolutely different but, for example, within the accuracy of the optical device, the same, this can satisfy such a criterion of essentially the same angles.

[0118] Such criteria apply below to corresponding specifications of angles and / or other properties of lighting directions 91 or the optical device.

[0119] To describe the geometric properties of the illumination directions, it can be helpful to define them using an illumination vector. The illumination vectors can be defined with respect to an origin of the optical device, such as the object, and / or an intersection of a focal plane with the optical axis. The length of the illumination vectors can, for example, correspond to the amplitude of the illumination from the respective illumination direction; however, when subsequently discussing the orientation of different illumination vectors, it may be unnecessary to consider their length. The angle that an illumination vector makes with the optical axis can then correspond to the angle of the respective illumination direction.

[0120] For example, it may be desirable for the illumination vectors of a pair of illumination directions to form an angle with each other greater than 10°, preferably greater than 20°, and particularly preferably greater than 40°. Alternatively or additionally, it would also be possible for the illumination vectors of a pair of illumination directions to each form an angle with the optical axis greater than 5°, preferably greater than 10°, and particularly preferably greater than 20°. In this way, it can be achieved that a difference vector between the two illumination vectors of a pair of illumination directions 91 has a significant component perpendicular to the optical axis; this can significantly increase the phase contrast in the phase contrast image.

[0121] In particular, it may be possible to transform the illumination vectors of two illumination directions of a pair of illumination directions into one another by rotating them about the optical axis of the optical device by an angle greater than 25°, preferably greater than 50°, and most preferably greater than 85°. This results in a particularly large difference vector.

[0122] The two illumination directions of a pair of illumination directions can also be arranged such that the associated illumination vectors intersect each other by rotation about the optical axis by an angle of 160° to 200°, advantageously by 175° to 185°, and particularly advantageously by 180°. It would also be possible for the associated illumination vectors to be transformed into each other by rotation about the optical axis by an angle of 70° to 110°, advantageously by 85° to 95°, and particularly advantageously by 90°. In other words, the two illumination vectors of a pair of illumination directions 91 can lie in a plane and be arranged symmetrically or substantially symmetrically with respect to the optical axis. The optical axis can lie in this plane (be contained by this plane), for example, if a rotation by 180° transforms the two illumination vectors into each other.In this way, a comparatively large phase contrast component can be obtained in the phase contrast image, because the two illumination directions of a pair are arranged in such a complementary way to each other.

[0123] In general, it can be desirable to use a larger number of illumination directions to obtain the phase contrast image. In particular, with appropriate arrangement of the different illumination directions, the phase contrast component in the phase contrast image can increase. For example, it would be possible to consider several pairs of illumination directions. For example, it would be possible to illuminate the object sequentially from 2, 4, 6, 8, or more illumination directions. For example, a first pair of illumination directions could determine a first difference vector of associated illumination vectors. A second pair of illumination directions could correspondingly determine a second difference vector of associated illumination vectors. The first and second difference vectors could form an angle with each other, e.g.an angle of 70° to 110°, advantageously of 85° to 95°, particularly advantageous of 90°.

[0124] It would also be possible for a first plane to be defined by the illumination vectors of a first pair of illumination directions. A second plane could, for example, be defined by the illumination vectors of a second pair of illumination directions. The first and second planes could, for example, form an angle of 70° to 110° with each other, advantageously from 85° to 95°, and particularly advantageously from 90°. The planes could, for example, be defined by the fact that the respective illumination vectors lie in the plane. It would also be possible for the planes to be defined by a normal vector oriented parallel to a difference vector of the respective illumination vectors; the optical axis could lie in the plane.

[0125] Thus, the difference vectors of the illumination vectors of the two pairs of illumination directions 91 can enclose a comparatively large angle of up to 90° with each other; this can increase the phase contrast in the phase contrast image along various image directions. For example, the phase contrast component in the phase contrast image can be particularly large along those image directions for which the illumination vectors of a pair of illumination directions have a component perpendicular to the optical axis. In particular, the phase contrast component in the phase contrast image can be particularly large along those directions for which the difference vector of the illumination vectors of a pair of illumination directions has a component perpendicular to the optical axis. Therefore, it can be desirable to use complementary and / or symmetrically arranged illumination directions.To create an isotropic phase contrast in the phase contrast image, it may be desirable for the illumination directions to enclose uniformly distributed angles with the optical axis.

[0126] Such lighting directions 91 or lighting vectors as described above can be implemented by a suitable arrangement and / or extension of the light sources 111 on the support 101.

[0127] In summary, the above describes techniques for angle-selective illumination using lighting modules. These techniques allow for flexible combination of angle-selective illumination with various optical devices.

Claims

1. Laser scanning microscope (200), comprising: - a first illumination module (211) with a laser light source, - a second illumination module (100) with a carrier (101) and with a plurality of light sources (111), which are arranged on the carrier (101), - a scanning optical unit (213), - an objective (214), - a specimen holder (215), which is configured to immobilize a specimen object in a beam path (261) defined by the laser light source, - a detector (230) and - a computing unit (299), which is configured to actuate the plurality of light sources (111) to separately and / or sequentially illuminate an object from a plurality of illumination directions (91), wherein in relation to the first illumination module (211), the scanning optical unit (213) and the objective (214) are arranged upstream of the specimen holder (215) and in the beam path (261), wherein at least one light source of the plurality of light sources is arranged between a centre of the carrier and a light-transmissive region (112) formed in the carrier or the detector (230) arranged on the carrier, wherein the centre is determined by a point on the carrier defined by a central ray of the beam path (261), wherein the second illumination module (100) is arranged in such a way that direct light of the at least one light source (111) penetrates into the objective (214).

2. Laser scanning microscope (200) according to Claim 1, wherein the at least one light-transmissive region (112) is embodied as an aperture that is arranged on the carrier (101) in off-centred fashion.

3. Laser scanning microscope (200) according to Claim 2, wherein the detector (230, 241) is at least partly arranged in the aperture.

4. Laser scanning microscope (200) according to any of the preceding claims, wherein the second illumination module (100) is arranged downstream of the specimen holder (215) or upstream of the specimen holder (215) in relation to the first illumination module (211).

5. Laser scanning microscope (200) according to any of the preceding claims, wherein the second illumination module (100) is arranged between the scanning optical unit (213) and the objective (214) in the beam path (261).

6. Laser scanning microscope (200) according to any of the preceding claims, wherein a detection spectrum of the detector (230) is at least partly different from an emission spectrum of the light sources (111) of the second illumination module (100).

7. Laser scanning microscope (200) according to Claim 6, furthermore comprising: - a further detector (241, 1230), having a detection spectrum which at least partly overlaps with an emission spectrum of the light sources (111) of the second illumination module (100).

8. Laser scanning microscope (200) according to any of the preceding claims, wherein the light sources (111) are arranged on a surface (110A, 110B) of the carrier (101) with a geometric fill factor that is no greater than 90%, preferably no greater than 50%, particularly preferably no greater than 30%.

9. Laser scanning microscope (200) according to Claim 1, wherein the light-transmissive region (112) is formed from a light-transmissive solid-state material.

10. Laser scanning microscope (200) according to any of the preceding claims, wherein the computing unit (299) furthermore is configured to combine a plurality of measurement images associated with the illumination directions (91) to obtain a result image, which has a phase contrast.