Controller for laser microdissection system, laser microdissection system, and method for laser microdissection

Abstract

To improve known laser microdissection systems. [Solution] A controller 136 for the laser microdissection system 100 is configured to receive a first image and a first coordinate set of a sample 102 having a plurality of image tiles stitched together, the first coordinate set defining at least one segment to be removed from the sample 102. The controller 136 also controls the laser microdissection system 100 to generate at least one second image of a portion of the sample 102, each generating at least one second coordinate set corresponding to at least a subset of the first coordinate set, and based on the at least one second coordinate set, controls the laser microdissection system 100 to remove at least one segment from the sample 102.

Description

Technical Field

[0001] The present invention relates to a controller for a laser microdissection system and a laser microdissection system. The present invention further relates to a method for laser microdissection.

[0002] Background of the Invention A laser microdissection system uses a laser beam to separate small portions, called dissectates, from a specimen. The specimen can be, for example, a thin tissue section that is cut to isolate specific cells or other microscopic regions of interest. The regions of interest here can be determined, for example, in an overview image of the specimen acquired by a slide scanner. In particular, these regions of interest can be determined as a set of coordinates that define the regions of interest with respect to the coordinates in the overview image. In order to make these coordinates usable in a laser microdissection system, it is necessary to convert these coordinates, for example, into the coordinate system used by the laser microdissection system.

[0003] Summary of the Invention An object of the present invention is to provide a controller for a laser microdissection system, a laser microdissection system, and a method for laser microdissection that improve known systems and methods.

[0004] The above object is achieved by the subject matter of each independent claim. Further embodiments are defined in each dependent claim and in the following description.

[0005] The proposed controller for a laser microdissection system is configured to receive a first image of a sample having a plurality of image tiles stitched together with each other, and a first set of coordinates in the coordinate system of the first image. The first set of coordinates defines at least one segment to be removed from the sample. The controller is also configured to control the laser microdissection system to generate at least one second image of a portion of the sample, and to generate at least one second set of coordinates in the coordinate system of the second image, wherein each of the second sets of coordinates corresponds to at least a subset of the first set of coordinates, and to control the laser microdissection system to remove at least one segment from the sample based on the at least one second set of coordinates. The controller is further configured to determine at least two reference points on the sample that are visible in the first image and at least one second image, to determine original coordinates corresponding to the coordinates of the reference points in the coordinate system of the first image, to determine target coordinates corresponding to the coordinates of the reference points in the coordinate system of at least one second image, and to generate at least one second set of coordinates based on the original and target coordinates.

[0006] The first image may be an overview image of a sample acquired by, for example, a slide scanner, a multi-space imager, or a fluorescence microscope. The sample may include a carrier, such as a membrane placed in a frame, a microscope slide, or a cell culture dish, and one or more specimens placed on it. The actual specimen may only cover a portion of the carrier, meaning that the first image may have one or more image tiles that do not contain the specimen. These image tiles are substantially empty and featureless, and it is difficult to accurately stitch them together because the pixel rows and columns cannot be accurately correlated. For example, in fluorescence images, image tiles that do not contain a portion of the actual specimen are black, and it is extremely difficult to correctly align them for stitching. It is recognized that this can cause problems when converting segments defined in the coordinate system of the overview image to the coordinate system used by the laser microdissection system. For example, a fixed reference point placed on the edge of the carrier is used. In this case, there may be many empty image tiles between the fixed point and the actual specimen, which can cause misalignment of the segments when converted to the coordinate system used by the laser microdissection system due to inaccurate stitching. Even a small misalignment of about 1 μm can cause a laser microdissection system to miss cell nuclei that are, for example, about 2 μm to 10 μm in size.

[0007] The proposed controller addresses the potential misalignment problem between the coordinate system of a first image, which may be an overview image acquired by a slide scanner, and the coordinate system of a second image, which is acquired by the laser microdissection system itself, by using a dynamic reference point. The reference point may be, for example, a structure of the actual specimen that is visible in both the first and second images. From the coordinates of the reference point in the first image (i.e., the original coordinates) and the coordinates of the reference point in the second image (i.e., the target coordinates), the controller determines the relationship between the coordinate systems of the first and second images. From this relationship, the controller determines at least a second set of coordinates, i.e., the coordinates of the segment in the coordinate system of the second image. Based on at least one second set of coordinates, the segment determined in the first image can be precisely removed using the laser microdissection system.

