Method and system for extracting part of microscopic sample

Abstract

To provide a method for extracting a part of a microscopic sample.SOLUTION: A method includes: a step of applying a liquid cover to a microscopic sample to obtain a processed microscopic sample; a step (210) of generating at least one marker (282) for a part (258.2) of the processed microscopic sample (252.3) using an imaging system; a step (212) of removing at least a part of the liquid cover from the processed microscopic sample (252.3) to obtain an uncovered microscopic sample (252.4); and a step (216) of extracting a part (258.2) of the uncovered microscopic sample (252.4) on the basis of at least one marker to obtain an extracted part (258.3).SELECTED DRAWING: Figure 2b

Description

Technical Field

[0001] The present invention relates substantially to a method for extracting a part of a microscopic sample, a corresponding system, and a computer program.

Background Art

[0002] Laser microdissection is a technique for visualizing a microscopic sample, cutting out (extracting) a part of the microscopic sample using a laser, and making such an extracted part available for subsequent molecular biology processes. For this purpose, the sample can be placed on a membrane-based slide, gently stained so as not to damage the biomolecules in the sample to be examined later, visualized, and marked for extraction (cutting). Such microdissection with a corresponding workflow is described, for example, in US Patent No. 10533931.

Summary of the Invention

Means for Solving the Problems

[0003] Considering the above situation, improvement in the extraction of a part of a microscopic sample is necessary. According to embodiments of the present invention, a method, a system, and a computer program having the features of the independent claims are proposed. Another advantageous development forms the subject matter of the dependent claims and the following description.

[0004] One embodiment of the present invention relates to a method for extracting a portion of a microscopic sample. The method comprises, namely, applying a liquid coating to the microscopic sample to obtain a processed microscopic sample. In one embodiment, the microscopic sample may be processed, for example, stained, before applying the liquid coating to the microscopic sample. In one embodiment, the microscopic sample is placed on a slide. The slide is positioned on a stage. Furthermore, an imaging system such as a microscope is used to generate at least one marker (or extraction marker) for a portion of the processed microscopic sample. Such markers may preferably be or have one or more individual markers (such as dots), or one or more shapes, lines, circles, ellipses, crosses, numbers, etc. Such shapes may indicate, for example, where to cut. Furthermore, at least a portion of the liquid coating is removed from the processed microscopic sample to obtain an uncoated microscopic sample. Depending on the specific workflow used, for example, the microscopic sample may be removed from the stage and then repositioned there. Furthermore, based on at least one marker, the portion of the uncoated microscopic sample is extracted to obtain an extracted portion. Extraction specifically refers to the physical extraction of a portion from a microscope sample, and it should be noted that the extracted portion can be transferred, for example, into or onto a container by means of dropping, ejection, or adhesion.

[0005] Applying a liquid coating to a microscopic sample improves marker generation, particularly due to improved visualization using the imaging system described above. Applying a liquid coating specifically refers to applying a liquid to the surface of the microscopic sample so that the liquid covers and / or penetrates the sample. The liquid in the coating has a positive effect on light reflected or emitted by the sample. Therefore, this improves the visualization of the microscopic sample, thus improving marker generation and the extraction of parts of the microscopic sample.

[0006] Such a liquid coating (or liquid coating slip) comprises polystyrene and xylene in one embodiment of the present invention. For example, polystyrene and xylene can be supplied as a mixture having equal amounts of the two components. Such a mixture is easily applyable, and the visualization process is greatly improved by such a mixture. For example, it may contain a particularly small amount of another component.

[0007] However, it has been found that such liquid coatings can negatively affect subsequent processes of the extracted portion. Removing these liquid coatings from such extracted portions is difficult and time-consuming. In particular, for (extremely) small portions, it may not even be possible to handle the portion to remove the liquid coating. Now recognized by the inventors, this difficulty can be overcome by removing at least partially the liquid coating from the processed microscopic sample after marker generation and before extracting a portion from the microscopic sample. Removing such liquid coatings from uncut (and therefore larger) samples is considerably easier. In one embodiment of the present invention, xylene is used (as a remover) to remove the liquid coating from the processed microscopic sample. For example, xylene can be applied to the processed microscopic sample to wash away the liquid coating, or the processed microscopic sample can be placed in a container filled with xylene.

[0008] In one embodiment of the present invention, the generation of at least one marker for the portion of a processed microscopic sample comprises, or is based on, the following: visualizing the processed microscopic sample using the imaging system, and determining at least one marker based on the visualized processed microscopic sample. This enables, for example, user monitoring. The user can also make inputs to generate at least one marker, taking into account the visualized microscopic sample.

[0009] According to another embodiment of the present invention, at least one of the markers is digitally generated and stored for later use in extracting the portion of the processed microscope sample. This allows, for example, the imaging system to be used for another task while the liquid coating is being removed.

