Automated method for cutting biological materials

Abstract

The present invention relates to an automated method for cutting biological material from a region of interest (30) within a tissue sample disposed on a planar substrate, using a cutting tool comprising an internal cavity and a scraping blade (125) disposed at an inlet of the internal cavity. The method comprises: ·identifying the boundaries of the region of interest; ·calculating a scraping path for the scraping blade based on the identified boundaries, such that the scraping blade engages all the material within the ROI and scrapes all the material within the ROI; and ·controlling the position of the cutting tool relative to the planar substrate such that the scraping blade follows the calculated scraping path, whereby the calculated scraping path includes one or more individual scraping operations in which the blade (125) is pressed against the planar substrate at a starting location (61, 62) within the identified boundaries and is moved forward through the ROI until it reaches a stopping location (71, 72) and is lifted from the planar substrate. The method of the present invention includes calculating the scraping path such that the stopping location of each individual scraping operation is positioned within the already scraped area within the identified boundaries.

Description

Technical Field

[0001] The present invention relates to an automated method for cutting biological material from a sample disposed on a planar substrate such as a slide glass using a cutting tool having a scraping blade and a tool body into which the scraped material is collected during cutting.

Background Art

[0002] Such a method, and an apparatus for performing the method, are known from International Publication No. 2020 / 054250. The position of the cutting tool relative to the sample is controlled such that the scraping blade selectively engages the material within an identified region of interest (ROI). The blade is brought into contact with the slide glass and pushed forward through the biological material within the ROI to scrape the material and collect it within the tool. Further examples are disclosed in International Publication No. 2022 / 063695, where the blade is disposed in an orifice and suction is applied during cutting, and the sample material separated by scraping is drawn into the orifice and the internal cavity of the tool and collected on the back side of a filter element spanning the internal cavity. The sample material is then transferred to a collection tube by generating a pressure pulse that seals a tube around the tool orifice and discharges the material into the tube.

[0003] To optimize the quality of subsequent analysis, it is important that only the material from the ROI is collected and that the material of the ROI is not left on the slide glass. The ROI material must be torn in order to be collected within the cavity of the tool. At the end of the scraping operation, the blade is lifted from the slide. Any scraped material that remains connected to the material on the slide at the location where the blade is lifted can be drawn out of the cavity. The inventors have found that when using suction, it can be reliably torn by applying a sufficiently large suction force during cutting. However, this has the drawback of increasing the adhesion of the collected material on the back side of the filter and making reliable discharge into the collection tube more difficult.

[0004] Therefore, there is still room for improvement in defining a scraping method that does not require high suction force and overcomes the problem of sample material being left behind on the slide. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] International Publication No. 2020 / 54250 [Patent Document 2] International Publication No. 2022 / 063695 [Overview of the Initiative] [Means for solving the problem]

[0006] The present invention relates to an automated method for cutting biological material from a region of interest (ROI) in a sample placed on a planar substrate using a cutting tool having a scraping blade, the scraping blade being positioned at the opening of an internal cavity within the tool from which the scraped material is collected during cutting. The method is: • A step to identify the boundaries of the area of ​​interest; The steps include: calculating a scraping path for a scraping blade based on identified boundaries, such that the blade engages with all the material in the ROI and scrapes off all the material in the ROI; - A step of controlling the position of the cutting tool relative to the planar substrate so that the scraping blade follows a calculated scraping path. Includes.

[0007] A scraping path comprises one or more individual scraping operations in which the blade is pressed against a planar substrate at a starting point within or on the identified boundary and moves forward through the ROI until it reaches a stopping point and is lifted from the planar substrate. According to the present invention, the scraping path is calculated such that the stopping point of each individual scraping operation is located in an already scraped area within the identified ROI boundary.

[0008] In one embodiment, the calculated scraping path includes at least one scraping operation in which the identified ROI boundary is scraped. In one example, this is a first scraping operation. The blade has a leading edge that moves forward during cutting to scrape material from a planar substrate, such as a glass slide. The blade further has an inner and outer edge that cuts open a scraping lane through the sample material. When scraping the ROI boundary, the position of the blade is controlled so that the outer edge of the blade is at the ROI boundary and used to cut material from the adjacent sample material. The blade follows the identified ROI boundary, generating a first scraping lane. The blade returns to its starting position and is only retracted after advancing beyond the starting position, so that the stopping position is located on the first scraping lane, i.e., the already scraped portion of the ROI boundary.

[0009] The cutting tool is preferably mounted on the tool carrier of the cutting apparatus performing this method so as to rotate around a vertical rotation axis perpendicular to the slide glass on which the sample is placed. The vertical rotation axis preferably coincides with the outer edge of the scraping blade. As understood, the apparatus further comprises actuators that allow control of the blade's movement relative to the planar substrate in the x and y directions during scraping, and actuators that allow lowering and raising the blade.

