Method, microscope system, and computer program product for recording composite microscope images

Abstract

To provide a method, a microscope system, and a computer program product for recording a merged microscope image that has high optical quality and image clarity and enables high-speed and cost-effective imaging.SOLUTION: A method 100 includes: a step 110 including a process 111 of capturing a first image in which a sample is illuminated in a structured manner in order to microscope-capture at least a first fused image and a second fused image, a process 112 of capturing a second image in which the sample is uniformly illuminated, a process 113 of fusing the first image and the second image, the first image being used for low-frequency image information located in the focus and the second image being used for high-frequency image information located in the focus, the second fused image depicting a different position on the sample than the first fused image; and a step 120 of merging at least the first fused image and the second fused image with the merged microscope image.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method, a microscope system, and a computer program product for recording a composite microscope image. [Background technology]

[0002] Due to the wide range of applications, a large number of different microscopes are known, which can differ considerably in terms of their optics and their performance.

[0003] A drawback of microscopes is that the image section that can be captured in one image is very small.

[0004] Also, a commonality between optical systems is that the laws of optics dictate that the depth of field for microscopic imaging is very shallow, which, despite only observing a single focal plane, can lead to image capture being disrupted by radiation occurring outside the focal plane, which is subsequently perceived as a blurred luminous phenomenon on the captured image.

[0005] Various approaches to solving this problem are already known in the state of the art.

[0006] In a confocal microscope, only a portion of the object being imaged is illuminated at a time. The sample is then scanned one by one. The reflected or fluorescent light from the sample is measured at that position, and an entire sample image is constructed based on that. A pinhole aperture is installed in the beam path of the reflected or fluorescent light, allowing only light from the clearly imaged area to pass. This effectively reduces the effects of interference from radiation from layers outside the focal plane. The disadvantage of this setup is that it takes a long time to create a single image by scanning the object.

[0007] On the other hand, HiLo microscopy is also known as an alternative method. This method is described, for example, in Lim et al., Optics Letters-33:1819-1821 (2008) and Jerome Mertz, Nature Methods-8:811-819 (2011). The principle behind this imaging method is that two images are captured. In HiLo microscopy, the first image, with so-called speckle illumination, is effectively used as a filter to depict the low frequencies (Lo) in the image, and therefore the background. The second image (Hi), created with Gaussian illumination, contains the image information that is in focus. Using the first image as a filter allows for the removal of interfering radiation from planes outside the focal plane. Summary of the Invention [Problem to be solved by the invention]

[0008] The object of the present invention is to at least partially eliminate the above-mentioned drawbacks known from the prior art, in particular to provide a method, a microscope system and a computer program product for recording composite microscope images that allow fast and cost-effective imaging with particularly high optical quality, including in particular image sharpness, while at the same time having as large an image section as possible, in particular an image section of the entire sample. [Means for solving the problem]

[0009] The above-mentioned problem is solved by a method having the features of claim 1, a microscope system having the features of claim 14, and a computer program product having the features of claim 15. Further features and details of the invention will become apparent from the respective dependent claims, the description and the drawings. Features and details explained in relation to the method according to the invention naturally also apply in relation to the microscope system according to the invention and / or the computer program product according to the invention, and vice versa. Therefore, reference may be made or always made to individual aspects of the invention in relation to the present disclosure.

[0010] According to a first aspect of the present invention, there is provided a method for recording a composite microscope image of a sample, the method comprising: microscopically imaging at least the first fused image and the second fused image, in particular by means of an optical imaging device, - taking a first image, the sample being illuminated in a structured manner, in particular by a structured illumination device; taking a second image, the sample being uniformly illuminated, in particular by a Gaussian illumination device; - fusing a first image and a second image, the first image being used for low frequency image information located at the focus by applying a low pass filter, and the second image being used for high frequency image information located at the focus by applying a high pass filter; wherein the second fused image depicts a different location on the sample than the first fused image; combining, particularly by a computing device, at least the first fused image and the second fused image to form a combined microscopic image; Includes.