[0010] According to another embodiment of the present invention, the above portion of an uncoated microscope sample is extracted using laser microdissection. Laser microdissection is a technique that enables high-speed and accurate extraction (cutting) of even extremely small portions of a microscope sample.

[0011] In one embodiment, a system having the above-described imaging system may be used, which is configured to perform laser microdissection. Such a system may be a microscope system having a typical microscope for imaging and further configured to perform laser microdissection. Such a combined system enables fast and efficient operation. While such a combined system offers certain advantages, it should be noted that two different or separate systems, such as an imaging system like a microscope and a laser microdissection system, can be used. This allows, for example, the use of systems designated for corresponding tasks, such as imaging (including marker generation) and laser microdissection, respectively.

[0012] According to another embodiment of the present invention, the above portion of an uncoated microscopic sample is extracted using one of the following: namely, by cutting the uncoated microscopic sample with a needle and by applying a buffer to the uncoated microscopic sample. The process can be further simplified by applying the buffer (or buffer solution) using a template. Such techniques are different from laser microdissection but can be used instead of (or in addition to) laser microdissection, for example, depending on specific needs or specific microscopic samples. Techniques using needles are preferably performed automatically. Similar to laser microdissection, separate systems are available for these alternative techniques.

[0013] According to another embodiment of the present invention, the extracted portion has a maximum diameter of less than 500 μm. Alternatively, such a diameter may be less than 300 μm or less than 100 μm. Alternatively, the extracted portion is one of the following: an individual living cell, a population of several individual cells, an organelle of a living cell, a part of a living cell, or a cell embedding matrix. The organelle or part of a living cell may have, for example, a nucleus, or another specific structure such as a mitochondria or a cellular inclusion. The cell embedding matrix is ​​particularly, for example, an extracellular matrix, collagen, or connective tissue. Note that the size of the cell, organelle, or part of it may also be less than 500 μm in diameter. In any case, handling such small portions extracted from a microscopic sample is difficult, and therefore, removing the liquid coating before extraction, as proposed, is particularly advantageous.

[0014] For reliability and validation, a pure film of the same size without the sample can be covered with the same liquid on the same slide and then used as a negative control for downstream processes. This allows for the detection of contamination that may occur due to the liquid coating and / or removal agent, which could later alter the results of downstream analysis.

[0015] According to another embodiment of the present invention, the method further comprises generating at least one reference marker before removing at least the portion of the liquid coating from the processed microscopic sample. Such a reference marker can be generated on the processed microscopic sample or on the slide on which the sample is placed. In the case of two or more reference markers, one or more markers can also be generated on each sample and slide. The method further comprises, after removing (or having already removed) at least the portion of the liquid coating from the processed microscopic sample, correlating at least one of the reference markers with at least one of the markers in order to extract the portion of the uncoated microscopic sample. In this way, the (extraction) marker and the uncoated microscopic sample can be easily and appropriately associated, for example, if the uncoated microscopic sample is removed from the stage to remove the liquid coating, or if the uncoated microscopic sample is moved to a separate system, separate from the imaging system, for extraction.

[0016] Another embodiment of the present invention relates to a system having an imaging system. The system is configured as follows: the imaging system is configured to generate at least one marker on a portion of a processed microscopic sample, where the processed microscopic sample is a microscopic sample to which a liquid coating has been applied. In one embodiment, the system is also configured to (automatically) apply the liquid coating to the microscopic sample. Furthermore, the system is configured to remove at least a portion of the liquid coating from the processed microscopic sample in order to obtain an uncoated microscopic sample and to (physically) extract a portion of the uncoated microscopic sample based on at least one of the markers. Such a system may be, for example, a microscope system comprising a microscope and a device capable of transferring a microscopic sample from a stage to a container containing, for example, xylene, and returning it to the stage.

[0017] According to another embodiment of the present invention, the system is further configured to draw a user interface, and the system is further configured as follows: that the imaging system is used to visualize the processed microscopic sample on the user interface, to receive input data from the user interface relating to at least one mark, to receive input data relating to at least one marker, and to generate at least one marker for the portion of the processed microscopic sample based on the input data.

[0018] According to another embodiment of the present invention, the system is further configured to perform laser microdissection, where the aforementioned portion of the uncoated microscope sample is extracted using laser microdissection. Thus, even liquid coatings that cannot be laser-cut can be used, as the coating is removed before the laser is used.

[0019] For the advantages of the system and other embodiments, please refer to the description of the method as applicable herein.

[0020] Another embodiment of the present invention relates to a computer program having program code for performing the following steps when the computer program is running on a processor: namely, the steps of correlating at least one reference marker with at least one marker, and preferably controlling the system to extract the portion from an uncoated microscopic sample using laser microdissection. At least one of the markers is generated for a portion of the processed microscopic sample that is removed as described above. The processed microscopic sample is a microscopic sample coated with a liquid coating. The uncoated microscopic sample is a microscopic sample from which the liquid coating has been removed. This allows the liquid coating to be removed from the microscopic sample after the markers have been generated and before the portion of the microscopic sample has been extracted.