[0010] In automated cutting methods, accuracy is crucial, but optimization of speed and efficiency is also desirable. The most efficient solution is thought to be to lift the blade just before it returns to its starting position, leaving a distance smaller than the width of the blade, which is expected to leave a small amount of unscraped material. This can then be scraped by rotating the blade so that its leading edge faces the interior area of ​​the ROI, and then initiating a subsequent scraping motion at the unscraped portion of the identified ROI boundary.

[0011] As mentioned above, this creates a risk that the material may be pulled out of the tool's internal cavity when the blade is lifted. Furthermore, the inventors have found that during scraping, the leading edge of the scraping blade, which is thin, for example, 0.03-0.1 mm, but thicker than the typical tissue sample thickness of 0.003-0.01 mm, pushes the material forward. This material pushed forward can also be left behind if the blade is lifted before it reaches an already scraped area, further reducing the accuracy of the scraping process.

[0012] After the boundary is scraped, presumably after being scraped in the first scraping operation, the internal region of the ROI can then be scraped in several subsequent scraping operations, with the blade being brought into contact with the glass slide at a starting point on the generated first scraping lane and moving inward until it reaches a stopping point in a different area of ​​the first scraping lane.

[0013] In the example above, the first scraping operation of the determined scraping path generates a first scraping lane whose outer contour coincides with the ROI boundary.

[0014] In an alternative embodiment that can be advantageously applied when the tissue sample has high tear strength and exhibits flaking during scraping, the first scraping motion begins in the interior region within the identified ROI boundary. Flaking can occur in paraffin-embedded samples where the tumor tissue is dense and the paraffin embedding is locally incomplete. As a result, when the scraping blade is pushed forward through the sample material, the tissue sample may tear at relatively weak points without being precisely cut away from the adjacent material by the outer edge of the blade. Therefore, scraping motions that follow the outer contour of the ROI boundary carry the risk of collecting unwanted sample.

[0015] In an alternative embodiment, the starting point of the first scraping motion is within the ROI boundary. In one example, the blade is programmed to follow a circuit that completely encloses the internal region within the ROI boundary. As before, the blade is lifted only after returning to the starting point and passing through it. In a further example, the first scraping motion is programmed so that the blade scrapes the entire internal region of the ROI and then lifts from the slide at the already scraped portion of the aforementioned internal region.

[0016] In subsequent scraping operations, the blade starts on the ROI boundary, is substantially parallel to the local contour of the boundary, and is oriented so that its leading edge faces inward into the ROI. The stopping point of each subsequent scraping operation is preferably on the first scraping lane or on a previously generated scraping lane.

[0017] In embodiments where the first scraping operation defines a first scraping lane that bypasses the ROI area, the stopping point is positioned on the first scraping lane after the circuit is completed and the blade has returned to the starting point and passed through it for a predetermined distance. The predetermined distance is based on the known width of the blade; in the example where the blade width is 1.0 mm, the predetermined distance is between 20-150% of the blade width and is preferably greater than the positioning tolerance of the blade of the device used. In an example of a typical cutting device, the positioning tolerance is 0.1 mm.

[0018] In a further development, the scraping pathway includes a first scraping operation initiated within the ROI, and subsequent scraping operations in the scraping pathway are calculated only after it has been determined whether the tissue sample being cut exhibits flaking.

[0019] An apparatus used to perform automated cutting according to the present invention typically comprises an imaging system used to obtain an image of a slide. The system includes an imaging sensor having a position relative to a pre-calibrated scraping blade.

[0020] The method can further advantageously include the step of capturing an image of at least a portion of the first scraping lane generated during the first scraping operation, and the step of processing the captured image to determine whether the outer edge / boundary of the generated scraping lane portion coincides with the programmed path of the blade outer edge. It is preferable that the portion of the first scraping lane that functions as a "test lane" follows a straight line.

[0021] If it is determined that the boundary of the scraped portion described above is cleanly cut off from the adjacent material, i.e., the scraped lane does not contain sample material, then the scraping path is calculated to remove the remaining ROI sample material from the slide, whereby one of the subsequent scraping operations is programmed to scrape the outer contour of the identified ROI boundary, as described for the first embodiment.

[0022] As a result of image processing, when the boundary of the scraped portion deviates from the programmed path of the blade outer edge, the scraping path calculated to remove the remaining ROI material includes subsequent scraping operations where the blade is positioned on the identified ROI boundary in a direction parallel to the local contour and moved inward, as described above.

