DE102023104335B3 - Illumination module, microscope and procedure - Google Patents

Abstract

Described is an illumination module (100, 200) for a microscope (550), comprising a light source (102) which is designed to emit illumination light (104) into an illumination beam path (106), a focusing optical system (114) which is arranged in the illumination beam path (106) and is designed to direct the illumination light (104) into a sample (108), a light sheet generation component (116, 120, 230, 232) which is arranged upstream of the focusing optical system (114) in the illumination beam path (106) and is designed to shape the illumination light (104) into a light sheet-like light distribution (118) at least in cooperation with the focusing optical system (114) for the selective light sheet illumination of a plane of the sample, and an optical switching element (122) which can be switched between a first optical setting and at least one second optical setting. In the first optical setting, the optical switching element (122) enables the selective light sheet illumination of the plane of the sample (108). In the second optical setting, the optical switching element (122) forms the illumination light (104), at least in cooperation with the focusing optical system (114), into a volume-like light distribution (128) that illuminates a volume region of the sample (108).

Description

Technical area

The invention relates to an illumination module for a microscope. The invention further relates to a microscope with an illumination module and a method for image reconstruction using a microscope.

background

Microscope systems are known from the prior art that make it possible to switch between two illumination modes. In a first mode, the sample is illuminated, for example, with a light sheet-like light distribution (hereinafter also referred to simply as a light sheet), so that a plane within the sample is selectively exposed to the illumination light. The illuminated sample plane is then imaged onto a detector to generate a two-dimensional (2D) tomographic image. Such a tomographic image can be successively carried out for different sample planes in order to obtain a stack of tomographic images. In a second mode, the sample is exposed to a volume-like light distribution in order to illuminate and image an extended three-dimensional (3D) volume area of the sample. By switching between these two illumination modes, the sample area excited to fluorescence can be varied, for example in fluorescence microscopy.

To date, switching between 2D imaging and 3D imaging has usually been done by controlling two separate light sources, whose light is combined into a common excitation path via a dichroic beam splitter. This has the disadvantage that the two recording modes have to work with different illumination wavelengths.

A particularly powerful microscopy method for 3D imaging is light field microscopy, which makes it possible to record rapidly occurring biological processes in a volume over a longer period of time. The 3D information from the observed volume is mapped onto a 2D camera sensor in order to then perform a computational image reconstruction. The ability to scan entire sample volumes at a speed corresponding to the frame rate of the camera without the use of moving parts such as galvanometer scanners makes this technology one of the fastest microscopy methods.

The disadvantage, however, is that the reconstruction of the 3D volume is extremely computationally intensive due to the complexity of the point spread function (PSF) to be considered in light field microscopy. In light field microscopy, an arrangement of microlenses is usually used in front of the detector, resulting in a large number of individual point spread functions, which together result in a complex overall PSF that is used as the basis for image reconstruction.

In the publication WAGNER, Nils [ua]: Deep learning-enhanced light-field imaging with continuous validation. In: Nature Methods, Vol. 18, 2021, No. 5, pp. 557-563. ISSN 1548-7105 (E); 1548-7091 (P) it was shown that the speed of image reconstruction can be improved by several orders of magnitude if specially trained neural networks are used instead of classical reconstruction algorithms such as the Richardson-Lucy algorithm. However, this approach requires that the neural network must be retrained for different types of samples, for which so-called ground truth data is provided. This is data that allows the quality of the applied reconstruction model to be checked.

In the aforementioned publication by Wagner et al., a system is used that basically consists of two microscopes. The main microscope is a light field microscope based on a design such as that described in the publication by BROXTON, Michael [et al.]: Wave optics theory and 3-D deconvolution for the light field microscope. In: Optics Express. 2013, Vol. 21, No. 21, pp. 25418-25439. ISSN 1094-4087 (E). This light field microscope includes a laser with special optics that allow the entire sample volume imaged by the light field camera to be illuminated. In addition to the main microscope, a second microscope is used in which a sample plane is selectively illuminated with a dynamically generated light sheet and imaged onto a camera. Using a pair of galvanometer mirrors, the sample is then scanned along the optical axis of the detection objective with a light sheet generated by a second laser to sequentially generate a 3D image stack that serves as the basis for training the light field neural network.