[0011] The method steps / stages may be performed at least in part simultaneously and / or sequentially, whereby the order of the method steps / stages is not limited by the order specified, and individual steps / stages may be performed in different orders. Furthermore, individual or all steps / stages may be performed repeatedly.

[0012] In the context of the present invention, reference is made to both first and second images and first and second fused images, where first and second refer only to the number of images or fused images and not to the order of imaging.

[0013] In particular, the method may be a computer-implemented method.

[0014] In the context of the present invention, recording of a microscopic image can be understood as the means necessary to obtain image information in a microscopic region of a sample.

[0015] A composite microscope image can be understood as a combination of several images with at least partially overlapping fields of view, and can in particular be produced by the synthesis of segmented panoramic or high-resolution images.

[0016] It is contemplated that definitions of imaging parameters are provided, which may consist of at least an imaging area, a resolution or a color channel.

[0017] An imaging area may be defined, resulting in a composite microscope image. The imaging area may be divided into fields of view where microscopic imaging occurs. The imaging area may include at least one area in the z-direction, x-direction, or y-direction. An area imaged in the z-direction, x-direction, or y-direction may also be referred to as a block.

[0018] Additionally, at least one definition of the resolution in at least the x, y, or z direction can be provided. In particular, the z resolution can be specified as the number of faces in the xy plane. This has the advantage that the user can easily record how many layers the composite image will contain.

[0019] The imaging area may be divided into blocks based on a defined resolution, the size of the blocks being defined as a function of, among other things, at least the number of fields of view, the number of xy planes, or the storage size of a storage device of the microscope system.

[0020] Furthermore, at least one color channel can be defined. If multiple color channels are defined, the order of the color channels can be defined during microscope imaging. In particular, at least one exposure time or filter depth can be defined individually for each color channel.

[0021] It is also contemplated that microscopic pre-imaging may be provided. Pre-imaging may be understood to mean that imaging is performed according to defined imaging parameters, but is limited compared to microscopic imaging, at least in terms of imaging area, resolution, filter depth, or computational quality. This allows the user to quickly get an overall picture of the effect of defining imaging parameters, without having to take the corresponding time to capture a complete image for each adjustment.

[0022] In principle, the sample can be any object that can be observed through the microscope used. In particular, the sample can be a biological and / or medical sample, in particular a cell, tissue or organ. The sample can be placed on an object holder. The object holder can hold one or more samples.

[0023] In the context of the present invention, microscopic imaging can be understood as generating, with the aid of an imaging device, an image containing information on the microscopic scale of a sample.

[0024] The fused image can be composed of information from at least two microscope images, particularly those taken at the same location on the sample.

[0025] The second image contains both blurred and sharp content. The blurred content substantially contains localized low-frequency components. A high-pass filter can be applied to the second image to discard the blurred image information. The high-pass filter can be configured to select only high spatial frequency components and filter out low frequency components.

[0026] In the first image, the structured illumination causes negligible contrast in the imaged modulation relative to the object signal occurring outside of the focus, i.e. the measure of the local contrast of the imaged modulation is a measure of how well the object is in focus.

[0027] A measure of local modulation contrast of the first image may be provided. The measure of modulation contrast of the first image may be performed as a measure of at least local variance, single sideband modulation, or double sideband modulation. The measure of local modulation contrast may be coarse-grained.

[0028] The low-frequency image information derived from the first image can be configured to be supplemented with high-frequency image information derived from the second image. The fusion of the high- and low-frequency image content results in a fully resolved, sharp image that contains all frequency content within the frequency bandwidth of the microscope system. This essentially corresponds to a HiLo microscopy approach.

[0029] Structured illumination can be understood as illumination that is configured to generate radiation from the sample in response to the illumination, primarily in the focal plane. In particular, the intensity of the structured illumination can vary depending on the location. The intensity variation can be generated by a diffuser illuminated by a light source, in particular a coherent light source.

