Method for checking a dissection process in a laser microdissection system and means for carrying it out

Abstract

Method for verifying a dissection process in a laser microdissection system (100), comprising: Performing a dissection process to cut out (510) a dissection (210, 410) from an object (200, 400) in a first area of ​​the object (200, 400) using a laser beam; Acquisition (310, 520) of first image data of at least the first area after the dissection process with a first focal plane (222) which is parallel offset along the optical axis (240) to the plane (220) of the object (200, 400), acquisition (310, 520) of second image data after the dissection process with a second offset focal plane (224) which is parallel offset along the optical axis (240) and on the opposite side of the object (200, 400) with respect to the first focal plane (222), and Check (320, 530, 540) whether the first image data and / or the second image data show sharp structures within the area to be separated by the dissection process in order to determine whether the dissection process was successful.

Description

[0001] The present invention relates to a method for checking a dissection process in a laser microdissection system and means for carrying it out in the form of a control unit, a computer program and a laser microdissection system. background

[0002] Laser microdissection is a powerful and non-destructive method for obtaining individual cells or cell regions from specific areas of microscopic samples, which can then be subjected to a variety of analytical techniques. For an overview, see relevant textbooks, such as the chapter "Laser microdissection" by Diane L. Sterchi in: Theory and practice of histological techniques. 6th ed. Philadelphia, PA: Churchill Livingstone / Elsevier, 2008. pp. 575-583. - ISBN 978-0-443-10279-0.

[0003] DE 10 2013 209 880 A1 describes a method for laser microdissection in which at least one dissectible material is separated from at least one object to be dissected by means of a laser beam focused onto the object and collected in a dissectible material collection container, wherein at least one collection surface of the dissectible material collection container is imaged, at least section by section, generating image data. The collection surface of the dissectible material collection container is imaged at at least two times, and the image data generated are compared.

[0004] EP 1 985 987 B1 describes a method for laser microdissection in which, to optimize the setting of a laser parameter of a laser light source, a test section line is generated with the laser beam and an image of the test section line is recorded. The image of the test section line is compared with a reference section line in order to either change the value of the laser parameter or to perform a laser microdissection operation with the set value of the laser parameter, depending on the comparison result.

[0005] Laser microdissection largely preserves cell and tissue morphology, as well as the macromolecules within the cell. This allows for the analysis of the genetic information and proteins, lipids, metabolites, etc., of the extracted cells using appropriate methods. Various microscopic samples can be processed using laser microdissection, such as frozen samples, tissue sections embedded in paraffin or plastic, blood and other bodily fluid smears, and cell cultures. In addition to such biological samples, laser microdissection can also be used to process artificial materials or plastics, glass, and thin sections of metals, minerals, or rock samples. Furthermore, direct dissection of plant parts such as leaves, grasses, and roots is possible.

[0006] Tissue sections and other samples can be stained or otherwise labeled for use in laser microdissection using established staining techniques, such as hematoxylin / eosin, or they can be used unstained. In particular, immunohistochemical staining techniques can be used in conjunction with fluorochromes or chromogens, or fluorescence in situ hybridization (FISH) can be performed. The staining or labeling techniques used also depend on the type of subsequent investigations to be carried out.

[0007] The terms "sample," "object," "object of investigation," and the like are used synonymously below and can refer to any type of stained or labeled, or unstained or unlabeled, sample described above. In particular, objects of investigation for use in laser microdissection are, as is generally known, fixed or immobilized on a slide and, if necessary, covered with a membrane.

[0008] The general term "laser microdissection" is used for a variety of different process variants, which are usually referred to in the professional community using English technical terms and sometimes inconsistently. These process variants are extensively described in the literature and are only briefly summarized below. The present invention can, in principle, be used in conjunction with all of these process variants.

[0009] In all cases of laser microdissection, the positioning of the laser beam used for processing is achieved by a relative movement between the laser beam and the specimen. In certain embodiments, this relative movement can be achieved by a horizontal movement of the specimen stage. In a particularly advantageous embodiment, first described, for example, in DE 100 18 253 C2, the microscope stage is fixed during cutting or for (fine) positioning of the laser beam. In this embodiment, a laser scanning device is arranged in a reflected-light unit of the microscope used, through which the laser beam is directed onto the specimen via the microscope objective. This scanning device consists of two thick, transparent wedge plates inclined to the optical axis and rotatable independently of each other about the optical axis.

[0010] The wedge plates are preferably controlled by stepper motors, which cause a relative rotation of the wedge plates to one another. The laser beam is guided through the wedge plates, causing it to be deflected by an angle relative to the optical axis. By rotating the wedge plates around the optical axis, the deflection angle can be set and varied, with the arrangement being such that the laser beam always passes through the center of the posterior objective pupil. This is achieved in particular by a suitable selection of the thickness and inclination of the wedge plates. A corresponding laser microdissection system is also described with reference to the attached [reference to be added]. Fig. 1 explained further.

[0011] In the embodiment just described, the laser beam can be directed at any point in the plane of the object under investigation. For this purpose, the maximum deflection angle is dimensioned such that the laser beam is deflected only to the edge of the field of view. This applies simultaneously to all lenses, regardless of their magnification. By using a laser scanning device in the particularly advantageous embodiment just described, a complex motorized sample stage is no longer necessary. Since the sample stage remains stationary during the cutting process, the user can observe and control the cutting process within the specimen.

[0012] The previously mentioned different procedural variants differ in particular in the way in which areas to be examined are separated from the structure of the object under investigation and transferred to suitable collection containers or onto suitable carriers.

[0013] In one variant of the process, the laser beam, particularly in the form of a cutting line, is guided around the area to be examined and thereby separated. In this case, the object under investigation is located, in particular, on the underside of a coated or membrane-covered slide. The laser beam cuts through the object or a membrane connected to it, and the separated area can fall by gravity into a collection container located below the object. Alternatively, for example, a cutting line can be closed off using a finely focused laser beam, leaving one or more remaining ridges. Subsequent refocusing allows the laser beam to be widened and directed onto the remaining ridge(s).This prevents, for example, twisting and stress-induced deflection when falling out.

[0014] In contrast, laser pressure catapulting (LPC) involves catapulting the excised sample, specifically by precisely directing a suitable laser pulse at a previously described residual ridge of a section line or a cell to be isolated. Various physical phenomena are responsible for this catapult effect. The catapulted cells can then be collected, for example, in a sample container or adhered to a membrane coated with an adhesive layer.

[0015] In laser capture microdissection (LCM), cells are attached to a membrane while still on the substrate by the laser beam. By lifting or peeling back the membrane, the target areas can be detached from the substrate, remaining attached to the membrane. The detached target areas, along with the membrane, can then be transferred to a collection container. This procedure can be combined with prior dissection to prevent cell detachment.