From the documents EN 10 2019 214 929 A1 and HEDDE, PN ; GRATTON, E.: Selective Plane Illumination Microscopy with a Light Sheet of Uniform Thickness Formed by an Electrically Tunable Lens. In: Microscopy Research and Technique, 81, 2018, 924 - 928. - ISSN 1097-0029 , light sheet microscopes are known, in which the light sheet can be moved using an electrically tunable lens.

The document MADRID-WOLFF, J. [et. al.]: Light-sheet enhanced resolution of light field microscopy for rapid imaging of large volumes. In: Proc. SPIE Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XXV, 10499, 23.02.2018, 1 - 9. - ISSN 0277-786X reveals the combination of a light sheet microscope with a light field microscope.

Brief description

The object of the invention is to provide an illumination module for a microscope which makes it possible to quickly and flexibly change the illuminated sample area while maintaining the same illumination wavelength.

This object is achieved by the lighting module according to claim 1. Advantageous further developments emerge from the dependent claims and the following description.

An illumination module for a microscope comprises a light source which is designed to emit illumination light into an illumination beam path. The illumination module comprises an objective which is arranged in the illumination beam path and designed to direct the illumination light into a sample. The illumination module further contains a light sheet generation component which is arranged upstream of the objective in the illumination beam path and is designed to shape the illumination light into a light sheet-like light distribution, at least in cooperation with the objective for selective light sheet illumination of a plane of the sample. The illumination module further comprises an optical switching element which can be switched between a first optical setting and at least one second optical setting. In the first optical setting, the optical switching element enables selective light sheet illumination of the sample plane. In the second optical setting, the optical switching element shapes the illumination light, at least in cooperation with the focusing optical system, into a volume-like light distribution which illuminates a volume region of the sample.

It should be noted that the optical switching element is designed and suitably controllable in such a way that a light sheet-like light distribution in a plane of the sample can also be generated by the light sheet generating component shaping the illumination light in cooperation with the focusing optical system and the optical switching element into a light sheet-like light distribution for sample illumination. The focusing optical system can be, for example, an objective facing the sample. However, it is not limited to this.

The illumination module makes it possible to switch flexibly between 2D imaging based on light sheet illumination and 3D imaging based on volume illumination. In a preferred application, the 3D imaging is based on a light field microscopic image. Such a light field image can then be correlated with a light sheet microscopic image in a particularly simple manner using the illumination module. It is particularly advantageous that the illumination module is able to generate the different light distributions with one and the same wavelength via the optical switching element it contains. This also means that a dichroic beam splitter, as is often used in conventional systems, can be dispensed with.

In the first optical setting, the optical switching element enables the selective light sheet illumination of the sample plane by leaving the illumination light, which is formed into the light sheet-like light distribution by the light sheet generation component in cooperation with the focusing optical system, unaffected. Such an embodiment can be implemented, for example, by the optical switching element being able to be introduced into and removed from the illumination beam path by means of an actuator. The first optical setting then corresponds to an operating state in which the optical switching element is removed from the illumination beam path. In contrast, in the second optical setting, the optical switching element is introduced into the illumination beam path and thus contributes to generating the volume-like light distribution as a result of its optical effect.

In a preferred embodiment, the optical switching element is an element with a variable optical effect that is arranged in the illumination beam path in both optical settings. In this case, it is advantageously possible to control the illumination solely via the variable optical effect of the switching element.

The optical switching element is preferably an optical lens with a variable focal length. Such a lens can be designed as a liquid lens, for example. Alternatively, a deformable mirror or a spatial light modulator can be used as the switching element.

The optical switching element is designed, for example, to focus the illumination light in the rear focal plane of the focusing optical system or in the vicinity thereof in the second optical setting. In this embodiment, the illumination light is focused in the entrance pupil of the focusing optical system for the purpose of volume illumination. This largely neutralizes the otherwise light sheet-generating effect of the focusing optical system.

In an alternative embodiment, the optical switching element can also be designed to vary the distance between the light source and the entrance pupil of the focusing optical system.

In a further alternative embodiment, the optical switching element can have an arrangement of acousto-optical deflectors (AOD) which, by applying different radio frequencies or by switching these radio frequencies on and off, have the same effect as a lens with variable focal length. Depending on the design, the focal length can be different in the two lateral spatial directions. For example, reference is made to the publication of KIRKBY, Paul A. ; NADELLA, KM Naga Srinivas ; SILVER, R. Angus: A compact acousto-optic lens for 2D and 3D femtosecond based 2-photon microscopy. In: Optics Express (OpEx), Vol. 18, 2010, No. 13, pp. 13720-13744. - ISSN 1094-4087 (E) referred to.