[0030] Uniform illumination (also known as Gaussian illumination) can be defined as illumination that exposes the imaged area with a substantially uniform intensity. Uniform illumination always has some degree of unavoidable non-uniformity due to the characteristics of the light source and the optical system. Uniform illumination can be achieved by a rotating diffuser illuminated by a light source, particularly a coherent light source. It is particularly advantageous for the diffuser to be the same as the diffuser used to provide structured illumination, so that the diffuser is stationary when structured illumination is provided. Therefore, it is possible to switch between structured illumination and uniform illumination by stopping or rotating the diffuser.

[0031] Furthermore, the order in which the first and second images are captured may be configured such that the first image is followed by another first image after the position on the sample has moved, or the second image is followed by another first image after the position on the sample has moved. This has the advantage that it is not necessary to change the imaging mode of the imaging device for each image capture, enabling faster imaging.

[0032] Combining fused images (also called stitching) can be understood as combining the image information contained in the fused images, which are at least partially overlapping, so that, in particular when the images are combined, at least the overlapping regions can be identified and the images can be aligned or their appearance can be adjusted, in particular at least with respect to brightness, color or contrast.

[0033] The different locations on the sample may mean that the first fused image and the second fused image have at least one region that is not included in both images. In other words, this means that the two images may be captured at different locations. Microscopic imaging and merging may be performed several times, particularly once, for a given region of the image, particularly a block. A block may have one or more images at different xy positions and planes. Merging may first be performed using one set of fused images from the first block and additional blocks, using the fused image with the highest contrast, whereby additional fused images may be merged using this ratio. This allows the merging process to minimize image errors.

[0034] The method may comprise at least one of the following steps / stages, particularly in the order presented:

[0035] At least the sample or lens is moved to a first block. A first xy plane of the block is microscopically imaged, where a first and second image are captured and fused. A next xy plane of the first block is microscopically imaged, where a first and second image are captured and fused until all xy planes of the first block have been microscopically imaged. Microscopic imaging of the first block is repeated for one or more additional color channels. All fused images of all xy planes of the first block are intermediately saved.

[0036] At least the sample or the lens is moved to a further block. A first xy plane of the further block is microscopically imaged, thereby capturing and fusing a first and second image, and a next xy plane of the further block is microscopically imaged, thereby capturing and fusing a first and second image until all xy planes of the block have been microscopically imaged. The microscopic image capture of the next block is repeated for one or more further color channels. All fused images of all xy planes of the first block are intermediately saved.

[0037] The fused image of the first block, particularly the xy-plane with the highest contrast in the first color channel, is combined with the fused image of the second block from the same xy-plane. The "same xy-plane" may particularly refer to at least partially overlapping image content. Alternatively or additionally, it may refer to at least the same position in the z-direction relative to the lens or sample. All further fused images of the first block are also combined according to the relationship of the first combined image.

[0038] At least the sample or lens can then be moved to the next block and the steps / stages repeated. The composite images of the blocks can be combined with each other to create a composite microscope image of the sample.

[0039] Overall, the advantage of the method according to the present invention is that it allows fast, cost-effective imaging with particularly high optical quality, including image sharpness, while simultaneously having as large an image section as possible, particularly an image section of the entire sample. The fusion of the first and second images means that image information from planes out of focus of the lens can be discarded, thereby enabling images with increased image sharpness to be created very quickly. This method is particularly fast compared to scanning images, as in confocal microscopy, because it does not require scanning images, and only two images need to be captured. By combining at least two, particularly several, fused images, the imaging speed can be exploited to stitch together several images, thus generating images with particularly large amounts of information, such as segmented panoramic images or high-resolution images with a large depth of field.

[0040] Within the scope of the present invention, it is also conceivable that the structured illumination is configured as at least a speckle illumination, a periodic grating or a checkerboard pattern when capturing the first image.

[0041] Speckle illumination can be configured to generate scattered points of light. Speckle can be understood as individual point-like locations on the sample where the illumination intensity is higher relative to the rest of the sample. Speckle can be generated by interference. For this purpose, a monochromatic coherent light source combined with a diffuser can be provided.

[0042] The advantage of speckle illumination is that it produces particularly sharp fused images because the intensity of backscattered light outside the focal plane is significantly reduced.