[0023] The step of processing the captured image can preferably include detecting the boundary of the scraped portion and comparing the detected boundary with the programmed path, and calculating the deviation distance if there is a deviation distance between them. The threshold value may be set based on, for example, the average absolute deviation and / or the standard deviation of 0.1 mm, whereby if the threshold value is exceeded, it is determined that there is a deviation from the programmed path.

[0024] The scraping path is calculated based on the identified ROI boundary. Typically, the boundary includes local contour portions that intersect at an angle, and the method includes estimating the lengths of the intersecting portions and the angles at which they intersect. Advantageously, the scraping path is calculated according to one or more additional rules that improve the accuracy, speed, and efficiency of the cutting.

[0025] The leading edge of the scraping blade has a maximum effective width when oriented in a direction perpendicular to the forward translation direction. This creates a scraping lane of the corresponding width as the blade moves forward through the ROI material. The effective width can be reduced by changing the angle at which the leading edge of the blade faces with respect to the translation direction.

[0026] An additional rule that can be applied when calculating the scraping path is that if the blade width does not exceed the local width of the ROI, the leading edge of the blade is oriented in a direction perpendicular to the translation direction. Thus, the step of calculating the scraping path can include estimating the local width of the ROI to be scraped in a particular scraping operation. When the local width is wider than the maximum blade width, the leading edge of the blade is oriented in a direction perpendicular to the translation direction. This maximizes the efficiency of a particular operation. When the estimated local width is narrower than the maximum blade width, the blade is rotated about the vertical axis to adjust the orientation of the leading edge of the blade with respect to the translation direction and reduce the effective width as needed.

[0027] An additional rule that can be applied is that the operation of the blade does not stop at the local contour of the identified boundary with the leading edge parallel to and facing the local contour. This prevents the ROI material from being pushed outside the boundary.

[0028] When the identified boundary includes a corner, i.e., when it includes first and second local contour portions that intersect at an angle of 95-100 degrees or less, the intersection angle is estimated. In embodiments where the ROI boundary is scraped using the outer edge of the blade, a further rule that can be applied when calculating the relevant scraping motion of the scraping path is that the blade is rotated backward around a vertical rotation axis coincident with the outer edge before reaching the corner. As described above, the default orientation angle of the blade to the first local contour portion of the ROI boundary is 90 degrees, i.e., the leading edge is perpendicular to the first local contour portion. It is preferable to stop the forward movement of the blade before the inner edge of the blade reaches the second local contour portion, adjust the angle at which the blade is directed to the first local contour portion so that it is smaller than the estimated corner angle, and then continue the forward movement.

[0029] The subsequent scraping operations following the calculated scraping path are advantageous in generating straight, parallel scraping lanes. This helps optimize the speed and efficiency of the cutting process. For greater precision, the scraping path may be determined such that adjacent scraping lines slightly overlap each other, for example, by 5-20% of the lane width. As a further development, the method may include identifying the direction in which the region of interest has the maximum length, or the direction in which a local contour portion of the ROI boundary has the maximum length. The scraping path is then calculated so that scraping lanes are executed parallel to the identified maximum length direction.

[0030] The scraping blade of the cutting tool used in the method of the present invention has, for example, a straight leading edge having a width of 1.0 mm. Any of the cutting tools disclosed in International Publication No. 2022063695 can be used in the method of the present invention, and the contents of this document are incorporated by reference. The disclosed tools include a filter element extending through an internal cavity such that scraped ROI material drawn into the tool is captured on the back surface of the filter element. The scraped material is transferred to a collection tube by airtightly positioning a tube around the distal end of the cutting tool and generating a pressure pulse to discharge the material.

[0031] It is preferable that transfer be performed after all of the ROI material has been scraped off the slide. Depending on the size of the ROI, the filter may become somewhat clogged before all of the material is collected, thereby reducing the effectiveness of suction during cutting. For certain tools with filters, a threshold can be defined corresponding to the surface area of ​​the scraped material that would cause clogging. The scraped surface area is determined by the known blade width and the length advanced during scraping. A corresponding threshold may be determined for the length advanced by the blade. When the threshold is reached, it is beneficial to interrupt scraping and perform an intermediate transfer action.

[0032] Therefore, the method may further include calculating the length advanced by the blade during scraping and, after the completion of the scraping operation, interrupting the scraping path when the calculated length reaches a predetermined threshold. The tool can be withdrawn only after the blade has entered an area that has already been scraped, and can then move to a collection stage where, for example, the scraped material is transported. The tool is then returned to the slide and the remainder of the calculated scraping path is executed.

[0033] Those skilled in the art will understand that two or more of the above-described embodiments, models, and / or aspects of the present invention may be combined in any way deemed useful.