The light sheet generating component preferably comprises an astigmatic optical system which is arranged in the illumination beam path between the optical switching element and the focusing optical system. Such an astigmatic system can be implemented, for example, in the form of a cylindrical lens.

The optical switching element can be designed to focus the illumination light into the astigmatic optical system or in the vicinity thereof in the second optical setting. This focusing reduces the beam cross section of the illumination light at the location of the astigmatic optical system in order to switch from the light sheet illumination to the volume illumination.

The light sheet generating component preferably comprises a beam deflection unit which is designed to generate the light sheet-like light distribution by scanning the illumination light. This embodiment works according to the principle of a laser scanning microscope, in which the illumination light is guided over the sample in a scanning movement. However, in the present case, this scanning movement is so fast that the time required for the light beam to scan the entire sample area is shorter than the recording time of a camera onto which the sample area is imaged. A fast-moving scanning mirror is used as a beam deflection unit, for example.

The illumination module can further comprise relay optics that are arranged in the illumination beam path between the beam deflection unit and the focusing optical system and are designed to image the beam deflection unit onto the rear focal plane of the focusing optical system. Such relay optics include, for example, a scanning lens and a tube lens that are arranged in front of the focusing optical system in the direction of light propagation.

The beam deflection unit can be arranged downstream of the optical switching element in the illumination beam path. This ensures that the illumination light does not perform any scanning movement at the location of the optical switching element. Possible aberrations within the switching element therefore only have a minor impact on the quality of the illumination.

Alternatively, the beam deflection unit is arranged upstream of the optical switching element in the illumination beam path. This has the advantage that no focusing of the illumination light caused by the switching element occurs at the location of the beam deflection unit.

Preferably, the light source is controllable to vary the intensity of the illumination light depending on the optical setting of the optical switching element. This makes it possible to control the light exposure of the sample depending on the illumination mode. In particular, the intensity of the illumination light can be set higher in volume imaging mode than in light sheet imaging mode.

In a preferred embodiment, the optical switching element is controllable depending on the wavelength of the illumination light.

The light source may comprise a plurality of lasers whose laser light is combined in the illumination beam path to form the illumination light.

Preferably, the light sheet-like light distribution and the volume-like light distribution have the same wavelength.

The switching by an optical switching element between the first optical setting, in which the light sheet-like light distribution is generated, and the second optical setting, in which the volume-like light distribution is generated, preferably takes place within 100 ms, particularly preferably within 25 ms and very particularly preferably within 10 ms. Likewise, the switching by the optical switching element between the second optical setting, in which the volume-like light distribution is generated, and the first optical setting, in which the light sheet-like light distribution is generated, preferably takes place within 100 ms, particularly preferably within 25 ms and very particularly preferably within 10 ms.

According to a further aspect, a microscope with an illumination module of the type described above is provided. The microscope can have a light sheet microscopic functional unit which provides two separate objectives for illumination and detection, the optical axes of which are tilted relative to one another by a certain angle, preferably 90°. Alternatively, the light sheet microscopic functional unit can also work according to the principle of inclined plane microscopy, in which one and the same objective is used for illumination and detection.

Preferably, the microscope comprises a detection device with a first detection module that is assigned to the first optical setting of the optical switching element and a second detection module that is assigned to the second optical setting of the optical switching element. In this embodiment, the first detection module is used for light sheet imaging, while the second detection module is used for volume imaging.

In a preferred embodiment, the detection device comprises an objective lens provided separately from the focusing optical system of the illumination module.

In a particularly preferred embodiment, the second detection module comprises a light field camera. A microlens arrangement is arranged upstream of such a light field camera, for example, which makes it possible to generate the desired 3D image information. In principle, both light field cameras in which the microlens arrangement is arranged in an image or intermediate image plane and light field cameras in which the microlens arrangement is located in a plane conjugated to a pupil plane (“Fourier light field”) are suitable for this purpose.

According to a further aspect, a method for image reconstruction using a microscope of the type described above is provided. The method comprises the following steps: recording a stack of images of different planes of a sample using the first detection module in the first optical setting of the optical switching element; recording a volume image of the sample using the second detection module in the second optical setting of the optical switching element; training a neural network based on the stack of recorded images and the volume image; recording a further volume image of the sample or another sample using the second detection module in the second optical setting of the optical switching element; and evaluating the further volume image using the trained neural network.