[0043] Periodic grating or checkerboard patterns can also be provided with the aid of interference generating optics.

[0044] Furthermore, in the method according to the invention, it may be advantageous for the illumination during the acquisition of the first and second images to be configured as fluorescent illumination, in particular having at least two color channels.

[0045] In other words, the first and second images can be fluorescence images, particularly multi-channel fluorescence images. Fluorescence images are particularly susceptible to radiation that does not originate from the focal plane, since this radiation can cause a certain blurring effect, blurring otherwise sharp structures against a fairly dark background. Therefore, the method according to the invention in combination with fluorescence illumination is particularly advantageous.

[0046] Furthermore, in the method according to the present invention, the first and second images can be captured in at least first and second color channels, whereby the sample is first illuminated in a structured and uniform manner in the first color channel and then in a structured and uniform manner in the second color channel. When capturing multiple color channels, it can be particularly advantageous to always start the illumination of the first or second image from the illumination at the time the capture of the previous color channel ended. For example, in the case of three color channels (red, green, and blue), the order would be structured illumination of red, uniform illumination of red, uniform illumination of green, structured illumination of green, structured illumination of blue, and uniform illumination of blue. This means that the diffuser does not need to be accelerated and decelerated frequently, which saves time during the capture and reduces interference in the form of vibrations.

[0047] When there are several color channels and microscope imaging is performed in several xy-planes, it is possible to first perform microscope imaging in each xy-plane of each color channel before switching to the next xy-plane. This has the advantage that particularly high image quality is achieved when combining the individual channels, since the image is captured without moving the sample or the lens. It is also possible for each xy-plane to be traversed by a single color channel.

[0048] Furthermore, in the method according to the invention, it may be advantageous that the position on the sample varies at least in the sample z-direction, the sample x-direction or the sample y-direction, in particular that the position of the sample in at least the x-direction or the y-direction can be moved by an xy-stage on which the sample can be positioned.

[0049] In the context of the present invention, the z-direction can be understood as the depth of the sample on the optical axis of the imaging optics within the sample region. The x- and y-axes therefore extend in a plane perpendicular to the optical axis within the sample region.

[0050] By varying the z-position, the composite microscope image can essentially correspond to a tomographic image: individual layers can be displayed with particular clarity due to the defined depth of field.

[0051] The sample can be mapped by combining fused images in the x and / or y directions. The entire sample can be combined from the fused images in at least the x, y, or z directions. This has the advantage that a particularly large amount of information is available for evaluation, since the sample is completely recorded from one or more directions.

[0052] In the context of the present invention, it is also conceivable that the position of the sample in the second fused image differs from the position of the sample in the first fused image by 500 nm to 500 μm, in particular 1 μm to 300 μm in the z direction.

[0053] In other words, optical microscopes can measure very thick samples. With thick samples, conventional optical microscopes have the problem that radiation from planes that do not correspond to the focal plane is added, resulting in a blurred composite image. This is prevented by the method according to the invention, so that samples in a specified thickness range can also be imaged with particularly high quality.

[0054] It is also conceivable within the scope of the present invention to adjust the depth of field of the individual captured fused images.

[0055] This allows the fusion effect to be optimally adapted to the current imaging situation. A deeper depth of field allows more information to be obtained from areas that are not exactly in the focal plane. This prevents the elimination of object information in the case of objects that are far apart in the z-plane when the distance between the combined fused images is large or when the focal plane is not set perfectly correctly. On the other hand, a shallow depth of field ensures particularly sharp images and a good overall view with a small distance between the combined images in the z-direction.

[0056] It is also conceivable that the method according to the invention also provides for the output of a composite microscope image, whereby in particular the depth in the z direction can be set during the output.

[0057] This allows the user to select the optimal depth of field for each situation. As mentioned above, its value can vary even within a sample. Therefore, customization always ensures an optimal user experience and allows for accurate evaluation of the composite microscope image.

[0058] It is further conceivable that in the method according to the invention, knowledge is provided regarding a lens configured to capture at least the first image and the second image, and in particular the depth in the z direction is set based on knowledge regarding the lens.