[0021] Further advantages and embodiments of the present invention will become apparent from the description and accompanying drawings.

[0022] It should be noted that the features described above and those further described below can be used not only in the combinations shown, but also in other combinations or individually, without departing from the scope of the present invention. [Brief explanation of the drawing]

[0023] [Figure 1] This is a schematic diagram showing a system equipped with an imaging system that can be used to carry out a method according to one embodiment of the present invention. [Figure 2a] This is a schematic diagram of a method according to one embodiment of the present invention. [Figure 2b] This is a schematic diagram of a method according to one embodiment of the present invention. [Figure 3]A schematic diagram showing another system equipped with an imaging system that can be used to implement the method according to one embodiment of the present invention.

Embodiments for Carrying Out the Invention

[0024] In FIG. 1, a system 100 having an imaging system 110 is schematically shown. In one embodiment, the imaging system 110 may be a microscope, and thus the system 100 may be a microscope system. The system 100 can be used to implement the method according to one embodiment of the present invention. First, the system 100 will be described in more detail. The method will be described with reference to FIGS. 2a and 2b and also in relation to the system 100 of FIG. 1.

[0025] The imaging system 110 can be used to visualize the microscope sample 152. In one embodiment, the imaging system 110 has an illumination optical system 112 (e.g., having a light source) for generating an illumination light beam 114, and the illumination light beam 114 is directed to the objective lens 118 via a reflector 116 and then to the microscope sample 152. The light reflected from the microscope sample 152 is directed as an imaging light beam 122 to the detector 124 via the objective lens 118, the reflector 116, and another reflector 120. The imaging system 110 may further have an eyepiece 126 for the user to inspect the microscope sample.

[0026] Note that transmitted light illumination may be used instead of the incident illumination shown in FIG. 1. In the latter case, the illumination light beam will illuminate the sample from below the sample 152.

[0027] In one embodiment, the imaging system 110 further includes a stage (or microscope stage) 130. A microscope sample 152 can be placed on a slide 150, and the slide 150 (on which the microscope sample 152 is placed) can be placed on the stage 130 so that the microscope sample 152 can be visualized. In one embodiment, the slide is a glass slide on or on which a film is placed, or a frame slide (e.g., made of metal or plastic) on which a film is placed within a frame slide. The microscope sample 152 is then placed on the film.

[0028] In one embodiment, the system 100 further includes a controller or computer 140. The system 100 may also include, for example, a display 144, a keyboard 146, and a computer mouse 148. The system 100 can be configured to draw or provide a user interface or graphical user interface 142, exemplarily shown on the display 144. In this way, the controller 140 can receive images from the microscope sample 152 acquired by the detector 124 and display them to the user via the user interface 142 or the display 144.

[0029] Such a system 100 allows, for example, the user in the user interface 142 to visualize the microscope sample 152. The user can then use the user interface 142 to generate input to the system 100 to generate markers and / or reference markers for the microscope sample 152, which can then be extracted later.

[0030] In one embodiment, system 100 is further configured to perform laser microdissection. In this case, system 100 (or in particular its imaging system 110) may have a laser 132 which can generate a laser beam 134 that is directed to an objective lens 118 via a reflector 116 and then to a microscope sample 152. For example, a scanning mirror 136 may be included in the reflector 116 or at another suitable location to direct the laser beam 134 along a desired path around the microscope sample 152, for the purpose of extracting a portion of it. A controller 140 may also be used to control the laser 132, for example.

[0031] In one embodiment, the container 138 is located, for example, within or on the stage 130. In this way, a portion is extracted from the microscope sample and falls downward. Note that this may require a frame slide with a membrane. Depending on the particular type of laser microdissection apparatus (if laser microdissection is used anyway), a different type of container and / or a different location of the container may be used. The embodiment shown in Figure 1 is for illustrative purposes only.

[0032] It should be noted that System 100 is an exemplary system that can be used to carry out a method according to one embodiment of the present invention. Other systems or imaging systems can be used as well. In particular, it is not necessary to use a combined system that enables imaging and laser microdissection. A system for laser microdissection provided separately from the imaging system can also be used. Furthermore, extraction methods other than laser microdissection can also be used.

[0033] Figures 2a and 2b schematically illustrate a method according to one embodiment of the present invention using a workflow. Note that the steps begin in Figure 2a and continue in Figure 2b.