[0008] Using the proposed controller offers further advantages. The controller allows for the faithful conversion of segment coordinates from an external device to the laser microdissection system. This enables the user to pre-determine which segments to be removed from the sample using a device other than the laser microdissection system, which is more time-efficient than defining segments using the laser microdissection system itself. The controller uses dynamic reference points, all of which can be located within the actual sample. Therefore, for example, it is not necessary to capture the entire carrier as the first image in order to capture a fixed reference point placed on the carrier. This significantly reduces the number of image tiles that need to be captured to generate the first image. Since the memory requirements for each image tile are approximately the same regardless of whether the image tile contains useful information or not, the memory required to store the first image can also be significantly reduced.

[0009] The controller may comprise at least one processor and at least one memory element. The controller may also include an interface for connecting to remote memory elements, in addition to or as an alternative to local memory elements.

[0010] In one embodiment, the controller is configured to control the laser microdissection system to generate at least two second images, each second image containing a different portion of the sample, and to determine at least one of the reference points for each second image such that the reference points are visible in the first image and each of the second images. In such embodiments, the controller controls the laser microdissection system to image multiple different portions of the sample and determines at least one reference point for each portion. In this way, the reference points can cover a large area of ​​the actual sample, and it is possible to faithfully transform the coordinates of the segments within this large area all at once. In particular, having a grid of reference points that covers a large area of ​​the actual sample makes it possible to faithfully transform the coordinates of all segments all at once. This significantly reduces the time required to transfer the segment coordinates to the laser microdissection system.

[0011] In another embodiment, the controller is configured to control the laser microdissection system to generate at least two second images, each second image containing a different portion of the sample, and for each of the second images, to determine at least two of reference points such that the reference points are visible in the first image and each of the second images, and to generate one of a second set of coordinates for each of the second images. In such embodiments, at least two of the reference points are determinable for one of the second images before another of the second images is acquired. Using the reference points determined for one of the second images, the controller then transforms the segments in the portion of the sample that are visible in that second image, i.e., generates a second set of coordinates corresponding to the segments in the portion of the sample that are visible in that second image. This process is then repeated for at least one further portion of the sample. This allows the controller to transfer the segment coordinates section by section to the laser microdissection system, and the coordinate transformation can be performed in parallel with the acquisition of the second image. This further reduces the time required to transfer the segment coordinates to the laser microdissection system.

[0012] In another embodiment, the controller is configured to control the laser microdissection system to remove at least one of the segments from the sample based on one of the second coordinate sets before another image of the second image is generated. This makes it possible to remove segments from the sample section by section by removing a segment from one part of the sample before moving to the next part of the sample. This significantly speeds up the process of removing segments from the sample.

[0013] In another embodiment, the controller is configured to determine the original coordinates based on target coordinates, a first image, and a second image using an image matching method. In such embodiments, the controller determines the original coordinates, i.e., the coordinates of the reference point in the coordinate system of the first image, by matching, for example, features of the actual specimen in the first image and the second image. To match features, the controller employs an image matching method. For example, as part of the image matching method, the controller can use artificial intelligence such as a convolutional neural network or any other mathematical algorithm, such as machine learning algorithms like Scale-Invariant Feature Transform (SIFT), image analysis or pattern recognition methods, or a combination thereof. Furthermore, instead of visible images, image properties describing the corresponding region of interest can be used for matching.

[0014] In another embodiment, the controller is configured to determine a section of the first image that corresponds to at least one of the second images, and to determine the original coordinates based on the target coordinates and the section of the first image. In such embodiments, the controller matches the coordinates of reference points in the first and second images by superimposing the second image onto the first image. This is a robust method for determining the original coordinates from the target coordinates. The controller can be configured to use an image matching method to determine a section of the first image that corresponds to at least one of the second images.