[0059] By adapting to each lens, it is possible to set the z-depth that represents the optimum compromise between image sharpness and depth of field.

[0060] In the method according to the invention, it is also conceivable that the microscopic imaging of at least the first fused image and the second fused image is performed successively.

[0061] Taking the images to be fused successively has the advantage that the taken images can be generated particularly quickly.

[0062] In the context of the present invention, it is further contemplated that shading correction is provided when at least the first fused image and the second fused image are combined to form a composite microscope image.

[0063] Shading correction can achieve particularly good optical quality, especially in the transition between two images to be combined.

[0064] It is also conceivable in the method according to the invention that at least the first fused image or the second fused image is displayed as a live image, thereby setting at least one depth in the z-direction.

[0065] The live image allows the user to immediately see the effect of the z-depth setting and therefore set the optimum value for the measurement. The immediate feedback effectively prevents the initiation of a lengthy imaging process, only to later discover that the setting was not optimum.

[0066] Preferably, the method according to the invention comprises at least Loading microscope slides into the microscope system, Overview scans of microscope slides, Recording label information on microscope slides, Detection of samples on microscope slides, Identifying the image recording area based on the detection of the sample; Defining imaging parameters, microscope preview imaging with predefined imaging parameters, in particular microscope preview imaging according to the invention, Microscopic imaging with predefined imaging parameters, in particular microscopic imaging according to the invention, · Creating a focus map, Movement of the xy stage to the image recording area, Correction of lens defects, storing at least the first image, the second image, the first fused image, the second fused image, or the composite microscopic image; and Movement of the xy stage to the next specimen or microscope slide is further conceivable.

[0067] By recording the label information, the microscope can be optimally adjusted before taking an image, e.g., the z-depth can be adjusted to match the label information. For example, the label can contain information about the thickness or type of sample, so that a specific depth is advantageous.

[0068] An overview scan of a sample can be understood as microscopic imaging with at least lower resolution, fewer or different channels, a larger image area, or a lower magnification compared to microscopic imaging, such an overview scan allowing for rapid identification of the sample and the area of ​​interest for high-resolution imaging.

[0069] The next sample can be quickly picked up by moving the xy stage to the next sample. Furthermore, the next microscope slide, which may have one or more samples on it, can also be picked up in this way. The next microscope slide can also be inserted into the xy stage via the microscope slide mechanism.

[0070] Loading the microscope slide into the microscope system, overview scanning the microscope slide, identifying the image recording area, defining the imaging parameters, microscope imaging, saving at least a first image, a second image, a first fused image, a second fused image or a composite microscope image, or moving the xy stage to the next sample or the next microscope slide may be performed in a specifically specified order.

[0071] According to a further aspect of the present invention, there is provided a microscope system, in particular for carrying out the method according to the present invention, comprising: an optical imaging device adapted to provide microscopic imaging of at least a first fused image and a second fused image, a camera and lens configured to capture the first image and the second image; a structured illumination device configured to illuminate the sample in a structured manner when the first image is captured; a Gaussian illuminator adapted to uniformly illuminate the sample when the second image is taken; an optical imaging device including: a computing device adapted to fuse a first image and a second image, wherein the first image is used for low frequency focused image information and the second image is used for high frequency focused image information, the second fused image depicting a different location on the sample than the first fused image, and combining at least the first fused image and the second fused image into a combined microscope image; Equipped with.

[0072] The microscope system according to the invention therefore has the same advantages as those described above with reference to the method according to the invention.

[0073] The structured illumination device may comprise at least one coherent light source, in particular a laser, or a diffuser plate, whereby the diffuser plate is illuminated by laser light and generates a speckle pattern.

[0074] Furthermore, the diffuser plate can be configured to be rotatable, so that when illuminated by a Gaussian illuminator, it can be rotated to a degree that allows a uniformly illuminated image to be captured on the time scale of microscopy imaging. This arrangement allows the first and second images to be generated particularly quickly, one after the other, thereby significantly reducing the overall capture time of the composite microscopy image.