[0034] In step 200, slide 250 is prepared. In one embodiment, slide 250 has a glass slide or frame slide 256 with a film 254 positioned on top (or in the middle). Slide 250 may correspond to, for example, slide 150 in Figure 1. Furthermore, a microscopic sample 252.1 is placed on the film 254. Microscopic sample 252.1 may correspond to, for example, microscopic sample 152 in Figure 1. Note that instead of a glass slide, a frame slide with a film positioned inside may be used as described above. Such a frame slide may be made from, for example, a metal such as steel or aluminum. Such a frame slide may also be made from, for example, plastic.

[0035] Microscopic sample 252.1 has two parts 258.1 and 258.2 that may be particularly important in the microscopic sample, for example. For example, the user may want to (physically) extract one or both of these parts from the microscopic sample 252.1 for further analysis. These parts 258.1 and 258.2 may be organelles such as cells or nuclei, for example.

[0036] Step 202 involves processing the microscopic sample, for example, by staining it. This is shown using a container 260 filled with a staining material or staining solution 262. For example, such a staining solution 262 can be applied to the microscopic sample using a pipette 264. For example, such a staining solution changes the color of portions 258.1 and 258.2, as shown for step 202 in Figure 2a, but does not change the color of the rest of the microscopic sample.

[0037] After processing or staining, a processed microscopic sample 252.2 is obtained. Note that processing may be performed before placing the microscopic sample on a slide or membrane. Furthermore, note that processing (such as staining) can be performed on multiple microscopic samples in an automated process. Specific processing or staining equipment is available. The situation shown in Figure 2a for step 202 is for illustrative purposes only. Furthermore, note that multiple processing steps and / or different processing steps may be performed on the same microscopic sample.

[0038] In step 204, a liquid coating is applied to the (processed) microscope sample 252.3 to obtain the processed microscope sample 252.3. This is indicated by a container 270 filled with the liquid coating or liquid 272. To coat the microscope sample with such liquid 272, the liquid 272 can be applied to the microscope sample using, for example, a pipette 274. Note that the application of the liquid coating may be performed before placing the microscope sample on a slide or membrane. In this regard, note that step 202 is not an essential step, but is often used, for example, to highlight specific parts of the microscope sample by staining.

[0039] Furthermore, it should be noted that the application of the liquid coating may be carried out in an automated process and on the microscopic sample. Specific coating apparatus or devices are available. In one embodiment, such a coating apparatus or device may be part of, or incorporated into, system 100, as shown in Figure 1. The situation shown for step 204 in Figure 2a is for illustrative purposes only.

[0040] Such a liquid coating or liquid 272 (also referred to as a liquid coating slip) may, in one embodiment, have polystyrene and xylene. For example, polystyrene and xylene can be supplied as a mixture in which the two components are equal or nearly equal in amount (or proportion). Another component may also be included, particularly in small amounts. For example, given that the sum of the two proportions must not exceed 100%, polystyrene may be supplied in a proportion of 30% to 70%, and xylene may also be supplied in a proportion of 30% to 70% (another component may be present). The ratio is ideally about 50% to 50%. In another embodiment, the liquid coating or liquid coating material 272 may have water and / or ethanol in a ratio of 25% water and 75% ethanol or a greater amount of ethanol up to 100%. Instead of ethanol, another liquid such as water, polystyrene, xylene, or acetone may be used.

[0041] In step 206, the processed microscope sample 252.3 is visualized. This can be done by using an imaging system, for example, the microscope shown in Figure 1. In particular, slide 250, which has the processed microscope sample 252.3, can be placed on a stage such as stage 130 shown in Figure 1. Note that slide 250 may be placed on the stage together with the microscope sample before applying the liquid coating. This may depend on the exact method of applying the liquid coating.

[0042] Visualizing the processed microscope sample 252.3 may involve, for example, acquiring an image (in particular, a real-time or live image) of the processed microscope sample 252.3 and displaying or presenting it on the display of the user interface 242. The user interface 242 shown in Figure 2a may correspond, for example, to the user interface 142 in Figure 1. In this way, the user can determine the locations where markers are to be created or generated for extraction, as will be described later.

[0043] Step 208 generates at least one reference marker. In the embodiment shown in Figure 2a, two reference markers, 280.1 and 280.2, are generated, one in the upper left of slide 250 and the other in the lower right of slide 250. For example, such reference markers can be generated by using the laser of system 100 to cut the film 254 of slide 250 at the positions indicated by the reference markers 280.1 and 280.2, as shown in Figure 1. Such reference markers can be used to better or more accurately associate the (extraction) marker with the microscopic sample (this will be discussed later). This is particularly important in cases where the slide 250 needs to be rearranged on the stage if it must be removed from the stage for one of the subsequent steps.

[0044] It should be noted that the association between (extraction) markers and microscopic samples is generally possible even without such reference markers. Therefore, step 208 is an optional step. It should also be noted that step 208 may be performed before step 206.