[0015] In another embodiment, the laser microdissection system is configured to generate at least one second image using at least one imaging technique. The controller can further be configured to generate an artificial first image based on the first image, simulating the visual characteristics of at least one imaging technique, and to determine the original coordinates based on the target coordinates and the artificial first image. The artificial first image is a simulation of what the first image would look like if it were generated using at least one imaging technique employed by the laser microdissection system. For example, the laser microdissection system can be configured to employ transmitted light microscopy as at least one imaging technique. The first image may be an image generated from multiple different fluorescence channels, a so-called multiplex fluorescence image. The controller can be configured to generate an artificial first image from the first image, for example, by simulating hematoxylin and eosin staining patterns using known methods. Simulating how the first image would look if it were generated using at least one imaging technique employed by the laser microdissection system helps in matching the first and second images, thereby enabling a more robust determination of the original coordinates based on the target coordinates. By determining the original coordinates more robustly, the segment can be transferred to the laser microdissection system with greater fidelity.

[0016] In another embodiment, the controller is configured to determine a coordinate transformation from the coordinate system of a first image to the coordinate system of at least one second image, and to generate at least one second set of coordinates based on said coordinate transformation. The coordinate transformation may be a linear transformation, for example, corresponding to rotation, inversion, and / or stretching or compression. Two reference points are sufficient for the controller to determine the coordinate transformation as a linear transformation. The controller can use three or more reference points to determine the coordinate transformation as a nonlinear transformation, which may also take into account nonlinear effects such as geometric distortions induced by the optical system, such as spherical aberration.

[0017] The present invention also relates to a laser microdissection system comprising at least one laser light source configured to generate a laser beam, an optical system configured to guide the laser beam onto a sample, and the aforementioned controller.

[0018] The laser microdissection system has the same advantages as the controller described above. In particular, the laser microdissection system can be supplemented by the features described herein in relation to the controller. Furthermore, the controller described above can be complemented by the features described herein in relation to the laser microdissection system.

[0019] In one embodiment, the laser microdissection system includes a scanning unit configured to move a laser beam within the field of view of the objective lens of the optical system. In this embodiment, the sample can be kept stationary, and the scanning unit can be used to move the laser beam over the sample with minimal effort. This reduces the number of moving parts in the laser microdissection unit and therefore improves the accuracy of separating the dissected pieces from the sample.

[0020] In another example, the scanning unit has two prisms rotatably positioned around the optical axis between the laser source and the objective lens. Here, the optical axis is, for example, the optical axis of the objective lens or its extension, via a beam splitter. Each prism deflects the laser according to the rotation of the prism. The beam deflections caused by each prism are vector-added. Therefore, by rotating the two prisms, the laser beam can be moved within the field of view of the objective lens. In particular, the rotation of the prisms also causes a change in the beam offset at the output of the scanning unit. This beam offset compensates for the lateral deflection of the laser beam separately generated in the plane of the objective lens pupil. As a result, the laser beam always passes through the pupil of the objective lens, regardless of the deflection angle.

[0021] In some embodiments, the scanning unit may also include at least one of a scanning mirror device, such as a digital mirror device, and a spatial light modulator. Each of the scanning mirror device and the spatial light modulator can be configured to provide at least equivalent functionality compared to a scanning unit comprising two prisms.

[0022] In another embodiment, the laser microdissection system includes a sample positioning unit configured to move the sample relative to the optical axis of the objective lens of the optical system. The sample positioning unit may include a movable microscope stage, such as an xy stage. The sample positioning unit enables the positioning of the sample within the field of view of the objective lens. When the laser beam is kept stationary, the sample can be moved using the sample positioning unit to cut the dissection in a manner similar to a table saw.

[0023] In another embodiment, the laser source includes at least one pulsed laser. The pulsed laser generates pulsed laser light, which consists of a series of laser light pulses interrupted by intervals in which no laser light is emitted. The duration of the intervals in which no laser light is emitted may be adjustable. The laser beam formed from such pulsed laser light can be used to cut the sample without damaging the rest of the sample. Furthermore, at least one pulsed laser can be controlled to generate short laser pulses. These short laser pulses can be formed as a laser beam defocused on the sample. These laser beams can be used to separate the cut pieces from the sample using the radiation pressure exerted on the sample by the laser beam.

[0024] In another embodiment, the laser light source includes at least one UV laser light source. The UV laser light source is configured to generate UV laser light, which forms a laser beam. The UV laser light has a short wavelength that enables high-precision cutting and reduces the possibility of thermal diffusion, thereby minimizing damage to areas adjacent to the cut piece.