[0075] According to a further aspect of the invention there is provided a computer program product comprising instructions which, when the program is executed by a computer, in particular a computing device of a microscope system according to the invention, cause the computer to carry out a method according to the invention.

[0076] The computer program product according to the invention therefore has the same advantages as those described above with reference to the method according to the invention and / or the microscope system according to the invention.

[0077] Further advantages, features and details of the invention will become apparent from the following description in which embodiments of the invention are described in detail with reference to the drawings. The features set out in the claims and in the description may be essential to the invention individually or in any combination. [Brief explanation of the drawings]

[0078] [Figure 1] FIG. 1 illustrates a method according to the present invention. [Figure 2] FIG. 10 is a diagram illustrating an example of synthesizing a fusion image. [Figure 3] FIG. 10 is a diagram illustrating another example of synthesizing a fusion image. [Figure 4] 1 is a diagram showing a microscope system according to the present invention; DETAILED DESCRIPTION OF THE INVENTION

[0079] In the following description of several embodiments of the present invention, the same reference signs are used for the same technical features in different embodiments.

[0080] FIG. 1 shows a method 100 for recording a composite microscope image 200 of a sample 300. The method 100 includes: A step 110 of microscopically capturing at least a first fused image 210 and a second fused image 220, a step 111 of taking a first image 230, in which the sample 300 is illuminated in a structured manner; capturing 112 a second image 240, wherein the sample 300 is uniformly illuminated; and A step 113 of fusing a first image 230 and a second image 240, where the first image 230 is used for low frequency image information located at the focus and the second image 240 is used for high frequency image information located at the focus, wherein the second fused image 220 depicts a different location on the sample 300 than the first fused image 210; a step 120 of combining at least a first fused image 210 and a second fused image 220 into a combined microscope image 200; Includes.

[0081] Overall, the method 100 according to the present invention achieves the advantage of enabling fast, inexpensive imaging with the largest possible image section, particularly an image section of the entire sample 300, while at the same time having particularly high optical quality, including particularly high image sharpness. The combination of the first image 230 and the second image 240 means that an image can be created very quickly, allowing image information from planes not in the focus of the lens 412 to be discarded, thereby increasing image sharpness. This method is particularly fast compared to scanning through images with a confocal microscope, since it does not require image scanning and only captures two images 230, 240. By combining at least two, particularly several, fused images 210, 220, the imaging speed can be exploited to stitch together several images, resulting in a particularly information-rich image 200, such as a segmented panoramic image or a high-resolution image with a large depth of field.

[0082] Furthermore, further steps / stages may be provided in the method according to the invention.

[0083] For example, output 130 of composite microscope image 200 may be provided, inter alia, on a screen. In particular, the depth in the z-direction at which image information from composite microscope image 200 is discarded may further be adjustable during output 130. In this way, a user can optimally adapt the depth to the evaluation and / or imaging requirements.

[0084] 2 and 3 show how the fused images 210, 220 can be combined 120.

[0085] FIG. 2 shows a top view of a sample 300. Dashed lines indicate the points where a first fused image 210 and a second fused image 220 are captured. Each dotted square corresponds to a first image 230 and a second image 240, which are then combined 113. In other words, the microscope system 400 captures two images 230, 240 four times in the illustrated example. The fused images 210, 220 overlap in each case. The fused images 210, 220 are then combined 120 to generate a composite microscope image 200. This composite microscope image 200 covers a much larger area of ​​the sample 300 in the xy plane than the individual fused images 210, 220. In particular, the entire sample 300 and / or preselected sections of the sample may be covered by the composite microscope image 200.

[0086] Figure 3 shows a further variation for synthesizing 120 the fused images 210, 220. In the example of Figure 2, the positions of the fused images 210, 220 on the sample vary in the xy plane, whereas in the example of Figure 3, variations in the z plane are shown. In other words, it is also possible to synthesize 120 the fused images 210, 220 that vary in the z axis, which is parallel to the optical axis of the imaging device 410 (see Figure 4).