[0034] Next, the present invention will be further described with reference to the embodiments described below. [Brief explanation of the drawing]

[0035] [Figure 1] This is a diagram of an example of a tissue sample containing a region of interest to be cut for analysis. [Figure 2a] This is a schematic diagram of an apparatus for carrying out the automated cutting method according to the present invention. [Figure 2b]This is a cross-sectional side view of an example of a cutting tool that engages with a tissue sample on a slide, which can be used in the method of the present invention. [Figure 2c] This is a cross-sectional side view of a further example of a cutting tool that can be used in the method of the present invention. [Figure 3a] This is a schematic diagram of a part of the scraping operation that does not follow the method of the present invention. [Figure 3b] This is a schematic diagram of a part of the scraping operation that does not follow the method of the present invention. [Figure 4a] This is a diagram of the tissue sample shown in Figure 1, after the first and second scraping operations have been performed according to the first embodiment of the method of the present invention. [Figure 4b] This diagram shows an example of a portion of an ROI boundary that includes a corner, illustrating the different positions of the scraping blade as it passes through the corner. [Figure 4c] This diagram shows an example of a portion of an ROI boundary that includes a corner, illustrating the different positions of the scraping blade as it passes through the corner. [Figure 5] This diagram illustrates another example of an ROI boundary that includes a relatively narrow section, showing the different positions of the scraping blade before entering the narrow section, while inside the narrow section, and after exiting the narrow section. [Figure 6] This is a diagram of the tissue sample from Figure 1 after the first and second scraping operations have been performed according to a second embodiment of the method of the present invention. [Modes for carrying out the invention]

[0036] It should be noted that items with the same reference number in different figures have the same structural features and functions, or the same signals. If the function and / or structure of such items is described, there is no need to repeat those descriptions in the detailed description.

[0037] Pathological diagnostic studies of biological materials such as tissues and cells form the basis for many treatment decisions, particularly in oncology. For example, genome-based testing is performed to inform treatment options for individual patients diagnosed with cancer. Biological material / tissue can be obtained from a biopsy and then, for example, embedded in paraffin, cut into thin slices, and fixed on a glass slide. These thin slices are called tissue samples. Other methods for obtaining and preparing biological material are known. An example of a tissue sample arranged on a glass slide 10 is shown in Figure 1.

[0038] The tissue sample 20 has a region of interest (ROI) 30 containing material within the boundary 35 of the ROI to be examined. This material must be physically separated from the slide and the unwanted sample material 25 outside the boundary. The ROI and associated boundary 35 can be identified by staining, or the pathologist may mark them on a reference slide after microscopic analysis. The ROI can also be identified through processing of digital images of the sample. Once the ROI is identified, the material is removed / cut from the slide and then transferred to the analysis construct. Typically, the material is transferred to a collection tube, after which steps of the sample preparation process, such as cell lysis, purification, and amplification, as well as any further necessary processing steps, are performed. As is understood, the reliability and accuracy of the analysis are optimized by ensuring that only material cut from the ROI is present, but also by maximizing the amount of ROI material collected.

[0039] This invention defines an automated method for cutting biological material from an ROI in a sample placed on a planar substrate such as a glass slide, using a tool having a scraping blade that is pressed against a slide and moved forward through the sample to scrape off the ROI material and collect it in a cavity within the tool. In the first step, the boundaries of the ROI are identified. Then, based on the identified boundaries, a scraping path for the blade is calculated. The position of the tool is controlled relative to the slide so that the scraping blade follows the calculated scraping path. In the method of this invention, the scraping path is calculated according to one or more rules designed to optimize the accuracy of the cutting by ensuring that no material from the region of interest is left behind on the glass slide.

[0040] Figure 2a shows a schematic arrangement of the configuration for carrying out the method of the present invention.

[0041] The apparatus 100 includes a platform 110 for supporting a glass slide 10 on which a tissue sample, such as the sample 20 shown in Figure 1, is placed. The apparatus includes a cutting tool 120 having a scraping head / scraping blade 125. The cutting tool 120 is preferably fixedly connected to a robotic stage 130 which has a series of actuators for performing the movements required during cutting. The robotic stage 130 preferably includes the following: - A rotary actuator that rotates the tool around a vertical axis R perpendicular to platform 110. - XY stage for translational motion, and - Z-stage for vertical motion. The Z-stage is equipped with hinge bearings for position control, ensuring that a constant and precise downward force is applied during cutting.

[0042] To be understood, it is also possible that one or more of the required actuators can be connected to platform 110.