[0025] The present invention further relates to a method for laser microdissection. The method includes at least the following steps: a) receiving a first image of a sample having a plurality of image tiles stitched together and a first set of coordinates in the coordinate system of the first image, wherein the first set of coordinates defines at least one segment to be removed from the sample; b) generating a second image of at least one portion of the sample using a laser microdissection system; c) generating a second set of coordinates in the coordinate system of the second image, wherein the at least one second set of coordinates corresponds to at least a subset of the first set of coordinates; and d) controlling the laser microdissection system to remove at least one segment from the sample based on the at least one second set of coordinates. The method further includes determining at least two reference points on the sample that are visible in a first image and at least one second image; determining original coordinates corresponding to the coordinates of the reference points in the coordinate system of the first image; determining target coordinates corresponding to the coordinates of the reference points in the coordinate system of at least one second image; and generating at least one second set of coordinates based on the original and target coordinates.

[0026] The method has the same advantages as the controller and laser microdissection system described above. In particular, the method can be supplemented by the features described herein in relation to the controller and / or laser microdissection system. Furthermore, the controller and laser microdissection system described above can each be supplemented by the features described herein in relation to the method.

[0027] In one embodiment, the method further includes generating at least two second images using a laser microdissection system, each second image having a different portion of the sample. The method may further include, for each of the second images, determining at least one of the reference points such that the reference point is visible in the first image and each of the second images. In such embodiments, a plurality of different portions of the sample are imaged. At least one reference point is determined for each of the portions. Thereby, a grid of reference points is generated, and the grid of reference points can cover a large area of the actual specimen. This enables a faithful transformation of the coordinates of the segments in such a large area and significantly reduces the time required to transfer the coordinates of the segments to the laser microdissection system. In particular, in such embodiments of the method, it is possible to faithfully transform the coordinates of all segments at once.

[0028] In another embodiment, steps b)-d) are repeated for different portions of the sample. In such embodiments, a second image is generated for each of the different portions of the sample, and at least two reference points are determined within the second image. Based on the at least two reference points, the segments within the current portion of the sample are transferred and the segments are removed from the sample. Steps b)-d) can be repeated until all segments defined by the first set of coordinates are removed from the sample.

[0029] In another embodiment, the sample includes a tissue microarray section. A tissue microarray includes a plurality of tissue samples embedded in another material, typically paraffin. A tissue microarray section is a thin section cut from a tissue microarray that includes a plurality of tissue sections. Since the tissue microarray section includes a plurality of tissue sections separated by sections that are substantially uncharacteristic of the embedding material, there is a risk of misalignment of the segments transferred to the laser microdissection system. Thus, the proposed method is advantageously used with a sample that includes a tissue microarray section and can prevent misalignment of the segments.

[0030] Hereinafter, embodiments will be described with reference to the drawings.

Brief Description of the Drawings

[0031] [Figure 1] It is a schematic diagram of a laser microdissection system according to an embodiment. [Figure 2] It is a diagram showing a flowchart of a method for laser microdissection according to an embodiment. [Figure 3] It is a schematic diagram of an exemplary first image. [Figure 4] It is a schematic diagram of an exemplary second image.

[0032] Detailed Description of the Drawings FIG. 1 is a schematic diagram of a laser microdissection system 100 according to an embodiment. The laser microdissection system 100 is configured to remove a small portion of a sample 102 using a laser beam. The removed portion is hereinafter referred to as the excised piece 104. The sample 102 can include a specimen, such as a biological specimen like a tissue section, disposed on a carrier, such as a film on a frame. For example, specific cells, cell clusters, or other microscopic features of the sample 102 can be separated from the sample 102 and collected as the excised piece 104. The region of interest corresponding to the portion of the sample 102 to be removed is hereinafter also referred to as a segment.

[0033] In the embodiment shown in FIG. 1, the excised piece 104 is collected within a well 106 of a collection device 108 disposed below the sample 102. The collection device 108 is illustratively shown as a multi-well plate. The well 106 of the collection device 108 can also be formed by a PCR tube that can be disposed within a frame, by one or more Petri dishes, or by a similarly suitable container to facilitate easy handling. The collection device 108 and / or the individual wells 106 can be removable, whereby the excised piece 104 can be further processed.

[0034] The laser microdissection system 100 includes a laser source 110 configured to generate a laser beam. Using the laser beam, the laser microdissection system 100 separates the detached pieces 104 from the sample 102. For example, the laser microdissection system 100 can use a focused beam to cut the detached pieces 104 from the sample 102, or it can use a defocused beam to tear the detached pieces 104 from the sample 102. The laser source 110 may include one or more pulsed lasers for generating pulsed laser light that forms the laser beam.