[0087] The dashed line indicates the depth in the z-direction at which image information from the second image 240 that is not in focus of the second image 240 is discarded. This depth may be adjustable. The distance between the fused images 210, 220 may be selected as shown so that the fused images 210, 220 do not overlap in the depth direction. The fused images 210, 220 may also at least partially overlap at these depths.

[0088] Of course, it is also possible to provide a composite microscope image 200 by combining 120 fused images 210, 220 that vary in the xy plane as well as fused images 210, 220 that vary on the z axis. As a result, the user of the microscope system 400 has particularly large amounts of image information available for evaluation, whereby the method 100 according to the present invention generates a composite microscope image 200 very quickly, despite the large amount of information.

[0089] 4 shows a microscope system 400 for carrying out the method 100 according to the invention. The microscope system 400 comprises: an optical imaging device 410 adapted to provide microscopic imaging 110 of at least a first fused image 210 and a second fused image 220, a camera 411 and a lens 412 adapted to capture 111 the first image 230 and the second image 240; a structured illumination device 413 adapted to illuminate the sample 300 in a structured manner when the first image 230 is taken, and a Gaussian illuminator 414 adapted to uniformly illuminate the sample 300 when the second image 240 is taken; an optical imaging device 410 including: a computing device 420 adapted to fuse 113 a first image 230 and a second image 240, where the first image 230 is used for low frequency focused image information and the second image 240 is used for high frequency focused image information, and where the second fused image 220 depicts a different location on the sample 300 than the first fused image 210, and the computing device 420 adapted to combine at least the first fused image 210 and the second fused image 220 into a combined microscope image 200; Equipped with.

[0090] 4 illustrates a microscope system viewed in the x-z plane. Light is directed from a coherent light source 430 through a structured illuminator 413 and / or a Gaussian illuminator 414, through a beam splitter 450 and a lens 412, onto a sample 300 placed on a stage 415. Reflected or fluorescent light is reflected back through the beam splitter 450 and onto a camera 411. In the illustrated embodiment, the structured illuminator 413 and the Gaussian illuminator 414 include a diffuser 440 configured to be rotatable. When the diffuser 440 is not rotated, speckle illumination is generated by the structured illuminator 413 to capture 111 a structured illumination image 230. When the diffuser 440 is configured to rotate, a uniformly illuminated image 240 can be captured 112. The control device 420 may control at least the image capture device 410 , the camera 411 , the lens 412 , the structured illumination device 413 , the Gaussian illumination device 414 , the stage 415 , the coherent light source 430 or the diffuser 440 .

[0091] The microscope system 400 according to the invention therefore has the same advantages as those described above with reference to the method 100 according to the invention.

[0092] The structured illumination device 413 may comprise at least one coherent light source 430, in particular a laser, and a diffuser 440, which is illuminated by the laser light and generates a speckle pattern.

[0093] Furthermore, the diffuser 440 is configured to be rotatable, and when illuminated by the Gaussian illuminator 414, can be rotated to such an extent that uniformly illuminated images are captured on the time scale of the microscope imaging 110. Such an arrangement allows the structured and uniformly illuminated images 230, 240 to be generated particularly quickly, one after the other, resulting in a significant reduction in the overall imaging time of the composite microscope image 200.

[0094] The foregoing description of the embodiments is intended to illustrate the invention purely by way of example, and it goes without saying that the individual features of the embodiments can be freely combined with one another, where technically expedient, without departing from the scope of the invention.

Claims

1. A method (100) for recording a composite microscope image (200) of a sample (300), comprising: Microscopically imaging (110) at least a first fused image (210) and a second fused image (220), comprising: a step (111) of taking a first image (230), wherein the sample (300) is illuminated in a structured manner; capturing (112) a second image (240), wherein the sample (300) is uniformly illuminated; a step (113) of fusing the first image (230) and the second image (240), wherein the first image (230) is used for low frequency image information located at the focal point and the second image (240) is used for high frequency image information located at the focal point; wherein the second fused image (220) depicts a different location on the sample (300) than the first fused image (210); combining (120) at least the first fused image (210) and the second fused image (220) into the composite microscopic image (200); Including, setting a depth of field of each of the first fusion image (210) and the second fusion image (220) captured; Method (100).