[0016] In another variation, for example, living cells in a sterile culture dish or on a suitable slide can be covered with a light-absorbing membrane. Using a laser beam, cells of interest can be cut around the membrane beneath the surface. When the membrane is removed, the cells remain in the culture dish or on the slide, and unwanted cells can be removed along with the membrane. This method is also known as cell ablation.

[0017] Laser microdissection typically uses laser light in the ultraviolet wavelength range, with the laser beam usually being pulsed. Focusing the laser beam achieves high energy density while simultaneously narrowing the local focus of the incident laser light. High pulse frequencies can be used, for example, for rapid cutting or tack welding, as well as for creating fine cut lines.

[0018] While the sectioning and collection of the dissected specimens or samples can be fully automated in this way if desired, it is still possible that a section may not be performed optimally for various reasons. Therefore, there is a need to improve the reliability of automated or semi-automated laser microdissection. Summary

[0019] To solve this problem, a method for verifying a dissection process in a laser microdissection system and means for carrying it out, in the form of a control unit, a computer program, and a laser microdissection system with the features of the independent claims, are proposed. Advantageous embodiments are the subject of the dependent claims and the following description.

[0020] A method for verifying a dissection process in a laser microdissection system is proposed. This method involves first performing a dissection in a first region of an object using a laser beam to cut out a section. Then, initial image data, at least of the first region, are acquired after the dissection. For acquiring these initial images, a first focal plane is selected that is parallel to and offset along the optical axis from the plane of the object. Furthermore, second images are acquired after the dissection using a second, offset focal plane, which is parallel to and offset along the optical axis and located on the opposite side of the object from the first focal plane. Depending on the laser microdissection system used, the dissection process can consist of either just the actual cutting operation using appropriately designed laser beams or...The dissection process includes laser pulses and can optionally include further steps, such as the active or passive transport of the sectioned dissectant from the object. Thus, the dissection process can also include steps like applying one or more laser pulses to transport the sectioned dissectant into a collection container. Similarly, lifting a membrane containing the sectioned dissectant to remove it from the object can be part of the dissection process. The term "dissection process" is intended to describe the corresponding steps independently of their success.

[0021] The first and second image data sets are then evaluated to determine whether the dissection process was successful, i.e., whether the dissected material was completely cut out and removed from the object. In particular, it can be checked whether the image data exhibit sharp contours within the first area to determine the success of the dissection process. The invention ensures that dissected material protruding from the plane of the object is detected on both sides of the sample. This test can be performed fully automatically by suitable image processing, i.e., based on appropriate software modules capable of performing such an evaluation of acquired image data.

[0022] If sharp structures are present in a displaced focal plane, it can be concluded that at least part of the dissected portion extends into this focal plane, and that the dissected portion is therefore still attached to the object. If sharp structures are found in the original object plane, it can be concluded that the dissection process was unsuccessful and that at least part of the dissected portion is still located in the plane of the object in its original position.

[0023] As a result, measures can be taken automatically, if necessary, to successfully complete the dissection process, such as recutting and / or further processes that also result in the removal of an already cut dissection, e.g., the application of one or more laser pulses to transport the cut dissection.

[0024] By defocusing to a focal plane offset along the optical axis, parts of a dissected specimen that protrude from the plane of the object can be detected. This allows, for example, the detection of protruding dissected specimens resulting from an incomplete laser cut.

[0025] In one variant, it is possible to define a multitude of offset focal planes, positioned parallel to and offset from the object's plane along the optical axis, and to acquire image data at each of these multiple offset focal planes. This acquired image data is then analyzed to determine whether the dissection and / or separation of the dissected tissue was successful. Using multiple offset focal planes at varying distances from the object, and with appropriate plane selection, ensures that any potentially folded or protruding dissected tissue will be visible at one of the focal planes.

[0026] For example, the offset focal planes can be controlled sequentially by gradually changing the focus of an optical system that generates the image data. This effectively sweeps through a broad focus range step by step or continuously, evaluating the image data in the process.

[0027] Preferably, an end distance from the plane of the object can be defined, with the plurality of further offset focal planes lying between the plane of the object and the end distance. This end distance thus defines the focal area in at least one direction; end distances can also be defined on both sides of the object to extend the focal area to both sides. The end distance can be freely chosen, with one possible embodiment having a maximum end distance of half the largest dimension of the dissected part in the plane of the object. With this choice, it is expected that every folded-back dissected part that protrudes into the area of ​​the cut hole can be detected.

[0028] In addition to the aforementioned possibilities, image data acquired after the dissection process can be compared with reference image data to determine whether the dissection was successful. This allows identification of whether the area to be cut out appears "empty" in the image, or whether any change is discernible compared to the image data before the dissection.

[0029] Depending on the specific implementation, reference image data can be generated in various ways. For example, reference image data can be acquired by capturing initial image data of the first area before the dissection process, i.e., an image of the area that has not yet been cut. This reference image data can optionally be acquired with the first and / or second offset focal plane, i.e., with a focus outside the object plane, but also with a focus placed directly within the object plane. Furthermore, reference image data can be generated by capturing image data of a background area without an object. Another possibility is to generate reference image data by defining threshold values ​​for parameters of the image data, where the parameters include at least one of the following for at least a subset of the image data: brightness, contrast, frequency distribution, grayscale distribution, and hue.Furthermore, reference image data can be acquired using various contrast methods (e.g., brightfield, phase, DIC, IMC, fluorescence). This yields theoretical reference image data that can reproduce typical properties of an empty background or a captured cross-section. In addition, reference image data can be generated by acquiring image data of the object outside the initial area. All these methods can also be combined as desired.

[0030] Furthermore, in one embodiment, reference image data can be acquired before the dissection process in a second area of ​​the object, which encompasses the first area. Then, third image data of the second area can be acquired after the dissection process. Finally, the acquired third image data can be compared with reference image data of the second area to determine whether the cut-out dissectible portion is located on the object. Thus, overview images of a larger image section can be used to locate, for example, dissectible portions that were correctly cut out but then did not end up in the intended collection container.

[0031] In all variants, it is possible to repeat the dissection process, at least partially, if it was previously determined that the dissection process was unsuccessful. This can include, for example, a partial re-cut only in individual areas, a complete re-cut along the entire cut line, one or more individually applied laser pulses for separating and / or transporting the dissected tissue, or other partial steps of the dissection process.