[0035] The laser microdissection system 100 also includes an optical system 112 configured to guide the laser beam along beam paths O,O'. In the embodiment shown in Figure 1, the optical system 112 includes an optical detection system 114 for acquiring an image of the sample 102. The optical detection system 114 comprises an objective lens 116, a tube lens 118, and a detector 120. Further optical elements such as lenses, filters, and apertures may be part of the optical detection system 114. The objective lens 116 is oriented toward the sample space 122 in which the sample 102 is placed. In this embodiment, the objective lens 116 focuses the laser beam into the sample space 122, for example, toward the sample 102. The objective lens 116 is further configured to receive detection light from the sample 102. The detection light is oriented by the objective lens 116 toward the detector 120 via the tube lens 118. The detector 120 is configured to generate an image of the sample 102 from the detection light.

[0036] In the embodiment shown in Figure 1, the laser microdissection system 100 also includes an illumination system 124 configured to irradiate the sample 102. The illumination system 124 is exemplary positioned below the sample 102. The illumination system 124 may also be positioned above the sample 102 and configured to irradiate it with incident light. The optical system 112 can further be configured to irradiate the sample 102 via an objective lens 116.

[0037] In this embodiment, a beam splitter 126 is positioned between the objective lens 116 and the tube lens 118. The beam splitter 126 is configured to direct the detection light towards the detector 120 via the tube lens 118. The beam splitter 126 may be, for example, a dichroic beam splitter 126. The beam splitter 126 divides the beam path O, starting from the sample 102, into two branches O' and O'', with one branch O' extending toward the laser light source 110 and the other branch O'' extending toward the detector 120. In this embodiment, the laser light is incident on the objective lens 116 via the beam splitter 126. Thus, the beam splitter 126 makes the objective lens 116 usable for both imaging and separating the cut piece 104.

[0038] To move the laser beam within the sample space 122, the laser microdissection system 100 according to this embodiment has a scanning unit 128. The scanning unit 128 is exemplary as part of an optical system 112 between the laser light source 110 and the objective lens 116. The scanning unit 128 exemplary includes two prisms 130 positioned in the beam path between the laser light source 110 and the beam splitter 126. The two prisms 130 are rotatable about the optical axis O' of the beam path and are configured to deflect the laser beam as they rotate. Thus, by rotating the two prisms 130, the laser beam can be moved relative to the sample 102 within the field of view of the objective lens 116. The scanning unit 128 further includes drive units 132 for each of the two prisms 130. The two drive units 132 are configured to rotate the prisms 130 independently of each other.

[0039] In this embodiment, the laser microdissection system 100 also includes a sample positioning unit 134 located within the sample space 122. The sample 102 is placed on the sample positioning unit 134. In the embodiment shown in Figure 1, the sample positioning unit 134 is exemplary configured as a microscope stage with an aperture, so that the dissection pieces 104 can fall into the well 106 of the collection device 108 under the influence of gravity. The sample positioning unit 134 is configured to move the sample 102 relative to the optical axis O of the objective lens 116. In particular, the sample positioning unit 134 is configured to move the sample 102 in a plane perpendicular to the optical axis O of the objective lens 116, i.e., in the x and y directions, and can also be configured to move the sample 102 in the direction of the optical axis O, i.e., in the z direction. The sample positioning unit 134 allows the sample 102 to be automatically and accurately positioned within the field of view of the objective lens 116. This brings a specific region of the sample 102 from which one or more dissection pieces 104 are removed into the field of view.

[0040] The laser microdissection system 100 further includes a controller 136, an input unit 138, and an output unit 140. The controller 136 is configured to receive user input via the input unit 138 and to display visual information to the user via the output unit 140. The input unit 138 is shown exemplary to include a keyboard. However, the input unit 138 may also include a computer mouse, a stylus for use with a touchscreen, or other suitable input device. The output unit 140 is shown exemplary as a monitor. The input unit 138 and the output unit 140 may also be a single element, such as a touchscreen. The controller 136 further includes an external interface 142 and is configured to receive data via the external interface 142. The external interface 142 may also have connectors for storage devices, such as flash drives, and / or connections to computer networks such as a local area network or the internet.