In this method, the neural network is trained using images taken of the sample to be analyzed or another sample. This other sample is preferably a reference sample that is similar to the sample actually being examined. The volume image to be evaluated is not limited to a single image. Rather, a whole series of volume images can also be evaluated in this way. Multiple image stacks and/or multiple volume images can also be used to train the neural network.

The method makes it possible to simplify image reconstruction by using image information obtained from a stack of images acquired using light sheet microscopy when evaluating the light field microscopic image.

Short description of the characters

• 1 ein Beleuchtungsmodul gemäß einem Ausführungsbeispiel in zwei orthogonalen Schnittansichten, wobei sich ein optisches Schaltelement in einer ersten optischen Einstellung befindet, in der eine lichtblattartige Lichtverteilung erzeugt wird;
• 2 das Beleuchtungsmodul aus 1 in zwei orthogonalen Schnittansichten, wobei sich das optische Schaltelement in einer zweiten optischen Einstellung, befindet, in der eine volumenartige Lichtverteilung erzeugt wird;
• 3 zwei unterschiedliche Betriebsmodi eines Beleuchtungsmoduls nach einem weiteren Ausführungsbeispiel, wobei sich das optische Schaltelement in 3(A) in der ersten optischen Einstellung, in der die lichtblattartige Lichtverteilung erzeugt wird, und in 3(B) in der zweiten optischen Einstellung befindet, in der die volumenartige Lichtverteilung erzeugt wird;
• 4 zwei unterschiedliche Betriebsmodi eines Beleuchtungsmoduls nach einem weiteren Ausführungsbeispiel, wobei sich das optische Schaltelement in 4(A) in der ersten optischen Einstellung, in der die lichtblattartige Lichtverteilung erzeugt wird, und in 4(B) in der zweiten optischen Einstellung befindet, in der die volumenartige Lichtverteilung erzeugt wird;
• 5 ein Mikroskop in einer Schnittansicht, welches das Beleuchtungsmodul aus den 1 und 2 enthält, wobei sich das optische Schaltelement in der ersten optischen Einstellung zur Erzeugung der lichtblattartigen Lichtverteilung befindet;
• 6 das Mikroskop aus 5 in einer Schnittdarstellung, wobei sich das optische Schaltelement in der zweiten optischen Einstellung zur Erzeugung der volumenartigen Lichtverteilung befindet; und
• 7 ein Flussdiagramm zur Veranschaulichung eines beispielhaften Verfahrens zur Bildrekonstruktion unter Verwendung des Mikroskops aus den 5 und 6.

In the following, embodiments are explained in more detail with reference to the figures. They show:

• 1 a lighting module according to an embodiment in two orthogonal sectional views, wherein an optical switching element is in a first optical setting in which a light sheet-like light distribution is generated;
• 2 the lighting module 1 in two orthogonal sectional views, wherein the optical switching element is in a second optical setting in which a volume-like light distribution is generated;
• 3 two different operating modes of a lighting module according to a further embodiment, wherein the optical switching element is in 3(A) in the first optical setting, in which the light sheet-like light distribution is generated, and in 3(B) in the second optical setting in which the volume-like light distribution is created;
• 4 two different operating modes of a lighting module according to a further embodiment, wherein the optical switching element is in 4(A) in the first optical setting, in which the light sheet-like light distribution is generated, and in 4(B) in the second optical setting, in which the volume-like light distribution is created;
• 5 a microscope in a sectional view showing the illumination module from the 1 and 2 wherein the optical switching element is in the first optical setting for generating the light sheet-like light distribution;
• 6 the microscope 5 in a sectional view, wherein the optical switching element is in the second optical setting for generating the volume-like light distribution; and
• 7 a flow chart illustrating an exemplary method for image reconstruction using the microscope from the 5 and 6 .

1 shows a schematic representation of an illumination module 100 intended for a microscope in two orthogonal sectional views. Referring to a rectangular coordinate system xyz, the illumination module 100 is shown in the upper part of the image (A) of the 1 in an xz-section and in the lower part (B) of the 1 shown in a yz-section.

The illumination module 100 comprises a light source 102 which is designed to emit illumination light 104 into an illumination beam path 106 which leads to a sample 108 to be illuminated. The light source 102 comprises, for example, one or more lasers 110 whose laser light is coupled into an optical fiber 112. The light exit end of the optical fiber 112 represents a point source which feeds the illumination light 104 to the illumination beam path 106. For the sake of simplicity, the coupling of the laser 110 to the optical fiber 112 is only shown in the upper part of the image (A) of the 1 shown and omitted in the other figures.