[0032] Furthermore, a control unit for a laser microdissection system is proposed, configured to execute one, several, or all of the process steps as described above. Likewise, a computer program with program code for carrying out a process is proposed, which can, for example, instruct a processor or a suitable control unit to perform such process steps. Implementation as program code allows for easy extension of existing and new systems.

[0033] Finally, a laser microdissection system is proposed, comprising a laser configured to perform a dissection process, at least one camera configured to capture image data of the object, and a control unit capable of executing the described process steps.

[0034] Further advantages and features of different embodiments will become apparent from the description and the accompanying drawing.

[0035] It is understood that the aforementioned features and those to be explained below can be used not only in the combination specified, but also in other combinations or on their own.

[0036] The term “and / or” encompasses all combinations of one or more of the associated listed elements and can be abbreviated with “ / ”.

[0037] The following section describes exemplary embodiments in more detail with reference to the drawing. Character description Fig. Figure 1 shows an exemplary laser microdissection system which can be used to carry out embodiments; Fig. 2 shows a schematic side view of an incompletely sectioned dissection specimen with different focal planes for image acquisition; Fig. Figure 3 shows a schematic sequence of a procedure according to an exemplary embodiment; Fig. Figures 4a to 4c show exemplary image data captured before and after a section of the object; and Fig. Figure 5 shows a schematic procedure according to an extended embodiment. Exemplary embodiments

[0038] In Fig. Figure 1 is a laser microdissection system according to an embodiment of the present invention, or a laser microdissection system that can be used to carry out a method according to an embodiment of the present invention, shown schematically and designated collectively by 100. The laser microdissection system 100 corresponds in essential parts to that disclosed in EP 1 276 586 B1, to which express reference is made hereto.

[0039] A Cartesian coordinate system, which illustrates the axes or directions x, y and z mentioned below, is shown in Fig. Figure 1 is shown in the lower right. In this coordinate system, the x-axis is perpendicular to the plane of the paper, and the y-axis and z-axis lie in the plane of the paper. The x-axis, y-axis, and z-axis are each perpendicular or orthogonal to each other. In this example, the z-axis corresponds to the optical axis in the area of ​​the sample.

[0040] The laser microdissection system 100 comprises a microscope 102. In the illustrated example, an illumination unit 106, only partially depicted here, is provided in a microscope base 104 of the microscope 102. The illumination unit 106 can, for example, include a light source (not shown) for providing illumination light, as well as means for influencing the illumination light, such as filters and / or apertures. For transmitted light illumination and for setting suitable contrast or observation methods, a condenser unit 108, greatly simplified in the illustrated example, is provided.

[0041] For example, a user interface 110, which may be a touchscreen, can be arranged on the microscope base 104, allowing a user to enter and / or read viewing and / or processing parameters. The microscope 102 has a control knob 112. This knob is used to operate a coarse and a fine focus knob for adjusting the height of a microscope stage 114.

[0042] An object 116, located on a slide 118 which in turn is held in a holder 120, for example a section prepared on the slide, can thereby be brought into the object plane of a lens 122. The lens 122 is mounted in a lens turret 124 along with other, unspecified lenses. A protective cover 126, in particular a transparent one, may be provided for protection against laser radiation.

[0043] The observation light emitted from the object 116 travels along an observation beam path 128. In a tube unit 130 with suitable coupling devices 132, a preferably variable portion of the observation light, for example by 60°, can be coupled out and presented to a user by means of an eyepiece pair 134. Another portion of the observation light can be coupled into a digital image acquisition unit 136, such as a camera, and captured. An image processing unit 138 can be assigned to the image acquisition unit 136, either locally in a control unit 136 or a control computer (see below), or in another spatial arrangement.

[0044] The laser microdissection system 100 comprises a laser unit 140 with a laser light source 142. A laser beam 144 provided by the laser light source 142, which can be, for example, a UV laser light source, is deflected in a reflected light unit, which is designated here as 146, by a first deflecting mirror 148 and a second deflecting mirror 150 and focused by the objective 122 onto the object 116.

[0045] In the laser microdissection system 100 illustrated here, the point at which the laser beam 144 strikes the object 116 can be adjusted in several different ways. On the one hand, a manual adjustment device 152 can be provided, by means of which the microscope stage 114, designed as a cross stage, can be adjusted in the x and y directions (i.e., perpendicular and parallel to the paper plane, respectively). In addition to the adjustment device 152, electromechanical positioning devices can also be provided, which can be controlled, for example, by the control unit 138, or whose position can be detected by the control unit 138.

[0046] The control unit 138 can also control any other motorized functions of the laser microdissection system 100 and, in particular, provide an interface to an external control computer 160, which can be connected via appropriate wireless or wired connections.

[0047] For laser microdissection, a laser deflection device 154 can be provided. By means of the laser deflection device 154, the laser beam 144 can be deflected relative to an optical axis running between the first deflection mirror 148 and the second deflection mirror 150. The laser beam can therefore strike the second deflection mirror 150 at different positions, which can, for example, be configured as a dichroic splitter. The arrangement is such that the laser beam 144 passes through the posterior objective pupil. By means of appropriate deflection, the laser beam 144 is focused on the object 116 at different positions. A deflection using a laser deflection device 154 of the type shown here is described in detail, for example, in EP 1 276 586 B1. It should be emphasized that different possibilities for deflecting a laser beam b or b are presented here.The invention can be used to position the object 116 in relation to the laser beam 144. The invention is not limited to the example shown.

[0048] In the illustrated example, the laser deflection device 154 has two solid glass wedge plates 156, which are inclined relative to the optical axis and rotatable independently of each other about it. For this purpose, the wedge plates 156 are mounted on ball bearings (not specifically designated). Each wedge plate is connected to a gear (also not specifically designated). The gear can be rotated by means of an actuator, which can be supplied with a corresponding control signal. The actuators can be equipped with position sensors, and a position detected by these sensors can be transmitted to the control unit 138.

[0049] Focusing of the microscopic image acquired via the observation beam path 128 and the laser beam 144 is achieved, on the one hand, by raising or lowering the microscope stage 114, thereby aligning the focus positions of the microscope objective 122 and the laser beam 144. Image processing of the microscopic image acquired via the observation beam path 128 can then be performed, for example, in the control unit and / or the control computer 152. Motorized control of the microscope stage 114 thus implements an autofocus system, which is illustrated separately in Figure 190.

[0050] Instead of the system shown here, differently designed laser microdissection systems can also be used, in which, for example, different optical paths are employed for observation and image acquisition as well as for the laser beam used for cutting. Similarly, in another variant, the upright microscope shown here could be replaced by an inverted microscope, in which the objectives and the associated optical elements are located below the specimen.