[0041] Furthermore, the controller 136 is configured to perform at least some steps of the method for laser microdissection. To perform the method, the controller 136 can be configured to control at least one of the following elements: namely, the laser light source 110, the optical system 112, the irradiation system 124, the scanning unit 128, and the sample positioning unit 134. The method will be described in more detail below with reference to Figures 2, 3, and 4.

[0042] Figure 2 is a flowchart of a method for laser microdissection according to one embodiment. Hereinafter, the method will be described with reference only to the laser microdissection system 100 shown in Figure 1. The method can be performed, at least in part, by, for example, the controller 136 of the laser microdissection system 100 shown in Figure 1.

[0043] Before the method is initiated, sample 102 can be prepared by placing at least one specimen, such as a tissue section or tissue microarray section, on a carrier, such as a frame or microscope slide. Furthermore, a first image of sample 102 is generated before the method is initiated, and this first image has multiple image tiles stitched together. For example, the first image can be acquired using a slide scanner or fluorescence microscope in multiple imaging steps. In some embodiments, the first image may be a multiplexed fluorescence image generated from multiple different fluorescence channels. An exemplary first image 300 is shown in Figure 3. In the first image, segments are defined before the method is initiated. The segments correspond to small portions of sample 102 that are to be removed from sample 102. For example, the segments may correspond to specific cells, cell clusters or other minute features of the specimen. The segments are defined by a first set of coordinates in the coordinate system of the first image. In some embodiments, the segments may be manually defined by the user. In other embodiments, the segments may also be automatically defined using known methods, such as machine learning methods.

[0044] In step S200, the method is initiated. In step S202, a first image is received along with a first coordinate set. For example, the first image and the first coordinate set can be received by the controller 136 of the laser microdissection system 100 via the external interface 142. In step S204, the laser microdissection system 100 is used to generate at least one second image of a portion of the sample 102. For example, the controller 136 controls the laser microdissection system 100 to acquire at least one second image. The second image is generated using at least one imaging technique, such as transmitted light microscopy, reflected light microscopy, or fluorescence microscopy. An exemplary second image 400 is shown in Figure 4.

[0045] In step S206, at least one second coordinate set is generated in the coordinate system of the second image. The at least one second coordinate set corresponds to at least a subset of the first coordinate set and therefore corresponds to at least a subset of the segments defined before the method is initiated. In some embodiments, the at least one second coordinate set corresponds to segments located in the portion of the sample 102 that is visible in the second image. In other words, in step S206, at least one subset of the segments defined in the first image is transferred to the laser microdissection system 100. The at least one second coordinate set can be generated, for example, by the controller 136.

[0046] To determine at least one second set of coordinates, at least two reference points on the sample 102 are determined, which are visible in the first image and at least one second image. In some embodiments, the reference points may be features of the sample 102 that are visible in both the first and second images. For example, specific biological structures of the specimen can be used as reference points. Cell nuclei, in particular cell nuclei stained using, for example, DAPI, membranes, organelles, especially stained organelles, other cellular structures, or extracellular matrix can all be used as one of the reference points. Furthermore, artificial structures, such as references or structures on the membrane on which the actual specimen is placed, can be used as one of the reference points. The reference points can be manually selected by the user in at least one second image. For example, the user can view at least one second image on the output unit 140 and input the reference points via user input through the input unit 138. The reference points can also be automatically determined by, for example, the controller 136. In some embodiments, at least one of the reference points can be automatically determined as a point on the sample 102 located at the center, corner, or predetermined coordinates of at least one second image.

[0047] Next, the original coordinates corresponding to the coordinates of the reference point in the coordinate system of the first image and the target coordinates corresponding to the coordinates of the reference point in the coordinate system of at least one second image are determined. To determine the original coordinates based on the target coordinates, first, a section of the first image corresponding to at least one second image can be determined. Known image matching methods can be used to determine this section. To facilitate the determination of the section, for example, the controller 136 can generate a virtual first image by simulating what the first image would look like if it were generated using at least one imaging technique employed by the laser microdissection system 100.

[0048] Finally, based on the original coordinates and target coordinates, at least one second set of coordinates is generated. In some embodiments, a coordinate transformation that associates the coordinate system of the first image with the coordinate system of the second image can be determined, for example, by the controller 136. This coordinate transformation can be used to transform the first set of coordinates or a subset thereof into at least one second set of coordinates.