The illumination module 100 further comprises a focusing optical system arranged in the illumination beam path 106, e.g. an objective 114, which directs the illumination light 104 onto the sample 108. In the illumination beam path 106 there is a light sheet generating component 116 which is arranged upstream of the objective 114 in the direction of light propagation. The light sheet generating component 116 serves, in cooperation with the objective 114, to form the illumination light 104 into a light sheet-like light distribution in order to selectively illuminate the sample 108 in a sample plane. In the embodiment shown, a light sheet 118 is thus generated in the sample 108, the extent of which in the x-direction is many times greater than in the y-direction, as shown in the right-hand part of the 1 In the example shown in 1 The light sheet plane is therefore in the xz-plane.

The light sheet generation component 116 comprises, for example, an astigmatic optical system such as a cylindrical lens 120, the light entry surface of which is curved only in the x-direction, while it is flat in the y-direction. The cylindrical lens 120 thus expands the beam cross-section of the illumination light 104 passing through it in the x-direction, as shown in partial image (A) of the 1 In contrast, the beam cross section of the illumination light 104 remains essentially unaffected when passing through the cylindrical lens 120 in the y-direction, as shown in the partial image (B) of the 1 shows.

The illumination module 100 further comprises an optical switching element 122 in the illumination beam path 106. As explained in detail below, the optical switching element 122 has the function of switching between a 2D and a 3D illumination of the sample 108. In the 1 In the embodiment shown, a collimator 124 is arranged upstream of the optical switching element 122, which collimates the illumination light 104, which the optical fiber 112 emits as a divergent beam, and directs it onto the optical switching element 122.

The optical switching element 122 can be switched between two optical settings. In a first optical setting, which is 1 As shown, the optical switching element 122 enables the selective light sheet illumination of a sample plane. In the embodiment according to 1 This is achieved in that the optical switching element 122 leaves the illumination light 104, which is formed into the light sheet 118 by the cylindrical lens 120 in cooperation with the objective 114, largely unaffected. This means that the optical switching element 122 does not exert any refractive effect on the illumination light 104 in the first optical setting.

In a second optical setting, which is 2 is shown, the optical switching element 122 ensures that the illumination light 104 is no longer formed into a light sheet-like light distribution, but into a volume-like light distribution 128. This is shown in the right part of the 2 shown that the light distribution 128 in the y-direction has a significantly larger extent than that in 1 shown light sheet. As in 2 As illustrated, the optical switching element 122 in its second optical setting has a focusing effect on the illumination light 104, which falls on the optical switching element 122 as a collimated beam.

In the example after 2 the optical switching element 122 is designed to focus the illumination light 104 in the second optical setting into the rear focal plane of the objective 114 or in the vicinity thereof. This means that the optical switching element 122 focuses the illumination light 104 in this example essentially into the entrance pupil of the objective 114. This also results in a significant focusing of the illumination light 104 in the area of the cylindrical lens 120. This results in a switching of the 1 shown 2D light sheet illumination to the one in 2 This switching is initiated by a control unit 126, which controls the optical switching element 122 accordingly. To simplify the illustration, the control unit 126 is again only shown in the partial image (A) of the 1 shown and omitted in the other figures.

In the embodiment according to the 1 and 2 the optical switching element 122 is an optical lens, e.g. a liquid lens, whose focal length can be varied via the control unit 126. However, such an embodiment is only to be understood as an example. The optical switching element 122 can also be designed, for example, as a deformable mirror or spatial light modulator (SLM), whose effect on the illumination light 104 can be varied via the control unit 126.

Apart from the specific design of the optical switching element 122, the illumination light 104 for the purpose of switching between the light sheet illumination and the volume illumination can also be implemented in a fundamentally different manner than in the 1 and 2 shown embodiment. For example, the optical switching element 122 can also be designed such that when switching between the two switching states, it changes the distance between the light source 102 and the entrance pupil of the lens 114 in a suitable manner.

In 3 A lighting module 200 is shown, which represents a further embodiment. Those components of the lighting module 200 which correspond to the components of the 1 and 2 shown embodiment are designated by the same reference numerals.