[0051] There is a possibility that a dissected fragment will not be completely separated from the object along the cut lines and will still be attached to the object at least at one edge. Likewise, a completely cut-out dissected fragment, especially in a non-contact system, might not end up in the designated collection container where the dissected fragments are gathered for further processing, but instead, for example, due to external influences, land on the surface of the sample or the sample carrier.

[0052] To ensure that the desired sections were performed correctly and that the dissected tissue was collected in the corresponding collection containers, one embodiment allows for monitoring of the dissection area by capturing image data.

[0053] In one possible embodiment, the area relevant for observation or image acquisition can be defined—that is, the area in which the section will take place. For this purpose, a predetermined coordinate range could be specified, or, for example, after the section lines have been defined by a user or by the control of the dissection system, the corresponding area within the section lines could be defined as the relevant observation area. Likewise, a first observation area can also be defined that is larger than the desired dissection specimen, for example, by defining a circular or rectangular observation area that reliably encloses the dissection area, or optionally multiple dissection areas, in all directions of extent.Furthermore, an observation area could also be defined that takes into account a specified additional border area beyond the dissection area, i.e., such that a widened border is created along the contours of the dissectate with an arbitrarily chosen border width, which forms the observation area.

[0054] The definition of the image area can also depend on the preset image section that a camera used for image acquisition and the optical system employed can capture. If a dissection area to be inspected cannot be completely captured in a single image section with the required or desired resolution due to its size or shape, the described image inspections can be performed across several image sections offset in the xy-plane, which are then evaluated individually or together.

[0055] Within this defined observation area, image data of this observation area can then be captured or recorded, at least after a laser dissection process has been carried out in which a defined area of ​​the object was cut out and removed using a laser beam.

[0056] According to one embodiment, such image data can now be used to verify whether a cut was completed and the dissected material was actually separated. It is conceivable that while a dissected material was largely cut out, the cut was not completed along one or more sections of the cut-out perimeter, leaving a border of the dissected material still attached to the object. This could be due to sample-specific properties, unsuitable cutting parameters, or errors in the cutting process.

[0057] Fig. Figure 2 shows a side view of a dissected sample 200, in which a dissected portion 210 was not completely cut out and removed, but remains connected to the rest of the sample 200 at an edge region 214 and folds downwards due to gravity. Depending on the angle at which the partially cut dissected portion folds down, the cut hole 212 may appear empty or partially filled. The dimensions and distances shown are only a schematic representation to illustrate the process steps and do not correspond to the actual conditions.

[0058] To detect dissected specimens 210 with such incompletely truncated edge regions 214, image data are therefore acquired and evaluated in focal planes other than the object plane. If a dissected specimen 210 protrudes upwards or downwards in a direction and thus extends beyond the object plane 220, an image acquired with a focal plane shifted at a distance from the object plane 200 along the optical axis 240 will show sharp structures in at least one of these areas. Conversely, if a dissected specimen is completely cut out, an image acquired with a focal plane 222, 224 outside the object plane 220 will not show sharp structures. The dashed lines in Fig. Figure 2 shows exemplary focal planes in which image data could be acquired. Accordingly, an image in a focal plane that intersects the hanging partial dissectible section 210 will show a sharp structure at this intersection line with the focal plane. In the example shown, each of the focal planes 222a, 222b, 222c, 222d would therefore show a sharp structure through the hanging dissectible section 210. In contrast, in this example, all focal planes 224, 234, which lie on the opposite side of the dissectible section relative to the optical axis, would not show sharp structures. Likewise, focal planes that are further away from the object plane 220 than the extent of the folded-down dissectible section, for example, focal planes 222e and 232 in Figure 2, will not show sharp structures. Fig. 2. Do not show any sharp structures.

[0059] Methods for examining image areas for sharpness through suitable automatic image processing are generally known and can be applied to the examination of image data. For example, the captured image data can also be further processed or converted for this purpose, such as to enable an evaluation of the data in the frequency domain.

[0060] Preferably, the system knows the location of the expected cut edges, i.e., the coordinates along which the cut was made in object 210. The information obtained, such as the location of the blurred areas in the xy-plane and / or their distance in the z-direction from the sample, can then be used together with the known cut coordinates to identify where the dissected section is attached and was not cut correctly. This can be achieved either by narrowing down the specific edge segment or by using predefined sections of the dissected section or the cut line, such as a division into four quadrants, so that it can be determined in which of the predefined sections the cut was incomplete. This information can then be used both for further image examinations and for a subsequent recutting of the desired dissected section.The data can also be stored and processed for other purposes. The search for sharp structures can be limited to the area of ​​the section line or to a defined boundary area around the section line, where the boundary area can be defined both inwards from the section line into the area of ​​the cut hole and outwards. This saves computing power during image processing, enabling rapid automatic dissection.

[0061] Since an attached dissectible section can protrude from a sample both upwards and downwards, at least two images are acquired, each with at least one first and one second focal plane. At least one focal plane is selected on each side of the sample along the optical axis, and one of these images is captured with this plane. Preferably, only the focal plane is changed, while the other parameters, such as image area and brightness settings, remain constant. Alternatively, a preferred side of the sample could be selected for the first focal plane. For example, if a system is used in which the excised areas fall downwards into a collection container by gravity without contact, then in most cases even a partially excised dissectible section is expected to fold downwards.This allows the first focal plane to be selected as a plane that is offset downwards by a predefined distance from the object plane along the optical axis. The image captured in this first focal plane can then be evaluated first. Only if the result does not show sharp structures at this point can a second or further images be taken, in which the focal plane is located on the opposite side of the sample, i.e., offset upwards by a predefined distance along the optical axis from the object plane. Alternatively, both images in the first and second focal planes can be acquired first and then evaluated.It is also conceivable to take both images, but to stop the evaluation as soon as a sharp structure is found indicating that at least part of the dissection protrudes from the sample plane and has therefore not been cut off along the entire section line.

[0062] The distance of the first and / or second focal plane from the object plane 220 (along the z-axis) can be selected in various ways. For example, half the minimum diameter of the expected dissected specimen can be set as the distance from the object plane to one or both offset focal planes, or a distance that is at most equal to the minimum diameter of the dissected specimen. With these values, it can be assumed that a dissected specimen attached perpendicularly or almost perpendicularly at an edge and otherwise almost completely cut out will be reliably detected by the focus check outside the object plane. Similarly, a focal distance 222a, 224a could be selected that is only slightly offset from the object plane, so that any folded-off object is expected to be detected.

[0063] The dimensions of the dissection can be obtained, for example, from the manually or automatically created section line coordinates in the system, or from an image taken before the section, which serves to identify or select relevant areas in the sample that are to be cut out later.