[0049] In step S208, the laser microdissection system 100 is controlled to remove at least one segment from the sample 102 based on at least one second set of coordinates. For example, a controller 136 can perform step S208. In some embodiments, in step S208, a segment located in the portion of the sample 102 that is visible in the second image is removed. The method then terminates in step S210.

[0050] In some embodiments of the method, at least steps S204 and S206 are repeated for different portions of the sample 102. In these embodiments, the sample 102 is imaged section by section. A subset of segments, including segments located in the portion of the sample 102 that is visible in the current second image, is transferred before the next second image is generated. Step S208 is similarly repeatable, meaning that segments located in the portion of the sample 102 that is visible in the current second image are removed from the sample 102 before the next second image is generated. In other embodiments, multiple second images can also be generated before at least one second set of coordinates in the coordinate system of the second images is generated, i.e., before at least a subset of segments is transferred.

[0051] Figure 3 is a schematic diagram of an exemplary first image 300. The first image 300 is stitched together from a plurality of image tiles 302, each image tile 302 corresponding to an image of a different part of a sample 102. The sample 102, exemplaryly, includes a plurality of tissue sections as a specimen. As seen in Figure 3, the first image 300 does not have empty image tiles 302, i.e., image tiles 302 that do not contain at least a portion of the actual specimen. This reduces the computational power required for stitching, as well as the amount of memory required to store the first image 300. The rectangles in Figure 3 indicate sections of the first image 300 that correspond to the second image shown in Figure 4. Figure 3 further shows several exemplary segments 304 defined in the coordinate system of the first image 300. Some of the segments 304 are located within sections of the first image 300 that correspond to the exemplary second image 400 shown in Figure 4.

[0052] Figure 4 is a schematic diagram of an exemplary second image 400. The second image 400 shown in Figure 4 was generated using the optical detection system 114 of the laser microdissection system 100. The view of the second image 400 includes sections of sample 102 corresponding to four of the image tiles 302 that make up the first image 300 shown in Figure 3. Figure 4 is transformed into the coordinate system of the second image 400 and thus further shows a subset of segments 304 available to the laser microdissection system 100 using the method described above. Based on the transferred segments 304, the laser microdissection system 100 can be used to remove the detached pieces 104 from the sample 102.

[0053] Elements that function identically or similarly are indicated by the same reference numeral in all figures. As used herein, the term "and / or" includes all possible combinations of one or more of the related items and may be abbreviated as " / ".

[0054] While several embodiments have been described in the context of the apparatus, it is clear that these embodiments also represent descriptions of the corresponding methods, where blocks or apparatus correspond to steps or features of steps. Similarly, embodiments described in the context of steps also represent descriptions of the corresponding blocks, items, or features of the corresponding apparatus. [Explanation of Symbols]

[0055] 100 Laser Microdissection Systems 102 samples 104 Separated piece 106 wells 108 Collection device 110 Laser light source 112 Optical Systems 114 Optical detection systems 116 Objective lens 118 Tube Lenses 120 detectors 122 Sample space 124 Irradiation System 126 Beam Splitter 128 scanning units 130 Prisms 132 Drive Unit 134 Sample positioning unit 136 Controllers 138 Input Units 140 output units 142 External Interfaces 300 images 302 Image Tiles 304 segments 400 images

Claims

1. A controller (136) for a laser microdissection system (100), The controller (136) Receiving a first image (300) of a sample (102) having a plurality of mutually stitched image tiles (302) and a first coordinate set in the coordinate system of the first image (300), wherein the first coordinate set defines at least one segment (304) to be removed from the sample (102), Controlling the laser microdissection system (100) to generate at least one second image (400) of a portion of the sample (102), To generate at least one second set of coordinates in the coordinate system of the second image (400), wherein each of the second sets of coordinates corresponds to at least a subset of the first set of coordinates, Controlling the laser microdissection system (100) to remove the at least one segment (304) from the sample (102) based on the at least one second coordinate set and It is configured to do the following: The controller (136) is Determining at least two reference points on the sample (102) that are visible in the first image (300) and the at least one second image (400), Determining the original coordinates corresponding to the coordinates of the reference point in the coordinate system of the first image (300), Determining target coordinates corresponding to the coordinates of the reference point in the coordinate system of at least one second image (400), To generate the at least one second set of coordinates based on the original coordinates and the target coordinates. A controller (136) is configured to perform the following actions.