[0045] Step 210 generates at least one marker or extraction marker 282. In one embodiment, this is based on a visualized processed microscopic sample 252.3, as shown on the right for step 210 in Figure 2b. Thus, step 206, which visualizes the processed microscopic sample 252.3, can be considered part of or a substep of step 210.

[0046] In the illustrated embodiment, a portion 258.2 of a microscope sample is extracted. Therefore, for example, a marker 282 is generated that surrounds portion 258.2 at a predetermined distance. In the illustrated embodiment, the marker 282 is a line. However, other types of markers, such as several parts including multiple points and / or lines or other shapes, are concessible. When extracting multiple portions, multiple lines can also be generated as markers. Such markers are particularly intended to indicate where the laser beam should be guided around the portion to be extracted (in the case of laser microdissection). Such markers should be generated so as to take into account the width of the laser beam with respect to the distance of the marker from the portion to be extracted. That is, the laser should not hit the portion to be cut, but should only cut the film adjacent to that portion.

[0047] The generation of such markers can be performed using the user interface 242. For example, the user can view a visualized processed microscope sample 252.3 on the user interface 242 or its display, and can draw lines (as markers) using the user interface 242 or its computer mouse. It should be noted that generating such markers does not necessarily require any input from the user. Such a process can also be automated and performed using, for example, image analysis to find the contours of stained areas.

[0048] In either case, the liquid coating applied to the microscope sample improves the image of the microscope sample, making it visible to the user or available for image analysis processing. This improvement in visibility is due, for example, to the liquid coating applied to the microscope sample having a positive effect on luminescence and / or reflection by the sample.

[0049] In one embodiment, the relative coordinates between the reference marker and the (extracted) marker can also be determined in step 210. These relative coordinates can later be used to correlate the reference marker and the (extracted) marker.

[0050] In practice, it should be noted that marker 282 is generally not visible on the microscope sample or slide itself, but only in the user interface. The marker 282 shown to the left of step 210 in Figure 2b is for illustrative purposes only. Rather, the marker or extraction marker 282 can be generated digitally. Once marker 282 is generated, it can be stored for later use in the extraction of the above portion of the processed microscope sample. For example, the marker can be stored in the controller or computer 140 or its storage device as shown in Figure 1.

[0051] In step 212, the liquid coating is removed from the processed microscopic sample to obtain an uncoated microscopic sample 252.4. Such a liquid coating can be removed, for example, by applying another liquid (removal agent) to the processed microscopic sample. This is indicated by a container 290 filled with the liquid or removal liquid 292. Such liquid 292 can also be applied to the microscopic sample using, for example, a pipette 294. Another preferred method is to immerse the processed microscopic sample in such liquid. For example, the processed microscopic sample may be immersed in the liquid 292 in the container 290. This step may involve removing the slide 250 from the stage along with the processed microscopic sample 252.3 placed on the slide 250.

[0052] In one embodiment, the liquid 292 is or contains xylene. The liquid coating can be removed from the microscopic sample by immersing the processed microscopic sample in xylene for a specific time, for example, 20 or 30 seconds. Additional steps may be taken to remove all remaining xylene from the microscopic sample. To achieve this, the sample can be heated or warmed to rapidly vaporize the xylene, although xylene also vaporizes at room temperature. Using a desiccant such as silica gel to dry the sample is also a suitable approach. However, generally, any remaining xylene does not adversely affect the subsequent examination of the microscopic sample or its extract by PCR-based methods for nucleic acids.

[0053] Furthermore, it should be noted that the removal of the liquid coating may be performed in an automated process and / or for multiple microscopic samples. Specific removal devices or apparatuses are available. In one embodiment, such a removal device or apparatus may be part of, or incorporated into, system 100, as shown in Figure 1. The situation shown by step 212 is for illustrative purposes only.

[0054] In step 214, the slide 250 can be rearranged on the stage along with the uncovered microscope sample. The previously stored marker 282 is loadable (for example, into a program running on the processor or controller 140 in Figure 1) and can be displayed, for example, via the user interface 242. Reference markers 280.1 and 280.2 are available and can be correlated with marker 282, which may be based, for example, on the relative coordinates identified in step 210.

[0055] These reference markers 280.1 and 280.2 can be used, in particular, to position marker 282 precisely relative to portion 258.2, just as previously defined. Note that in this regard, the position of the reference markers did not change with respect to the microscope sample and the portion to be extracted.

[0056] The correlation between the reference marker and the (extracted) marker can be performed, for example, within a program running on the processor or controller 140 in Figure 1, as described above.

[0057] Note that step 214 can be omitted if it is necessary to remove slide 250 from the stage along with the microscope sample in order to remove the liquid coating. Furthermore, note that if a separate system is used to generate markers and to extract portions as described above, slide 250 can be placed on a separate stage of the separate system along with the uncoated microscope sample 252.4. In such a case, the previously stored marker 282 can be transferred to such a separate system for extraction. The relative coordinates between the reference marker and the (extraction) marker can also be stored after being identified (in step 210) and then transferred as well.