[0043] The apparatus further comprises an imaging system 150 that can be used to identify the boundaries of the ROI. The imaging system includes an imaging sensor, and the position of the scraping blade relative to the sensor is pre-calibrated. The apparatus further comprises a processor for calculating an appropriate scraping path based on the identified boundaries, and a controller 140 that receives the calculated scraping path and controls the robotic stage accordingly, so that the scraping blade of the cutting tool is moved relative to the platform to scrape all the material within the identified ROI. Preferably, the apparatus further comprises a vacuum generator 160 that generates an uplifting air flow in the scraping blade 125 so that the scraped material is drawn into the internal cavity of the tool.

[0044] A cross-sectional side view of the tool is shown in Figure 2b. The illustrated example tool 120 comprises a thin-walled tube having an internal cavity 128 and a scraping head at the entrance to the internal cavity. The front or leading edge of the tube functions as a scraping blade 125 of the scraping head. The scraping blade has a bottom 125a that is in contact with the upper surface of the slide 10. The leading edge of the scraping blade is also in contact with the tissue sample 20 in a scraping zone formed by the bottom 125a and the opposing sides 125b, 125c of the tube in the contact area. Thus, when relative motion occurs in the X direction, material from the tissue sample is scraped from the slide 10 into the cavity 128 of the cutting tool. In the scraping zone, portion 125b defines the outer edge of the scraping blade; portion 125c defines the inner edge of the scraping blade, thereby the leading edge acts only as a whole. The scraping lane cut into the tissue sample 20 has a width w in the scraping region that corresponds to the effective width of the scraping blade between the outer edge 125b and the inner edge 125c.

[0045] Further examples of cutting tools that can be attached to a cutting apparatus carrying out the method of the present invention are shown in Figure 2c. The tool 220 comprises a body portion 226 formed from a single piece through which an internal cavity extends. A portion of the internal cavity 228 tapers in diameter toward the tool orifice 223. Further portions of the internal cavity are formed by conical recesses 450 adapted to connect the tool, precisely aligned with correspondingly shaped conical projections on the tool carrier of the cutting apparatus.

[0046] The tool body portion 226 further includes a seating portion 230 for mounting the filter element 229 in the axial direction. The tool's scraping blade 225 is provided on a second portion 227 joined to the body portion 226 and extends obliquely with respect to the longitudinal central axis of the tool's internal cavity. In the illustrated example, the second portion 427 may be overmolded onto the scraping blade 225 and irreversibly joined to the body portion 226 by form fit, adhesive bonding, or other suitable bonding method. In other examples, the entire tool body is overmolded onto the scraping blade.

[0047] The scraping path calculated for a particular scraping blade and a particular ROI involves several separate scraping operations in which the scraping blade is brought into contact with the slide glass at the starting point and moved relative to the slide until it reaches the stopping point and is lifted off the slide. As shown in Figure 3a, as the scraping blade 125 is moved forward through the ROI material, the material can be separated in the form of a ribbon 30a that is guided or sucked into the tool cavity during cutting. If the blade is lifted with the leading edge of the ribbon 30a still attached to the adjacent sample material, the ribbon of material can be pulled out of the cavity, as shown in Figure 3b. The front / leading edge of the scraping blade also pushes the sample material forward during cutting, which creates an accumulation 30b or "mound" of ROI material in front of the blade. This material may be left behind if the accumulation in front of the blade is not taken into account in the scraping path.

[0048] According to the present invention, the scraping paths are calculated so that each individual scraping operation ends at an already scraped location within the identified ROI boundary.

[0049] In a preferred embodiment, the scraping path begins with a first scraping motion in which the identified ROI boundary 35 is scraped away. Referring to Figure 4a, which shows the same tissue sample as depicted in Figure 1, the scraping blade 125 is brought into contact with the slide at the starting location 61 of the identified boundary. The boundary typically includes intersecting local contour portions, and one possible way to select a starting location is to identify the longest portions of the local contour portions and start at those locations. The blade is oriented so that its leading edge is substantially perpendicular to the boundary at that location, and further positioned so that its outer edge coincides with the outer contour of the boundary. The blade is then moved forward in a first translational motion indicated by arrow a. The blade then follows the boundary in several subsequent translational motions, thereby keeping the leading edge perpendicular to the local contour of the boundary as much as possible, and the outer edge is used to cut the ROI material from the unwanted sample material 25. When navigating corners with angles between 95 and 100 degrees, it is typically necessary to change direction, as illustrated with reference to Figures 4b and 4c.

[0050] In Figure 4a, the blade 125 is shown at various locations in the subsequent translation as the first scraping lane 51 following the boundary is generated. In the final translational motion, as indicated by arrow q, the blade returns to the starting position 61, moves beyond it, for example by 50% of the blade width, and is lifted from the slide at the stopping position 71, which is an area that has already been scraped.

[0051] Figures 4b and 4c show examples of ROI boundaries having local contour portions 35a, 35b that intersect at angles less than 90 degrees, thus generating a corner 38. Furthermore, the blade 125 is shown at several different positions 1-7 as it passes through the corner 38 during the first scraping operation.