[0064] In further embodiments, the process steps of which are exemplified in Fig. As shown in Figure 3, instead of a fixed distance for the first or second focal plane, where only a few individual images are evaluated, a focus area starting at the object plane 220 or a starting area spaced apart from it, extending to a predetermined final distance from the object plane, can be traversed on one or both sides, whereby images can be continuously captured and evaluated or at specific intervals. The focus area can therefore be... Fig. 2, for example, corresponds to the area that lies between the uppermost focal plane 234 and the lowermost focal plane 232, each at the same or different distance from the object plane 210.

[0065] In step 300, the optical system is used to defocus the image, selecting a first focal plane that is offset from the object plane. In step 310, image data is acquired in this offset focal plane and checked for sharp structures in step 320. If sharp structures are found, it can be assumed that part of the sample protrudes from the object plane and was therefore not completely removed and collected. This information, along with relevant coordinates and data about the dissected specimen, can then be saved or further processed in step 350. For example, this data can be used to repeat the section or part of it in process step 360.If no sharp structures are found, step 330 can first be used to check whether the desired endpoint of defocusing has already been reached, i.e., whether all desired focus planes have been traversed and—depending on the embodiment—the final distance or the object plane has been reached again. If all intended focus planes have been traversed and the respective acquired image data show no sharp structures anywhere, the sharpness check for this dissectible specimen can end in step 340. If not all focus planes have yet been traversed, defocusing can be performed again in step 300 by one further distance step, i.e., another offset focus plane is selected in which image data is again acquired and checked for sharp structures according to step 310.

[0066] In another embodiment, a focal plane can also be selected before the first offset focal plane in step 300. This focal plane lies directly in the object plane, and image data can be acquired with this focus (step 310). In step 320, this data is checked for sharp structures within the area where the dissection was to be separated. Since the focus setting is usually still in the object plane after the dissection process, an image can be acquired without further adjustment of the optical elements. If the dissection was not correctly cut or separated and is still partially or completely in the object plane, sharp structures will be detected. Thus, statements about the success of the dissection process can be made in this first step, and appropriate post-processing can optionally be initiated.Depending on the design, the sharpness test can then take place in further planes in any case, for example to find partially folded dissections, or it can be aborted if sharp structures are found.

[0067] The final distance 232, 234, up to which the focus area is traversed, can correspond, for example, to the first or second focal plane described above. Likewise, the selected final distance for continuously traversing and evaluating a focus area can also be significantly smaller or larger than the previously defined distance of a focal plane for a single image. For example, a focus area can be selected that extends to half the maximum diameter of a dissected specimen on both sides of the sample. Since a stuck sample typically still has marginal contact with a cut edge in the focus area, a selected final distance of half the maximum diameter should reliably ensure a sharp structure within this traversed focus area when the sample is tilted.Therefore, checking focal planes located further away from the object is generally unnecessary when scanning through a focus area. Nevertheless, the focus areas can of course be chosen arbitrarily; for example, the same focus area can be selected for all dissections to simplify the process. Fig. Figure 2 shows an example of a final distance 232, 234 that extends beyond the extent of the dissected section in the shown section plane; however, this representation is only schematic and should not be understood as a limitation. For example, the extent of the dissected section in a direction perpendicular or oblique to the image plane could extend significantly beyond the length of the folded-over section shown. Likewise, the final distance can also be chosen freely.

[0068] In this way, it can also be ensured that even an attached dissectible specimen, which is almost completely folded over and thus protrudes from the object plane only with a curved edge area and will therefore only show sharp structures in a focal plane relatively close to the object plane, is also detected by the sharpness test.

[0069] This type of design is particularly suitable when the image processing system has sufficient computing power. The number of images captured, or the rate at which image data is acquired while traversing the focus area, can be fixed or variable. For example, defocusing can be performed at a constant speed, during which image data is acquired at defined time intervals. The data can be acquired immediately after acquisition, and thus in parallel with further defocusing, so that the result of the image analysis from a previous focus plane does not necessarily have to be awaited. In this way, a large number of images in different focus planes are obtained. The acquisition rate can also be selected depending on the size of the dissectible specimen and thus on the chosen final distance of the focus area.

[0070] It goes without saying that the sequence for traversing the focus area can also be chosen arbitrarily. As already described, defocusing and image data acquisition in the offset focus planes can begin at the object plane and continue up to a selected final distance from the object plane. The corresponding process can then be repeated on the other side of the sample. However, it is also possible to begin the evaluation and image acquisition in the focus plane chosen as the maximum final distance, for example, with focus plane 234 above object plane 210, and then traverse the focus area step by step or continuously from there to the selected final distance on the opposite side of sample 200, i.e., to focus plane 232 below the sample. Image data can be acquired in all desired planes.Again, the side of the sample where the greater number of folded-off dissections is expected can preferably be chosen as the starting point.

[0071] In this embodiment, the evaluation or image acquisition can be stopped as soon as a sharp area outside the object plane is found in step 320. Alternatively, further images can be acquired or evaluated thereafter, from which, for example, three-dimensional information about the attached dissectible fragment can be obtained, such as the angle at which an attached fragment protrudes from the plane. The additional images and sharpness checks in this image data can also be used to verify the initial results, i.e., to ensure that a sharp area is indeed present.

[0072] It goes without saying that the number of captured image data points in different focal planes can be chosen arbitrarily, so that each configuration can be used ranging from individual images at a fixed distance from the object plane to quasi-continuous image acquisition at very small intervals. Likewise, the traversal of the focus area can be performed continuously or in steps for each image.

[0073] Additionally, in one possible embodiment, it is possible to generally check, for example even before checking the cut edges, whether the area to be cut out has actually been cut out. If the dissection was successful and the dissected specimen was completely cut out, the cut-out area is expected to appear differently in the image than the rest of the object. Therefore, a successful cut can be verified, among other things, by comparing the acquired image data, which includes at least the dissected area, with reference image data.

[0074] Fig. Figure 4 shows examples of various image data that can be acquired before and after a laser cut. It shows Fig. 4a a captured image of an undissected sample 400 and a contour 402. The contour can be, for example, the contour of a captured object or a section line drawn in the image data, along which a section is to be made in a subsequent step. Fig. 4b shows image data of the sample from Fig. 4a shows the same image section, but now the area to be cut out appears white and therefore empty. It can therefore initially be assumed that this area has been dissected. Fig. 4c again shows image data of the sample from Fig. 4a and Fig. 4b after a laser cut, where it is now visible that the white-appearing area is smaller. While an area can thus be identified as cut out, the dissected part 410 is still attached to the sample in a marginal area 414. The different hatching is intended to illustrate that the dissected part is folded at an angle relative to the plane of object 400 and protrudes obliquely from this plane. In this case, the previously described [context missing] with reference to the Fig. 2 and Fig. 3. Sharpness test described in an offset focal plane, e.g., plane 222c from Fig. 2, exhibit a sharp structure, as indicated by line 416.