[0058] In step 216, a portion 258.2 of the uncoated microscope sample 252.4 is (physically) extracted from the uncoated microscope sample 252.4. This is done based on the marker 282. In this way, an extracted portion 258.3 is obtained, which can be further inspected or processed. In the embodiment shown in Figure 2b, the extraction is performed by laser microdissection. In such a case, the laser beam 234 (which may correspond to the laser beam 134 in Figure 1) can be controlled to follow the marker 282 (which may be a line) and thereby cut out portion 258.2 from the microscope sample.

[0059] Depending on the specific type of system used for laser microdissection, for example, the disassembled or extracted parts 252.3 can be dropped into a collection container (as described in Figure 1) or transported into the container by another method.

[0060] As mentioned above, slide 250 may have, for example, a glass slide with a film placed on it. For such a glass slide, the film can settle on the glass and then adhere to the glass. For example, liquid (e.g., from a liquid coating or from a liquid used to remove a liquid coating) can be obtained between the film and the glass through pores in the film, and the film can adhere to the glass by capillary force. This is particularly useful in non-contact laser microdissection (LMD) systems. Such pores can be created by generating the reference markers described above.

[0061] Such adhesion of the film can, for example, prevent the extracted portion from falling off the slide or from being ejected. This can be overcome, for example, by supplying one or more defocused laser pulses to the center of a small resolution region (i.e., a cut-out portion, e.g., a portion with a diameter of 1–40 μm). Conversely, this can contribute to the inability to shift the film relative to the reference marker. The corresponding defocused laser pulses can be centered to facilitate acquisition.

[0062] Figure 3 schematically shows system 300 having an imaging system 310. System 300 corresponds to system 100 in Figure 1, and imaging system 310 corresponds to imaging system 110 in Figure 1. Therefore, the same reference numerals as in Figure 1 are used. For explanation, please refer to Figure 1.

[0063] The difference between system 300 and system 100 is the provision of a pipe 302 configured to apply a liquid to the microscope sample 152, for example, by spraying it. In particular, such a liquid may be a liquid coating liquid (e.g., liquid 272 in Figure 2a). Additionally or alternatively, such a liquid may be a liquid for removing the liquid coating (e.g., liquid 292 in Figure 2b). Another (additional) pipe may also be used for liquid removal. The controller 140 can be configured to control such a pipe 302 to apply such a liquid to the microscope sample 152. In this way, system 300 is configured to apply a liquid coating to a microscope sample and / or remove the liquid coating from the microscope sample.

[0064] It should be noted that System 300 is for illustrative purposes only, and other configurations of the system configured to apply and / or remove a liquid coating may be used. For example, such a system may have a robot configured to remove a slide from a stage, immerse it in the liquid, and reposition the slide on the stage.

[0065] As used herein, the term "and / or" includes all possible combinations of one or more of the related items and may be abbreviated as " / ".

[0066] 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.

[0067] Some embodiments relate to microscopes that include a system as described in relation to one or more of Figures 1 to 3. Alternatively, the microscope may be part of a system as described in relation to one or more of Figures 1 to 4, or may be connected to a system as described in relation to one or more of Figures 1 to 4. Figure 3 shows a schematic diagram of a system 300 configured to carry out the methods described herein. The system 300 includes a microscope 310 and a computer system 140. The imaging system or microscope 310 is configured to take images and is connected to the computer system 140. The computer system 140 is configured to carry out at least a portion of the methods described herein. The computer system 140 may be configured to run machine learning algorithms. The computer system 140 and the imaging system or microscope 310 may be separate entities, or they may be integrated within a single common housing. The computer system 140 may be part of the central processing system of the imaging system or microscope 310, and / or the computer system 140 may be part of a dependent component of the imaging system or microscope 310, such as a sensor, actor, camera, or lighting unit of the imaging system or microscope 310.

[0068] The computer system 140 may be a local computer device (e.g., a personal computer, laptop, tablet computer, or mobile phone) comprising one or more processors and one or more storage devices, or it may be a distributed computer system (e.g., a cloud computing system comprising one or more processors and one or more storage devices distributed to various locations such as local clients and / or one or more remote server farms and / or data centers). The computer system 140 may include any circuit or combination of circuits. In one embodiment, the computer system 140 may include one or more processors, which can be of any kind. As used herein, the processor may be intended to be any kind of computing circuit, such as a microprocessor for a microscope or microscopic component (e.g., a camera), a microcontroller, a composite instruction set computing (CISC) microprocessor, a reduced instruction set computing (RISC) microprocessor, a very long instruction word (VLIW) microprocessor, a graphics processor, a digital signal processor (DSP), a multicore processor, a field-programmable gate array (FPGA), or any other kind of processor or processing circuit. Other types of circuits that may be included in the computer system 140 may be custom circuits, application-specific integrated circuits (ASICs), etc., such as one or more circuits (communication circuits, etc.) used in wireless devices such as mobile phones, tablet computers, laptop computers, two-way radios, and similar electronic systems. The computer system 140 may also include one or more storage devices that may include one or more memory elements suitable for a particular application, such as main memory in the form of random access memory (RAM), one or more hard drives and / or one or more drives that handle removable media such as compact discs (CDs), flash memory cards, digital video discs (DVDs), etc.The computer system 140 may also include a display device, one or more speakers and a controller which may include a keyboard and / or mouse, trackball, touchscreen, voice recognition device, or any other device which enables a user of the system to input information into and receive information from the computer system 140.