In contrast to the previous embodiment, the lighting module 200 operates according to 3 with dynamic light sheet generation, in which the light sheet is generated by scanning the illumination light 104. Accordingly, instead of the astigmatic optical system 116, the illumination module 200 contains a beam deflection unit 230, which has, for example, a rapidly moving scanning mirror that moves the illumination light 104 in a scanning movement over the sample area to be illuminated. The time in which the illumination light 104 is moved over the sample area to be illuminated is significantly shorter than the typical recording time of a camera onto which the sample area is imaged.

In the embodiment according to 3 the illumination module 200 contains a relay optics 232, which is arranged between the beam deflection unit 230 and the objective 114. The relay optics 232 is formed, for example, from a scan lens 234 and a tube lens 236. The relay optics 232 serves to image the beam deflection unit 230 onto the rear focal plane of the objective 114.

In the 3 In the embodiment shown, the optical switching element 122 is located between the beam deflection unit 230 and the relay optics 232. In the first optical setting of the switching element 122, which is shown in 3 As shown in the upper part of the image (A), the switching element 122 leaves the illumination light 104 passing through unaffected. Thus, the light sheet 118, which is generated by the beam deflection unit 230 in cooperation with the lens 114, also remains unaffected by the optical switching element 122.

If, however, the switching element 122 is switched to the second optical setting, which is shown in the lower part of the image (B) of the 3 As shown, the switching element 122 focuses the illumination light 104 into the rear focal plane of the objective 114. Again, the optical switching element 122 can be designed, for example, as an optical lens with a variable focal length, so that the focal length is adjusted accordingly for the purpose of the above-mentioned focusing. By focusing the illumination light 104 into the rear focal plane of the objective 114, an almost collimated beam is created, which generates the desired volume-like light distribution in the sample 108. In the second optical setting, the beam deflection unit 230 is deactivated. The beam deflection unit 230 is then set so that the illumination light 104 reflected by it in the illumination stage beam path 106 propagated centrally along the optical axis.

In the embodiment according to 3 the optical switching element 122 is arranged downstream of the beam deflection unit 230 in the illumination beam path. This prevents the optical switching element 122 from generating a focus of the illumination light 104 in the second optical setting that is located near the beam deflection unit 230. However, in this configuration, the light beam moved by the beam deflection unit 230 falls on the switching element 122. As a result of this scanning movement, aberrations that occur in the optical switching element 122 can impair the precision of the sample illumination.

To avoid such interference, the lighting module 200 may be configured according to 4 In this modification, the optical switching element 122 is arranged in front of the beam deflection unit 230 in the direction of light propagation. The illumination light 104 therefore does not perform a scanning movement at the location of the switching element 122. Aberrations that may occur in the switching element 122 therefore have less influence on the quality of the illumination.

As mentioned above, the settings of the optical switching element 122 can be controlled in the desired manner via the control unit 126. In particular, it is possible to set the optical switching element 122 depending on the wavelength of the illumination light 104. If the switching element 122 is designed, for example, as a lens with a variable focal length, the light distributions required for sample illumination can be precisely set in the desired form, even if the wavelength of the illumination light is varied. Without such wavelength-dependent control of the switching element 122, undesirable aberrations could otherwise occur when the wavelength changes.

The control unit 126 can also be used to vary the intensity of the illumination light 104 emitted by the light source 102 depending on the setting parameters of the optical switching element 122. Thus, in a 3D imaging in which the sample 108 is illuminated with the volume-like light distribution, the intensity of the illumination light 104 will generally have to be higher than in the case of a 2D imaging in which the sample 108 is selectively illuminated in a plane with the light sheet.

It should be noted in particular that it is the 1 to 4 The configurations shown make it possible to generate the desired light distributions for 2D and 3D imaging at the same wavelength. This is what distinguishes these configurations from conventional solutions, which generally use two separate light sources to switch between two illumination modes, whose light of different wavelengths is combined into a common illumination path via a dichroic beam splitter. The solution proposed here, on the other hand, can use one and the same light source and, in particular, without a dichroic beam splitter to provide the different illumination modes.

In the 5 and 6 a microscope 550 is shown which contains an illumination module of the type described above. In the following it is assumed that the illumination module 100 of the type described in the 1 and 2 It goes without saying, however, that the configuration shown in the 3 and 4 shown configurations and variations thereof can be used in the microscope 550.

The 550 microscope enables 2D imaging based on light sheet illumination and 3D imaging based on volume illumination. The 3D imaging is based on the principle of light field microscopy, in which the 3D information from the illuminated sample volume is projected onto a 2D camera sensor and a computational image reconstruction is then carried out.