[0075] Fig. Figure 5 shows an exemplary procedure in which the dissection area is first checked to see if it appears empty. For this purpose, reference image data can be acquired or generated before a cut in step 500; these are described in more detail below. Subsequently, in step 510, the laser cut is performed along the specified cut line. In step 520, image data is then acquired after the cut has been made. This image data can be compared with the reference image data from step 500 in step 530. If the comparison of the image data shows that the area to be cut appears empty, a check for sharp structures in offset focal planes can be performed in step 540, which, in conjunction with Fig. 3 and the procedure shown therein is described in detail. If this examination in step 550 also shows that no sharp structures were found, the image examination for this dissected specimen is terminated in step 560 and the next sample area can be accessed. If, on the other hand, the area to be cut out is recognized as not empty in step 530, the laser cut 510 can be repeated. Preferably, the image acquisition and evaluation are also repeated after a repeated cut to verify the success.

[0076] For test 530, whether an area to be cut out is as in the Fig. 4b and Fig. Since the section 4c is empty, in one possible embodiment the same image data used for the subsequent sharpness check can be used; that is, image data acquired in a focal plane offset from the object plane by a certain distance along the optical axis. As already described, the distance of the offset focal plane to the object plane essentially corresponds to the dimension of the dissected specimen and can therefore be, for example, in the micrometer range. Because the sectioned area should be empty after a successful section, and thus any features still visible there (e.g., microscope background, collection container) are located far outside the object plane at a distance that typically exceeds the distance of the offset focal plane by several orders of magnitude, the choice of focal plane for image comparison based on parameters such as brightness or contrast is less critical.

[0077] In other embodiments, reference image data and image data for checking whether the cut-out area is empty can be acquired alternatively or additionally with a focal plane in the object plane. This variant is particularly suitable if the lenses for image acquisition and observation are the same as the lenses used to focus the laser beam for the cut. In this case, the focal plane is already fixed in the object plane immediately after a cut, without any further adjustment.

[0078] Several methods can be used to make an initial assessment of the sectioning. For example, background image data could be used as reference data, corresponding to the background against which the object and the observation area are photographed. With appropriate illumination of the observation area in a microscope, the background, and thus the completely sectioned area of ​​an object, can be expected to appear essentially bright or white in the image, while the remaining areas depicting the object's surface appear darker. Similarly, the sectioned area may exhibit a relatively uniform structure, while the object's surface typically displays more or less heterogeneous structures.Reference image data can then be, for example, background images taken before the sections were made. This can be achieved by capturing images of the area without an object or slide, preferably under the same lighting conditions as when the images were taken after the sections. Such a reference image can provide reference data for multiple objects and sections.

[0079] It is also possible to acquire reference image data in the observation area before laser cutting, so that image data of an unprocessed sample or an undissected object area is available as reference data for comparison before cutting. This image data can, for example, be acquired with the same offset focus settings that are subsequently used for sharpness testing. Alternatively, image data could also be acquired before cutting with a focal plane in the object plane, thus avoiding the need to reposition the optical elements.

[0080] After acquiring the reference image, the sectioning can then take place, and subsequently, image data can be acquired again in the same observation area. The image data can be stored in either a volatile or non-volatile memory element, thus enabling a comparison of the image data with the reference image data after the sectioning.

[0081] Such a comparison of at least two captured images can be carried out in various ways. Fundamental methods for comparing image data are well-known in the field; for example, a pixel-by-pixel comparison can be performed, in which contrast values, color values, brightness values, or other parameters are compared and / or aligned with threshold values.

[0082] In another embodiment, the reference image data can also be theoretical image data that corresponds to the expected image data in a cropped area. This data can be defined, for example, by the expected contrast or by specifications in the frequency domain. An area could be recognized as cropped, for instance, if a region within the object surface is identified that exhibits high brightness, low contrast, a predefined uniform structure, or a specific frequency distribution. One or more of these criteria can be used to define theoretical reference image data, or all of these criteria can be used in combination.The parameters used to define theoretical reference image data can also depend on the type of image acquisition device and optical imaging system used, for example, whether the image data is in grayscale or color, how the sample is illuminated, and other factors.

[0083] In further variations, in addition to a simple comparison with reference image data, information about the position and size of the object to be cut out, for example from the coordinates of the cutting lines, can also be included in the check. This allows it to be determined whether, for example, a cut was only partially executed correctly, so that part of the dissected material might have been folded back and this area appears empty in the image data check. In this case, the area identified as empty would be significantly smaller than the area defined by the cutting line. The cut can then be classified as completely successful if the area identified as empty corresponds to the area defined by the cutting line, and as partially successful if only a smaller area is identified as empty.

[0084] If this initial check reveals that the area to be cut out is not empty and therefore the cut was not performed correctly, the cut can be classified as faulty and automatically re-cut immediately or at a later time along the already defined cutting line. Optionally, instead of automatically re-cutting, the system can simply record or notify the user that the cut was not performed correctly. If the area to be cut out is not classified as empty or cut out, the subsequent check step involving defocusing and sharpness testing can be skipped.

[0085] However, if this initial check of the cutting area shows that the area appears empty and that at least part of the object has indeed been cut out, then as a second step the previously described procedure for defocused checking of the cutting edges with offset focus planes can be carried out in order to detect stuck dissections that, however, result in an empty area in the general image comparison.

[0086] In further embodiments, these image inspections can be supplemented by additional steps. For example, the described inspections for empty areas and sharp structures can be supplemented by an image inspection of the collection container. This can involve the use of additional cameras or image acquisition elements and sensors, which may be positioned directly on the collection container or beneath a transparent one. The image data for this inspection can also be acquired and checked individually after the cut, or they can be compared against reference image data acquired from the same collection container or a section thereof before the cut. It is also possible to acquire an overview image of several sections of a collection container after multiple cuts have been made.

[0087] A special case arises when an incompletely sectioned dissectible portion is folded entirely onto the object's surface and thus, within tolerances, still lies within the object plane. In this case, an examination of the edge regions at a defined focal distance from the object may not reveal sharp structures, and the sectioned area will also appear empty during an image inspection as described above. Therefore, in one embodiment, an overview image can be captured in which, in addition to the area to be dissected, at least a peripheral region around the area to be dissected is also captured and evaluated. Preferably, this overview image can then be compared with a previously captured reference image of the same image area, taken before the sectioning process.If the object is completely flipped onto the sample surface, a blank area will indeed appear in the region of the cut line when compared to the reference image, but the surrounding area will also show deviations compared to the reference image. The criteria for image matching may differ from the criteria for detecting a blank area.