[0069] Some or all of the steps may be performed by a hardware device (or by using a hardware device), such as a processor, microprocessor, programmable computer, or electronic circuit. In some embodiments, one or more of the most critical steps may be performed by such a device.

[0070] Depending on certain implementation requirements, embodiments of the present invention may be implemented in hardware or software. This implementation is feasible using a non-transient recording medium, which is a digital recording medium, etc., that stores electronically readable control signals and cooperates (or can cooperate) with a programmable computer system to carry out each method. Examples include floppy disks, DVDs, Blu-rays, CDs, ROMs, PROMs and EPROMs, EEPROMs, or FLASH memory. Thus, the digital recording medium may be computer-readable.

[0071] Some embodiments of the present invention include a data carrier having electronically readable control signals that can cooperate with a programmable computer system so as to carry out any of the methods described herein.

[0072] Generally, embodiments of the present invention can be implemented as a computer program product comprising program code, which operates to perform one of the methods when the computer program product is executed on a computer. This program code may be stored, for example, on a machine-readable carrier.

[0073] Another embodiment includes a computer program stored in a machine-readable carrier for carrying out any of the methods described herein.

[0074] Therefore, in other words, embodiments of the present invention are computer programs having program code for carrying out any of the methods described herein when the computer program is executed on a computer.

[0075] Accordingly, another embodiment of the present invention is a recording medium (or data carrier or computer-readable medium) containing a stored computer program for carrying out any of the methods described herein when executed by a processor. The data carrier, digital recording medium, or recording medium is typically tangible and / or non-transient. Another embodiment of the present invention is an apparatus, such as those described herein, comprising a processor and a recording medium.

[0076] Therefore, another embodiment of the present invention is a data stream or signal sequence representing a computer program for carrying out any of the methods described herein. The data stream or signal sequence may be configured to be transmitted, for example, over a data communication connection, such as the Internet.

[0077] Another embodiment includes processing means, for example, a computer or programmable logic device configured or adapted to carry out any of the methods described herein.

[0078] Another embodiment includes a computer having an installed computer program for carrying out any of the methods described herein.

[0079] Another embodiment of the present invention includes an apparatus or system configured to transfer (e.g., electronically or optically) a computer program for carrying out any of the methods described herein to a receiver. The receiver may be, for example, a computer, a mobile device, a storage device, etc. The apparatus or system may include, for example, a file server for transferring the computer program to the receiver.

[0080] In some embodiments, a programmable logic device (e.g., a field-programmable gate array) may be used to perform some or all of the functions of the methods described herein. In some embodiments, the field-programmable gate array may cooperate with a microprocessor to carry out any of the methods described herein. Generally, the methods are advantageously carried out by any hardware device. [Explanation of symbols]

[0081] 100,300 systems 110,310 Imaging Systems 112 Illumination optical system 114 Illumination beam 116 Reflector 118 Objective lens 120 Reflector 122 Imaging light beam 124 detectors 126 Eyepiece 130 stages 132 lasers 134,234 laser beams 136 Scanning Mirror 138 Container 140 controllers 142,242 User Interfaces 144 displays 146-key keyboard 148 Computer Mouse 150,250 slides 152,252.1 Microscope sample 200-216 steps 252.2 Processed Microscope Samples 252.3 Processed Microscope Samples 252.4 Uncoated microscope samples 254 Membrane 256 glass slides 258.1, 258.2 Part of the microscope sample 258.3 Extracted part 260 Container 262 Staining solution 264 pipettes 270 Container 272 Liquid coating 274 pipettes 280.1, 280.2 Reference markers 282 Marker 269 ​​Container 292 Removal liquid 294 pipettes 302 pipe

Claims

1. A method, wherein the said method is To obtain a processed microscope sample (252.3), the steps include applying a liquid coating (272) to the microscope samples (152, 252.1, 252.2) (204), Step (210) of generating at least one marker (282) on a portion (258.2) of the processed microscope sample (152, 252.3) using an imaging system (110), To obtain an uncoated microscope sample (252.4), the process includes the step (212) of removing at least a portion of the liquid coating (272) from the processed microscope sample (152, 252.3), To obtain an extracted portion (258.3), the step (216) is to extract a portion (258.2) of the uncoated microscopic sample (152, 252.4) based on at least one of the markers, A method of having.