In addition to the illumination module 100, the microscope 550 has a detection device 552, which serves to image the sample 108 illuminated in the respective mode in 2D or 3D. As explained above, the 2D/3D switching takes place via the optical switching element 122 contained in the illumination module 100. 5 illustrates the 2D imaging in the first optical setting of the switching element 122. In contrast, 6 the 3D imaging in the second optical setting of the switching element 122.

In the embodiment shown, the detection device 552 has an objective 554 that is provided separately from the objective 114 of the illumination module 100 and that collects detection light from the illuminated sample 108. In the embodiment shown, the two objectives 114 and 554 are arranged with their optical axes perpendicular to one another. A beam splitter 556 is arranged downstream of the objective 554 in the direction of propagation of the detection light. This is designed, for example, as a neutral splitter and lets part of the detection light through while reflecting the other part.

In the beam path of the transmitted detection light, the beam splitter 556 is followed by a tube lens 558 and a camera 560. The tube lens 558 and the camera 560 form a first detection module 562, which is functionally assigned to the first optical setting of the optical switching element 122.

A tube lens 564, a microlens arrangement 566 and a camera 568 are arranged one after the other in the beam path of the detection light reflected at the beam splitter 556. The tube lens 564, the microlens arrangement 566 and the camera 568 form a second detection module 570, which is assigned to the second optical setting of the optical switching element 122.

Alternatively, the beam splitter 556 can also be replaced by a mirror that can be motorized into the beam path and removed again. Depending on the switch position, the detection light is either completely reflected or transmitted.

In order to perform either 2D light sheet imaging using the first detection module 562 or 3D light field imaging using the second detection module 570 with the microscope 550, the optical switching element 122 is connected between the two in the 5 and 6 shown operating modes are switched.

An exemplary method for image reconstruction based on combined light sheet/light field imaging is shown in the flow chart of 7 shown.

In step S1, a stack of images of different planes of the sample 108 or of a sample similar to the sample 108 is first recorded. For each of these images, the optical switching element 122 is in the first optical setting, in which the illumination module 100 illuminates the sample with the light sheet 118. The respective image is recorded via the first detection module 562. In order to successively approach different sample planes, it is possible, for example, to move the sample 108 in the z direction. In addition to the image stack recorded with the first detection module 562 in the first optical setting of the switching element 122, a volume image of this sample is now recorded in the second optical setting of the switching element 122 with the second detection module 570.

Based on the image stack and the volume image acquired in step S1 (and possibly further image stacks and volume images acquired), a neural network is then trained in step S2.

In step S3, the illumination module 100 generates a volume-like light distribution in the sample 108. The optical switching element 122 of the illumination module 100 is in the second optical setting, in which the illumination light 104 is focused into the rear focal plane of the objective 114. In this way, a volume image of the sample 108 is generated using the light field technique using the second detection module 570.

Finally, in step S4, a volume image or a series of volume images is evaluated based on the neural network that was previously trained in step S2.

The term “and/or” includes all combinations of one or more of the associated listed elements and can be abbreviated with “/”.

Although some aspects have been described in the context of a device, it is clear that these aspects also represent a description of the corresponding method, wherein a block or device corresponds to a method step or a function of a method step. Analogously, aspects described in the context of a method step also represent a description of a corresponding block or element or a property of a corresponding device.

List of reference symbols

100

Lighting module

102

Light source

104

Illumination light

106

Illumination beam path

108

sample

110

Laser

112

Optical fiber

114

lens

116

Light sheet generation component

118

Light sheet

120

Cylindrical lens

122

Switching element

124

Collimator

126

Control unit

128

volume-like light distribution

230

Beam deflection unit

232

Relay optics

234

Scan lens

236

Tube lens

550

microscope

552

Detection device

554

lens

556

Beam splitter

558

Tube lens

560

camera

562

first detection module

564

Tube lens

566

Microlens array

568

camera

570

second detection module

Claims (21)