2. The controller (136) is Controlling the laser microdissection system (100) to generate at least two second images (400), wherein each second image (400) includes a different portion of the sample (102), For each second image (400), determine at least one of the reference points such that the reference point is visible in the first image (300) and each of the second images (400). A controller (136) according to claim 1, configured to perform the following:

3. The controller (136) is Controlling the laser microdissection system (100) to generate at least two second images (400), wherein each second image (400) includes a different portion of the sample (102), For each of the second images (400), at least two of the reference points are determined such that the reference points are visible in the first image (300) and each of the second images (400). To generate one of the second coordinate sets for each of the second images (400) and A controller (136) according to claim 1 or 2, configured to perform the following:

4. The controller (136) according to claim 3, configured to control the laser microdissection system (100) to remove at least one of the segments (304) from the sample (102) based on one of the second coordinate sets before another image of the second image (400) is generated.

5. The controller (136) according to any one of claims 1 to 4, wherein the controller (136) is configured to determine the original coordinates based on the target coordinates, the first image (300), and the second image (400) using an image matching method.

6. The controller (136) is Determining a section of the first image (300) that corresponds to at least one of the second images (400), The original coordinates are determined based on the target coordinates and the section of the first image (300). A controller (136) according to any one of claims 1 to 5, configured to perform the following:

7. The laser microdissection system (100) is configured to generate the at least one second image (400) using at least one imaging technique, The controller (136) is Based on the first image (300), an artificial first image (300) is generated that simulates the visual characteristics of at least one imaging technique. Determining the original coordinates based on the target coordinates and the artificial first image (300) A controller (136) according to any one of claims 1 to 6, configured to perform the following:

8. The controller (136) is Determining the coordinate transformation from the coordinate system of the first image (300) to the coordinate system of the at least one second image (400), To generate the at least one second set of coordinates based on the said coordinate transformation. A controller (136) according to any one of claims 1 to 7, configured to perform the following:

9. A laser microdissection system (100), A laser light source (110) configured to generate a laser beam, An optical system (112) configured to guide the laser beam onto the sample (102), A controller (136) according to any one of claims 1 to 8 and It is equipped with Laser microdissection system (100).

10. The laser microdissection system (100) according to claim 9, wherein the laser microdissection system (100) comprises a scanning unit (128) configured to move a laser beam within the field of view of the objective lens (116) of the optical system (112).

11. The laser microdissection system (100) according to claim 9 or 10, wherein the laser microdissection system (100) includes a sample positioning unit (134) configured to move the sample (102) relative to the optical axis (O) of the objective lens (116) of the optical system (112).

12. A method for laser microdissection, a) Receiving a first image (300) of a sample (102) having a plurality of image tiles (302) that are stitched together, and a first set of coordinates in the coordinate system of the first image (300), wherein the first set of coordinates defines at least one segment (304) to be removed from the sample (102), b) Using a laser microdissection system (100), generate at least one second image (400) of a portion of the sample (102), c) To generate at least one second set of coordinates in the coordinate system of the second image (400), wherein the at least one second set of coordinates corresponds to at least a subset of the first set of coordinates, d) Controlling the laser microdissection system (100) to remove the at least one segment (304) from the sample (102) based on the at least one second set of coordinates, Includes, The above method further, Determining at least two reference points on the sample (102) that are visible in the first image (300) and the at least one second image (400), Determining the original coordinates corresponding to the coordinates of the reference point in the coordinate system of the first image (300), Determining target coordinates corresponding to the coordinates of the reference point in the coordinate system of at least one second image (400), To generate the at least one second set of coordinates based on the original coordinates and the target coordinates. Methods that include...

13. The above method further, The method involves generating at least two second images (400) using a laser microdissection system (100), wherein each second image (400) includes a different portion of the sample (102), For each of the second images (400), determine at least one of the reference points such that the reference point is visible in the first image (300) and each of the second images (400). The method according to claim 12, including the method described in claim 12.

14. The method according to claim 12, wherein steps b) to d) are repeated for different parts of the sample (102).

15. The method according to any one of claims 12 to 14, wherein the sample (102) includes a tissue microarray section.

Images