2. Step (210) of generating at least one of the markers (282) for the portion of the processed microscope sample (152, 252.3) is: Using the imaging system (110), visualize the processed microscope samples (152, 252.3) (206), Based on the visualized processed microscope sample (152, 252.3), identify at least one of the markers (282), Having or being based on these, The method according to claim 1.

3. At least one of the markers (282) is digitally generated and stored for later use in extracting the portion (258.2) of the uncoated microscope sample (152, 252.4). The method according to claim 1 or 2.

4. The portion (258.2) of the uncoated microscope sample (152, 252.4) is extracted using laser microdissection. The method according to claim 1 or 2.

5. A system (100) having the imaging system (110), configured to perform laser microdissection, is used, and the portion (258.2) of the uncoated microscope sample (152, 252.4) is extracted using laser microdissection. The method according to claim 4.

6. The portion (258.2) of the uncoated microscope sample (152, 252.4) is extracted by either cutting the uncoated microscope sample (152, 252.4) with a needle or applying a buffer solution to the uncoated microscope sample (152, 252.4). The method according to claim 1 or 2.

7. The liquid coating (272) comprises polystyrene and xylene. The method according to claim 1 or 2.

8. At least the portion of the liquid coating (272) is removed from the processed microscope sample (152, 252.3) using xylene. The method according to claim 7.

9. The extraction portion (258.3) has a maximum diameter of less than 500 μm, or The extracted portion (258.3) is one of the following: an individual living cell, a cell population consisting of multiple individual cells, an organelle of a living cell, a part of a living cell, or a cell embedding matrix. The method according to claim 1 or 2.

10. The aforementioned microscope sample (152, 252.1) is placed on a slide (150), The slides (150, 250) include a glass slide (256) in which a film (254) is placed on a glass slide, or a frame slide in which a film is placed within a frame slide. The aforementioned microscope sample (152, 252.1) is placed on the film (254), The method according to claim 1 or 2.

11. Before the liquid coating (272) is applied to the microscopic sample (152, 252.1), the microscopic sample (152, 252.1) is processed (202) to obtain the processed microscopic sample (252.3). The method according to claim 1 or 2.

12. The method further comprises the step (208) of generating at least one reference marker (280.1, 280.2) before at least the portion of the liquid coating (272) is removed from the processed microscopic sample (152, 252.3), The method further comprises the step of correlating at least one of the reference markers (280.1, 280.2) with at least one of the markers (282) in order to extract the portion (258.2) of the uncoated microscopic sample (152, 252.4) after at least the portion of the liquid coating (272) has been removed from the processed microscopic sample (152, 252.3). The method according to claim 1 or 2.

13. A system (100, 300) having an imaging system (110), wherein the system (100, 300) is Using the imaging system (110, 310), at least one marker (282) is generated on a portion (258.2) of a processed microscope sample (152, 252.3), which is a microscope sample (152, 252) coated with a liquid coating (272). To obtain an uncoated microscope sample (252.4), at least a portion of the liquid coating (272) is removed from the processed microscope sample (152, 252.3). Based on at least one of the markers (282), the portion (258.2) of the uncoated microscopic sample (152, 252.4) is extracted. The system is configured as follows: (100, 300).

14. The system (100, 300) is further configured to draw a user interface (142, 242), and the system (100) is configured Using the imaging system (110), the processed microscope sample (152, 252.3) is visualized on the user interface (142, 242). The user interface (142, 242) receives input data related to at least one of the markers (282), Based on the input data, at least one of the markers (282) is generated for the portion (258.2) of the processed microscope sample (152, 252.3). It is further structured in the following way: The system according to claim 13 (100, 300).

15. The system (100, 300) is further configured to apply the liquid coating (272) to the microscope sample (152, 252.1, 252.2). The system according to claim 13 or 14 (100, 300).

16. The system (100, 300) is further configured to perform laser microdissection, and the portion (258.2) of the uncoated microscope sample (152, 252.4) is extracted using laser microdissection. The system according to claim 13 or 14 (100, 300).

17. When a computer program runs on a processor, the next step is, A step of correlating at least one reference marker (280.1, 280.2) with at least one marker (282), wherein at least one of the markers (282) is generated for a portion (258.2) of a processed microscopic sample (152, 252.3), and the processed microscopic sample (152, 252.3) is a microscopic sample (152, 252) coated with a liquid coating (272), A step of controlling the system (100, 300) to extract the portion (258.2) from an uncoated microscope sample (152, 252.4), wherein the uncoated microscope sample (152, 252.4) is the processed microscope sample (152, 252.3) from which the liquid coating has been removed. A computer program containing program code for executing something.

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