Illumination module (100, 200) for a microscope (550), comprising: a light source (102) which is designed to emit illumination light (104) into an illumination beam path (106), a focusing optical system (114) which is arranged in the illumination beam path (106) and is designed to direct the illumination light (104) into a sample (108), a light sheet generation component (116, 120, 230, 232) which is arranged upstream of the focusing optical system (114) in the illumination beam path (106) and is designed to form the illumination light (104) into a light sheet-like light distribution (118) at least in cooperation with the focusing optical system (114) for the selective light sheet illumination of a plane of the sample, and an optical switching element (122) which can be switched between a first optical setting and at least one second optical setting is, wherein the optical switching element (122) in the first optical setting enables the selective light sheet illumination of the plane of the sample (108) by leaving the illumination light (104) that is shaped by the light sheet generation component (116, 120, 230, 232) in cooperation with the focusing optical system (114) into the light sheet-like light distribution (118) uninfluenced, and the optical switching element (122) in the second optical setting shapes the illumination light (104) at least in cooperation with the focusing optical system (114) into a volume-like light distribution (128) that illuminates a volume region of the sample (108). Lighting module (100, 200) according to Claim 1 , wherein the optical switching element (122) is an element with variable optical power which is arranged in both optical settings in the illumination beam path (106). Illumination module (100, 200) according to one of the preceding claims, wherein the optical switching element (122) is an optical lens with variable focal length. Illumination module (100, 200) according to one of the preceding claims, wherein the optical switching element (122) is designed to focus the illumination light (104) in the second optical setting into the rear focal plane of the focusing optical system (114) or in the vicinity thereof. Illumination module (100, 200) according to one of the preceding claims, wherein the optical switching element (122) is designed to vary the distance between the light source (102) and the entrance pupil of the focusing optical system (114). Illumination module (100) according to one of the preceding claims, wherein the light sheet generating component comprises an astigmatic optical system (120) arranged in the illumination beam path (106) between the optical switching element (122) and the focusing optical system (114). Lighting module (100) after Claim 6 , wherein the optical switching element (122) is designed to focus the illumination light (104) into the astigmatic optical system (120) or in the vicinity thereof in the second optical setting. Lighting module (200) according to one of the Claims 1 until 5 , wherein the light sheet generating component comprises a beam deflection unit (230) configured to generate the light sheet-like light distribution (118) by scanning the illumination light (104). Lighting module (200) after Claim 8 , further comprising a relay optics (232) which is arranged in the illumination beam path (106) between the beam deflection unit (230) and the focusing optical system (114) and is designed to image the beam deflection unit (230) onto the rear focal plane of the focusing optical system (115). Lighting module (200) after Claim 8 or 9 , wherein the beam deflection unit (230) in the Illumination beam path (106) is arranged downstream of the optical switching element (122). Lighting module (200) after Claim 8 or 9 , wherein the beam deflection unit (60) is arranged upstream of the optical switching element (122) in the illumination beam path (106). Illumination module (100, 200) according to one of the preceding claims, wherein the light source (102) is controllable to vary the intensity of the illumination light (104) depending on the optical setting of the optical switching element (122). Illumination module (100, 200) according to one of the preceding claims, wherein the optical switching element (122) is controllable depending on the wavelength of the illumination light. Illumination module (100, 200) according to one of the preceding claims, wherein the light source comprises a plurality of lasers (110) whose laser light is combined in the illumination beam path (106) to form the illumination light (104). Illumination module (100, 200) according to one of the preceding claims, wherein the light sheet-like light distribution and the volume-like light distribution have the same wavelength. Illumination module (100, 200) according to one of the preceding claims, wherein the light sheet generating component (116, 120, 230, 232) is part of the focusing optical system (114). Microscope (550) with an illumination module (100, 200) according to one of the preceding claims. Microscope (550) by Claim 17 , comprising a detection device (552) with a first detection module (562) which is assigned to the first optical setting of the optical switching element (122) and a second detection module (570) which is assigned to the second optical setting of the optical switching element (122). Microscope (550) by Claim 18 , wherein the detection device (552) comprises an objective lens (554) provided separately from the focusing optical system (114) of the illumination module (100, 200). Microscope (550) by Claim 18 or 19 , wherein the second detection module (570) comprises a light field camera (566, 568). Method for image reconstruction using a microscope (550) according to Claim 20 , comprising the following steps: recording a stack of images of different planes of a sample (108) by means of the first detection module (562) in the first optical setting of the optical switching element (122), recording a volume image of the sample (108) by means of the second detection module (570) in the second optical setting of the optical switching element (122), training a neural network based on the stack of recorded images and the volume image, recording a further volume image of the sample (108) or of another sample by means of the second detection module (570) in the second optical setting of the optical switching element (122), and evaluating the further volume image by means of the trained neural network.

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