[0052] The step of identifying the ROI boundary 35 preferably includes estimating the angles of all corners within the boundary. In the illustrated example, corner 38 has an angle of 70 degrees. As the blade approaches the corner and moves forward between position 1 and position 2, the blade 125 is oriented perpendicular to the local contour 35a, and the outer edge of the blade 125b coincides with the outer edge of the local contour. This forward movement is preferably stopped at position 2 before the inner edge 125c of the blade meets the local contour 35b. This prevents the accumulated ROI material (such as schematically shown in Figure 3b) from being pushed outside the boundary. The blade is then rotated backward to position 3 by, for example, an angle of 30 degrees around a vertical rotation axis R (see Figure 2) that coincides with the outer edge of the blade 125b. At position 3, the blade has an orientation angle θ with respect to the local contour portion 35a, which is smaller than the estimated corner angle. Next, the blade is moved forward to position 4, where its outer edge is approximately at the intersection of contour portions 35a and 35b. As shown in Figure 4b, the blade is then rotated backward again to position 5, and then moved forward at an angle to position 6, where the blade is perpendicular to the local contour 35b, and the outer edge 125b of the blade coincides with the local contour 35b. The blade is then moved linearly forward to position 7.

[0053] During individual scraping operations, the blade remains in contact with the slide. While moving continuously or in stages, the blade can optionally be angled around the vertical axis of rotation while the linear motion is stopped.

[0054] After a first scraping lane 51 with an outer contour coinciding with the ROI boundary is generated, the interior of the ROI 30 can then be scraped in several subsequent scraping operations. Referring again to Figure 4a, a second scraping lane 52 can be generated by bringing the blade into contact with the slide 10 at a second starting location 62 on the first scraping lane 51 and moving the blade forward to a second stopping location 72 on the opposite side of the first scraping lane. The subsequent scraping operations are preferably parallel to each other and may slightly overlap each other by, for example, 10-20% of the blade width. The number of additional scraping operations depends on the blade width and the size of the area to be scraped.

[0055] The most efficient cutting method is to move the blade forward with the leading edge perpendicular to the translational direction so that the blade width is maximized. This is not always possible, for example, when going around a corner. It may also be necessary to change the blade's orientation to address the local width of the ROI.

[0056] Figure 5 shows a further example of an ROI 530 with a boundary 535. A portion of the ROI 530a has a local width s that is narrower than the maximum effective width w of the scraping blade 125. The blade is also shown in this case at several different positions 1-7 during the scraping operation. As the blade moves from position 1 to 2, the local width of the ROI is wider than w, and the blade is oriented so that its leading edge is perpendicular to the translational direction. As the blade advances forward to position 3, it is rotated backward around a vertical rotation axis that coincides with the outer edge 125b of the blade, and as the blade enters the narrow portion 535a, the orientation angle is further adjusted to reduce the effective width of the blade, and it advances to position 4. As shown in the figure, as the blade advances further to positions 5, 6, and 7, the orientation angle is adjusted based on the local width of the ROI.

[0057] Generally, the most efficient way to scrape a specific ROI is to start from the ROI boundary, as described above with respect to Figure 4a, and then scrape the interior area with several subsequent scraping movements. Depending on the type of tissue sample, for example, with high tear strength tissue samples as described above, there is a risk of flaking if the outer edge of the blade is used at the ROI boundary to cut the ROI from the adjacent sample material. In this case, it is prudent to use different types of scraping paths, which involve several scraping movements in which the blade is placed at the ROI boundary, with its leading edge substantially parallel to the local contour, and moved forward toward the interior area of ​​the ROI. If the length of the local contour portion is shorter than the width of the blade, the blade is not moved forward but is simply pressed against that portion to cut away the adjacent unwanted material. The cut portion can then be scraped away with subsequent scraping movements in which the outer edge of the blade follows the local contour.

[0058] According to the present invention, each scraping operation ends after the blade has reached an area that has already been scraped, i.e., the blade is lifted from the slide glass. The first scraping operation of the scraping path is then programmed to start from an internal region of the ROI.

[0059] Examples of possible first and second scraping lanes are shown in Figure 6. The blade is placed on the slide 10 at a starting point 661 inside the identified ROI boundary 635 and moved forward to generate a continuous first scraping lane 651 that completely encloses the region 630a of ROI material within the boundary. For simplicity, a substantially rectangular scraping lane 651 is depicted, but other enclosed shapes are also possible. The blade returns to the first starting point 661 and is moved in a “counterclockwise” direction until it passes through it, and then lifted at a stopping point 671. In the second scraping operation, the blade is placed at the ROI boundary 635 at a second starting point 662 and moved forward to generate a second scraping lane 652. The second scraping operation ends at a stopping point 672 which is part of the first scraping lane 681. In the subsequent scraping operation, the blade is returned to a new starting point on the ROI boundary and moved inward until it reaches the previously generated scraping lane. The process stops once all ROI material has been collected.