2. the structured illumination in the step (111) of capturing the first image (230) is implemented as at least a speckle illumination, a periodic grating, or a checkerboard pattern; The method (100) of claim 1.

3. the illumination in the step (112) of capturing the first image (230) and the second image (240) is configured as a fluorescent illumination, in particular having at least two color channels; The method (100) according to claim 1 or 2.

4. the steps (111) of taking the first image (230) and (112) of taking the second image (240) are performed using at least a first and a second color channel, in particular the sample (300) is first illuminated in a structured and uniform manner using the first color channel, and thereafter the sample (300) is illuminated in a structured and uniform manner using the second color channel, The method (100) according to claim 1 or 2.

5. The position on the sample (300) varies at least in the z-direction of the sample (300), the x-direction of the sample (300) or the y-direction of the sample (300), and in particular the position of the sample (300) in at least the x-direction or the y-direction is moved by an xy-stage (415) on which the sample (300) can be positioned. The method (100) according to claim 1 or 2.

6. the position of the sample (300) in the second fused image (220) differs from the position of the sample (300) in the first fused image (210) by 500 nm to 500 μm, in particular 1 μm to 300 μm in the z direction, The method (100) according to claim 1 or 2.

7. An output (130) of the composite microscope image (200) is further provided, characterized in that it is possible to set a depth in the z-direction in the output (130). The method (100) according to claim 1 or 2.

8. a recognition of a lens (412) configured for performing the step (111) of capturing at least the first image (230) or for performing the step (112) of capturing the second image (240) is provided, in particular a depth in the z-direction is provided based on the recognition of the lens (412), The method (100) according to claim 1 or 2.

9. wherein the step (110) of microscopically imaging at least the first fusion image (210) and the second fusion image (220) is performed consecutively. The method (100) according to claim 1 or 2.

10. wherein the step (120) of combining at least the first fused image (210) and the second fused image (220) to form the composite microscopic image (200) also provides shading correction. The method (100) according to claim 1 or 2.

11. At least the first fusion image (210) or the second fusion image (220) is displayed as a live image, and at least one depth in the z-direction is adjustable. The method (100) according to claim 1 or 2.

12. Furthermore, at least Loading the microscope slide into the microscope system (400); an overview scan of the microscope slide (310); recording label information on said microscope slide (310); detection of said sample (300) on said microscope slide (310); - Identifying an image recording area based on the detection of said sample (300); - Creating a focus map, - moving the xy stage (415) to the image recording area; - Correction of lens defects, storing at least the first image (230), the second image (240), the first fused image (210), the second fused image (220) or the composite microscopic image (200); Movement of the xy stage (415) to the next sample (300) or the next microscope slide (310) The present invention is characterized in that: The method (100) according to claim 1 or 2.

13. A microscope system (400) for carrying out the method (100) in particular according to claim 1 or 2, comprising: an optical imaging device (410) adapted to provide microscopic imaging (110) of at least a first fused image (210) and a second fused image (220), a camera (411) and a lens (412) adapted to capture (111) a first image (230) and a second image (240); a structured illumination device (413) adapted to illuminate the sample (300) in a structured manner when the first image (230) is taken; and a Gaussian illuminator (414) adapted to uniformly illuminate the sample (300) when the second image (240) is captured; an optical imaging device (410) including: a computing device (420) adapted to fuse (113) the first image (230) and the second image (240), wherein the first image (230) is used for low frequency focused image information and the second image (240) is used for high frequency focused image information, and the second fused image (220) depicts a different location on the sample (300) than the first fused image (210), and fuse at least the first fused image (210) and the second fused image (220) into a composite microscope image (200); Equipped with A microscope system (400).

14. The program comprises instructions that, when executed by a computer, in particular a computing device (420) of a microscope system (400) according to claim 13, cause the computer to carry out the method according to claim 1 or 2. Computer program products.

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