[0088] With a sufficiently large image section of such an overview image, it can also be used to identify dissected material that was correctly and completely excised but then, due to various influences, ended up back on the sample surface instead of being collected in a suitable container. Again, reference image data of a larger sample area, encompassing the dissection area or even several dissection areas, or of the entire sample, can be acquired before sectioning and then compared with corresponding image data acquired after sectioning.

[0089] In all embodiments, any data generated during the acquisition and evaluation of the image data can be used immediately, saved for later use, or transferred to other units and systems. For example, the coordinates of the cutting line along which the laser cut is made can be saved and used again during the evaluation of the image data to obtain information such as the location of the expected edge area, the dimensions of the expected dissectible portion, the endpoints of the cutting line, or other relevant information.

[0090] For further automation, the image data and evaluation results obtained from image analysis procedures as described above, across multiple dissected samples, can also be used for further adjustments. For example, it is possible to analyze and save where incompletely sectioned dissected samples occur in identical or similar samples. This analysis could, for instance, focus on identifying the part of the section line where dissected samples frequently adhere, or on determining which section of a larger sample exhibits more frequent sectioning problems when multiple dissected samples are obtained from a single sample. Furthermore, it could be determined what proportion of the automatically obtained dissected samples were not completely excised in the first section.

[0091] Depending on the results of these evaluations, the system can then adjust the cutting parameters. For example, in response to the detection of multiple cutting lines that are stuck in a similar area at the beginning of a continuous cutting line and are not completely cut through, a different laser focus or laser power could be selected for subsequent cuts in that section of the cutting line. Alternatively, a notification can be sent to the user indicating that an adjustment of the cutting parameter values ​​appears necessary. A threshold could also be set for the percentage of defective cuts, so that these adjustments are only made when the percentage of incomplete cut lines exceeds a certain percentage of the total cuts.

[0092] In general, it is also possible to supplement or even replace automatic, computer-aided image analysis with manual evaluation. For example, borderline cases can be defined in which, after automatic image analysis by appropriate software, a user is prompted via a suitable interface to visually verify the identified image features. If, for instance, an automatic check of a captured image for sharp areas yields a result within a predetermined tolerance range around a defined threshold, such that it cannot be determined with absolute certainty whether the area in question can be characterized as "sharp," a follow-up review by the user can be requested.In this case, the user could be shown the entire captured image area, or just the unclear areas, on a suitable user interface, such as a display screen or touchscreen, and offered a choice on how to handle these areas. The user could then, for example, choose whether to perform another cut along the predefined cut lines or whether no further processing is required.

[0093] Some embodiments relate to a microscope comprising a system such as in conjunction with one or more of the Fig. 1 to 5 described. Alternatively, a microscope can be part of a system, such as in conjunction with one or more of the Fig. 1 to 5 described, be, or be connected with the same. With renewed reference to Fig.In this context, system 100 comprises a microscope 102 and a computer system 160. The microscope 102 is configured to capture images and is connected to the computer system 160. The computer system 160 is configured to execute at least part of a procedure described herein. The computer system 160 may be configured to execute a machine learning algorithm. The computer system 160 and the microscope 102 may be separate units or integrated together in a common housing. The computer system 160 could be part of a central processing system of the microscope 102 and / or the computer system 160 could be part of a component of the microscope 102, such as a sensor, an actuator, a camera, or an illumination unit, etc.

[0094] The computer system 160 can be a local computing device (e.g., a personal computer, laptop, tablet computer, or mobile phone) with one or more processors and one or more storage devices, or it can be a distributed computing system (e.g., a cloud computing system with one or more processors or one or more storage devices distributed across different locations, for example, a local client and / or one or more remote server farms and / or data centers). The computer system 160 can comprise any circuit or combination of circuits. In one embodiment, the computer system 160 can comprise one or more processors, which can be of any type.In our usage, "processor" can mean any type of computing circuit, such as, but not limited to, a microprocessor, a microcontroller, a complex instruction set (CISC) microprocessor, a reduced instruction set (RISC) microprocessor, a very long instruction word (VLIW) microprocessor, a graphics processor, a digital signal processor (DSP), a multi-core processor, a field-programmable gate array (FPGA), e.g., of a microscope or a microscope component (e.g., a camera), or any other type of processor or processing circuit. Other types of circuits that may be included in Computer System 160 may be a custom-made circuit, an application-specific integrated circuit (ASIC), or the like, such as one or more circuits (e.g., a communications circuit) for use in wireless devices, such as...The computer system 160 may include mobile phones, tablet computers, laptop computers, two-way radios, and similar electronic systems. The computer system 160 may include one or more storage devices, which may comprise one or more memory elements suitable for the application, such as main memory in the form of read-only memory (RAM), one or more hard disks, and / or one or more drives that handle removable media, such as CDs, flash memory cards, DVDs, and the like. The computer system 160 may also include a display device, one or more speakers, and a keyboard and / or control system, which may include a mouse, trackball, touchscreen, voice recognition device, or any other device that allows a system user to input information into and receive information from the computer system 160.

[0095] Some or all of the process steps can be performed by (or using) a hardware device, such as a processor, a microprocessor, a programmable computer, or an electronic circuit. In some embodiments, one or more of the key process steps can be performed by such a device.

[0096] Depending on specific implementation requirements, embodiments of the invention can be implemented in hardware or software. Implementation can be carried out using a non-volatile storage medium such as a digital storage medium, for example, a floppy disk, DVD, Blu-ray disc, CD, ROM, PROM, EPROM, EEPROM, or FLASH memory, on which electronically readable control signals are stored. These signals interact (or can interact) with a programmable computer system to execute the respective method. Therefore, the digital storage medium can be computer-readable.

[0097] Some embodiments according to the invention include a data carrier with electronically readable control signals that can interact with a programmable computer system so that one of the methods described herein is carried out.

[0098] In general, embodiments of the present invention can be implemented as a computer program product with program code, wherein the program code is effective for executing one of the methods when the computer program product runs on a computer. The program code can, for example, be stored on a machine-readable medium.

[0099] Further embodiments include the computer program for carrying out one of the methods described herein, which is stored on a machine-readable medium.

[0100] In other words, an embodiment of the present invention is therefore a computer program with program code for carrying out one of the methods described herein when the computer program is running on a computer.