[0060] Examples, embodiments, or optional features, whether indicated as non-limiting or not, should not be understood as limiting the claimed invention. It should be noted that the embodiments described above are illustrative rather than limiting, and that those skilled in the art can design many alternative embodiments without departing from the scope of the appended claims.

[0061] In the claims, no reference numerals placed in parentheses should be construed as limiting the claims. The use of the verb "comprise" and its conjugations does not preclude the existence of elements or steps other than those described in the claims. The article "a" or "an" preceding an element does not preclude the existence of multiple such elements. The present invention can be implemented by hardware comprising several distinct elements and a appropriately programmed computer. In device claims listing several means, some of these means may be embodied by a single identical hardware item. The mere fact that a particular measure is described in different dependent claims does not indicate that these measures cannot be combined and used advantageously. [Explanation of Symbols]

[0062] 1-7 Position of the scraping blade at different moments during the scraping motion 10 microscope slides 20 Tissue samples 25 Unnecessary tissue materials 30. Area of ​​Interest (ROI) 30a Ribbon-shaped tissue material cut by scraping action 30b Accumulation of tissue material pushed forward by the scraping motion 35, 535, 635 ROI boundaries 35a, 35b Intersection of ROI boundaries 38. Corners within ROI boundaries (sections where intersections occur at angles of 90 degrees or less) 51, 651 First scraping lane 52, 652 Second scraping lane 61, 661 Starting point of the first scraping lane 62, 662 Starting point of the second scraping lane 71, 671 Stopping point of the first scraping lane 72, 672 Stopping point of the second scraping lane 100 cutting equipment 110 Platform for supporting glass slides 120, 220 Cutting Tools 125, 225 Cutting Tool Scraping Blades 125a Bottom of the tubular scraping blade 125b Outer edge of the scraping blade 125c Inner edge of scraping blade 128, 228 Internal cavities of cutting tools 130 Robotic stage for adjusting the blade position relative to the glass slide 140 controllers 150 Imaging Systems 160 Vacuum Generator 223 Cutting tool orifice 226 Cutting tool body 427 Second tool body 229 filter elements 230 Seat for holding the filter element 530, 630a ROI section a. The first translational motion of the scraping action q Final translational motion of the scraping action R Cutting Tool Vertical Rotation Axis s Local width of a portion of the ROI w width of scraping blade / scraping lane θ: Orientation angle of the blade relative to the local contour.

Claims

1. An automated method for cutting biological material from a region of interest (30, 530, 630) in a tissue sample (20) placed on a planar substrate (10), using a cutting tool (120, 220) comprising internal cavities (128, 228) and scraping blades (125) positioned at the entrances of the internal cavities, - A step to identify the boundaries of the region of interest (35, 535, 635), - A step of calculating a scraping path for a scraping blade based on identified boundaries, wherein the scraping blade engages with all the material in the region of interest and scrapes all the material in the region of interest, - A step of controlling the position of the cutting tool relative to the planar substrate so that the scraping blade follows the calculated scraping path. This includes, and by doing so, In an automated method, the calculated scraping path includes one or more individual scraping operations, in which the blade (125) is pressed against the planar substrate (10) at a starting location (61, 62, 661, 662) within an identified boundary and moves forward through the ROI until it reaches a stopping location (71, 72, 671, 672) and is lifted from the planar substrate, A method characterized in that the scraping path is calculated such that the stopping point of each individual scraping operation is located in an already scraped area within an identified boundary.

2. The method according to claim 1, wherein the cutting tool is mounted to rotate around a vertical rotation axis (R) that coincides with the outer edge (125b) of the scraping blade.

3. The method according to claim 2, comprising a scraping operation in which a calculated scraping path generates a scraping lane (51, 651) that completely encloses at least a portion of the region of interest, thereby returning the blade to the starting position (61, 661) and controlling the position of the blade (125) to follow the circuit continuing beyond the starting position, such that a stopping position (71, 671) is positioned in the already scraped portion of the generated scraping lane.

4. The method according to claim 3, wherein the stopping point is 20–150% of the width of the scraping blade away from the starting point, and this distance is greater than the positioning tolerance of the cutting device to which the cutting tool is mounted.