[0101] Another embodiment of the present invention is therefore a storage medium (or a data carrier or a computer-readable medium) comprising a computer program stored thereon for executing one of the methods described herein when executed by a processor. The data carrier, the digital storage medium, or the recorded medium is generally tangible and / or not seamless. Another embodiment of the present invention is a device as described herein comprising a processor and the storage medium.

[0102] Another embodiment of the invention is therefore a data stream or signal sequence that represents the computer program for carrying out one of the methods described herein. The data stream or signal sequence can, for example, be configured to be transmitted via a data communication connection, such as the Internet.

[0103] Another embodiment includes a processing means, for example a computer or a programmable logic device, which is configured or adapted to perform one of the methods described herein.

[0104] Another embodiment comprises a computer on which the computer program for performing one of the methods described herein is installed.

[0105] Another embodiment of the invention comprises a device or system configured to transmit (for example, electronically or optically) a computer program for executing one of the methods described herein to a receiver. The receiver may be, for example, a computer, a mobile device, a storage device, or the like. The device or system may, for example, include a file server for transmitting the computer program to the receiver.

[0106] In some embodiments, a programmable logic device (e.g., a field-programmable gate array, FPGA) can be used to perform some or all of the functionalities of the methods described herein. In some embodiments, a field-programmable gate array can cooperate with a microprocessor to perform one of the methods described herein. In general, the methods are preferably performed by any hardware device. Reference symbol list 100 laser microdissection system 102 Microscope 104 microscope foot 106 lighting units 108 condenser unit 110 User interface 112 Drive knob 114 Microscope stage 116 objects under investigation 118 slides 120 bracket 122 lens 124 lens turret 126 Protective cover 128 Observation beam path 130 tube unit 132 Disconnection device 134 pairs of eyepieces 136 image capture units 138 Image evaluation unit 140 laser units 142 Laser light source 144 Laser beam 146 incident light unit 148 Deflection mirrors 150 deflecting mirrors 152 Adjustment device 154 Laser deflection device 156 wedge plates 160 tax calculators 170 data connection 190 Autofocus system x-coordinate axis, spatial direction y coordinate axis, spatial direction z coordinate axis, spatial direction 200 samples or objects 210 incompletely separated dissection 212 Cutting hole 214 Edge area 220 object level 222a...222e offset focal planes below the sample 224a...224e offset focal planes above the sample 232 Final distance below the sample 234 Final distance above the sample 240 optical axis 300 Change focus Capture 310 image data Check 320 image data for sharp structures 330 Check end of focus range 340 End sharpness test Process 350 pieces of information further Re-cut 360 400 objects 410 incompletely separated dissection 412 Cutting hole 414 Edge area 416 sharp structure Generate 500 reference image data 510 Laser cutting Capture 520 image data after cutting 530 Comparison of image data against reference image data 540 Sharpness test 550 Dissection successful 560 Image Check Finish

Claims

[1] Method for verifying a dissection process in a laser microdissection system (100), comprising: Performing a dissection process to cut out (510) a dissection (210, 410) from an object (200, 400) in a first area of ​​the object (200, 400) using a laser beam; Acquisition (310, 520) of first image data of at least the first area after the dissection process with a first focal plane (222) which is parallel offset along the optical axis (240) to the plane (220) of the object (200, 400), acquisition (310, 520) of second image data after the dissection process with a second offset focal plane (224) which is parallel offset along the optical axis (240) and on the opposite side of the object (200, 400) with respect to the first focal plane (222), and Check (320, 530, 540) whether the first image data and / or the second image data show sharp structures within the area to be separated by the dissection process in order to determine whether the dissection process was successful. [2] Method according to claim 1, further comprising: Defining a multitude of further offset focal planes (222a, 222b, 222c, 222e; 224a, 224b, 224c, 224e) which are parallel offset along the optical axis (240) to the plane (220) of the object (200, 400); Acquire (310, 520) image data in each of the multiple offset focal planes (222a, 222b, 222c, 222e, 224a, 224b, 224c, 224e); and check (320, 530, 540) whether at least one of the image data has sharp structures to determine whether the dissection process was successful. [3] Method according to claim 2, wherein the offset focus planes (222a, 222b, 222c, 222e, 224a, 224b, 224c, 224e) are successively controlled by stepwise changing the focus of an optical system which generates the image data. [4] Method according to claim 2 or 3, further comprising: defining a final distance (232, 234) from the plane (220) of the object (200, 400), wherein the plurality of further offset focus planes (222a, 222b, 222c, 222e; 224a, 224b, 224c, 224e) lie between the plane (220) of the object (200, 400) and the final distance (232, 234). [5] Method according to claim 4, wherein the final distance (232, 234) is at most half the largest extent of the dissection (210, 410) in the plane (220) of the object (200). [6] Method according to any of the preceding claims, further comprising: comparing (530) image data acquired after the dissection process with reference image data to determine whether the dissection process was successful. [7] Method according to claim 6, further comprising: Generating (500) reference image data by acquiring initial image data of the first area prior to performing the dissection process. [8] Method according to claim 6 or 7, further comprising: Generating (500) reference image data by acquiring image data of the first area with the first and / or the second offset focal plane (222, 224) prior to performing the dissection process. [9] Method according to any one of claims 6 to 8, further comprising: Generating (500) reference image data by capturing image data of a background area without an object (200, 400). [10] Method according to any one of claims 6 to 9, further comprising: Generating (500) reference image data by setting threshold values ​​for parameters of the image data, wherein the parameters include at least one of the following for at least a subset of the image data: brightness, contrast, frequency distribution, gray value distribution, color values. [11] Method according to any one of claims 6 to 10, further comprising: Generating (500) reference image data by acquiring image data of the object (200, 400) outside the first area. [12] Method according to any one of claims 6 to 11, further comprising: Acquiring (500) reference image data prior to performing the dissection process in a second area of ​​the object (200, 400) which includes the first area; acquiring (520) third image data of the second area after the dissection process; and comparing (530) the acquired third image data with reference image data of the second area to determine whether the cut-out dissection is on the object. [13] Method according to any one of the preceding claims, further comprising: If it was determined that the dissection process was unsuccessful, the dissection process should at least be partially repeated. [14] Control unit (160) for a laser microdissection system (100), wherein the control unit (160) is configured to perform the process steps according to one of the preceding claims. [15] Laser microdissection system (100) comprising a laser (140) configured to perform a dissection process for cutting and separating a dissection (210, 410) from an object (200, 400), and at least one camera configured to capture image data of the object (200, 400), and a control unit (160) according to claim 14. [16] Computer program with program code for carrying out a method according to one of claims 1 to 13 using the laser microdissection system (100) according to claim 15, when the program code is executed by the control unit (160) of the laser microdissection system (100).

Images