5. - The outer contour of the generated scraping lane (51) coincides with the identified boundary (35). The method according to claim 3, wherein the position of the scraping blades (125, 225) is controlled during the scraping operation so that the outer edge (125b) of the scraping blade follows the identified boundary (35) and is used to cut the material at the boundary away from the adjacent material (25) of the sample (20).

6. The method according to claim 5, wherein the boundary identification step includes identifying local contour portions (35a, 35b) of the boundary that intersect at a corner (38) having an angle of 100 degrees or less, and estimating the intersection angle, and the position of the scraping blades (125, 225) is controlled during the scraping operation such that the leading edges of the scraping blades (125, 225) are rotated backward around a vertical rotation axis (R) so that they are directed at an angle (θ) smaller than the estimated intersection angle before reaching the corner (38).

7. The method according to claim 5, wherein a first scraping operation is a scraping operation in which the outer edge of the blade follows an identified boundary (35, 535), and the interior of the region of interest (30) is scraped by several subsequent scraping operations.

8. The method according to claim 1, wherein the first scraping operation and the associated first scraping lane (651) are located inside the identified boundary (635).

9. The method according to claim 8, wherein the calculated scraping path includes several subsequent scraping operations having a starting location (662) on an identified boundary, the leading edge of a blade (125, 225) is oriented parallel to the local contour of the boundary (635) at the corresponding starting location, thereby moving forward toward the interior of the region of interest until the blade reaches a stopping location (672) on a first scraping lane (651) or on a previously generated scraping lane.

10. The method according to claim 7, wherein a subsequent scraping operation generates mutually parallel scraping lanes (52, 652).

11. The method according to claim 7, wherein adjacent scraping lanes overlap each other by an amount equivalent to 5-20% of the width (w) of the scraping blade (125).

12. Identifying the direction in which the region of interest has the maximum length, or the direction in which the local contour portion of the ROI boundary (35) has the maximum length, and calculating the scraping path so that a scraping lane parallel to the identified maximum length direction is executed. The method according to claim 10, further comprising:

13. The method according to claim 1, wherein the step of calculating the scraping path further includes estimating the local width (s) of the region of interest to be scraped in a particular scraping operation, and orienting the leading edge of the scraping blade (125, 225) perpendicular to the translational direction, provided that the blade width (w) is not less than the estimated local width.

14. The cutting tool is attached to an apparatus comprising an imaging system (150) and a processor, and the method further includes the steps of capturing an image of at least a portion of a first scraping lane (651) and processing the captured image, wherein the processing step is - Detect the boundary of the first scraping lane section, - The detected boundary is compared with the programmed path of the corresponding edge (125a, 125b) of the scraping blade (125, 225), - To determine whether the detected boundary deviates from the programmed path, and if it is determined that a deviation has occurred, - If the calculated scraping path includes several subsequent scraping operations as defined in claim 9, and it is not determined that a deviation has occurred, - The calculated scraping path includes subsequent scraping operations as defined in claim 5. The method according to claim 8, further comprising:

15. - The cutting tool (220) includes a filter element (229) that extends through the internal cavity (228) of the scraping tool. A vacuum generator (160) is connected to the cutting tool to create an upward airflow at the inlet (223) of the tool cavity that draws the scraped material into the tool and holds it on the back surface of the filter element (229). The method includes the step of calculating the distance the scraping blade (225) has traveled during scraping, The method according to any one of claims 1 to 14, further comprising interrupting the scraping path if, after the completion of the scraping operation, the calculated distance exceeds a predetermined threshold, wherein the threshold corresponds to the maximum surface area of ​​the sample material that can be held on the back surface of the filter element (229) without adversely affecting the suction performance, and restarting the scraping path after the scraped material has been transferred to the collection tube.

16. A cutting tool having internal cavities (128, 228) and a scraping blade (125) positioned at the entrance of the internal cavities, - A platform (110) for supporting a flat substrate (10) on which a tissue sample (20) is placed. - Imaging system (150) for identifying the boundaries (35, 535, 636) of the region of interest (30) within the tissue sample. A processor for calculating the scraping path that will engage the scraping blades (125, 225) with all of the sample material within the identified boundaries. A series of actuators (130) for moving the cutting tool in the X, Y, and Z directions relative to the platform (110), and for rotating the tool around a vertical rotation axis (R), A controller (140) that receives a calculated scraping path and controls a series of actuators (130) so that the scraping blade follows the calculated scraping path. Equipped with, A cutting device (100) characterized in that a processor is configured to calculate a scraping path such that automated cutting is performed according to the method described in any one of claims 1 to 14.

17. The cutting apparatus (100) according to claim 16, further comprising a vacuum generator (160) for generating an updraft at an inlet (223) to an internal cavity (228) of the tool, the tool further comprising a filter element (129) extending through the internal cavity, and the apparatus being configured to perform the method of claim 15.

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