Apparatus and method for performing laser cutting and collection of target micro-region of tissue section on blade

Abstract

An apparatus for performing laser cutting and collection of a target micro-region of a tissue section on a blade. The apparatus comprises a carrier stage, a blade, a laser cutting system, a control module and a collector, wherein the laser cutting system comprises a laser device, a beam expander, a galvanometer scanner and an objective lens; and by means of adjusting a scanning angle θ of the galvanometer scanner and combining same with the objective lens, a scanned laser is focused to realize the cutting of a target micro-region on a tissue section and the ejection of the target micro-region. Laser microdissection is combined with mechanical sectioning, and non-contact collection of a specific micro-region of a tissue section placed on an inclined blade is achieved under the action of laser edge ejection combined with a dielectrophoretic force, such that the integrity of the morphology of a sample throughout an acquisition process is ensured, three-dimensional positional information of a target cell or cell population is accurately reserved, the cutting efficiency is improved, and non-contact cutting can also be realized to prevent the sample from being contaminated.

Description

Device and method for laser cutting and collecting target micro-area of ​​tissue slices on a blade Technical Field

[0001] The present invention relates to the field of photoelectric imaging, and in particular to a device and method for laser cutting and collecting a target micro-area of ​​a tissue slice on a blade. Background Art

[0002] Spatial omics analysis of heterogeneous cells or cell populations is of great significance in biomedical research. The isolation of specific cells from biological tissues is fundamental for biologists to study heterogeneous cell gene expression and further understand biological functions or disease mechanisms. Therefore, a high-precision, contamination-free method is needed to separate target cells or cell populations from surrounding biological tissues. Laser microdissection is a powerful technique that can image tissue sections and locate target cells, using micro-laser beams to achieve high-precision separation, effectively combining morphological and various molecular analyses.

[0003] However, the existing laser microdissection technology requires that the tissue block be cut into tissue slices by a tissue slicer before being implemented, and then cut using a laser microdissection system. For example, the invention patent with application publication number CN114923755A provides a method for separating Chinese cabbage tissue cells based on laser microdissection technology. After pre-treating the sample, the tissue slices are sliced, and the processed tissue slices are transferred to the stage of the laser microdissection microscope. The target cells are cut off using a laser, and then the sample is collected with a centrifuge tube cap. Usually, when the tissue slices are transferred to the stage of the laser microdissection microscope, the tissue slices need to be processed into an appropriate size and then mounted, and then the laser cutting operation is performed. This makes it difficult to accurately obtain the three-dimensional position information of the target cells or cell groups, and the efficiency of obtaining the target microregion is relatively low, which has a certain impact on the downstream analysis of RNA, protein, etc. in the target microregion. At the same time, when transferring tissue slices and collecting target cells, the sample is easily touched and contaminated. At the same time, in this type of method, the collection of target cells is performed manually. When multiple target cells need to be collected by classification, the collection efficiency is low and the sample is easily contaminated.

[0004] Summary of the Invention

[0005] Therefore, in order to solve the above problems, the present invention provides a device and method for laser cutting and collecting target micro-areas of tissue sections on a blade.

[0006] The present invention is achieved through the following technical solutions:

[0007] The target micro-area laser cutting and collection device of tissue sections performed on a blade includes:

[0008] A carrier table, used for placing the sample tissue block and controlling its movement;

[0009] A blade is tilted and set on one side of a carrier table by a fixture. The tilt angle of the blade is φ (please provide an angle range and explain the reason / beneficial effect of setting this angle). The blade is stationary and is moved relative to the sample tissue block on the carrier table by moving the carrier table to complete mechanical sectioning and obtain a tissue section. After cutting is completed, the tissue section is located on the upper surface of the blade, and the blade serves as the base of the tissue section.

[0010] The laser cutting system includes a laser for providing pulsed laser light, a beam expander for expanding the laser light emitted by the laser, a galvanometer for controlling the scanning angle and scanning speed of the expanded laser light, and an objective lens for focusing the scanned laser beam. The system achieves the cutting of tissue slices and target micro-regions on the tissue slices, as well as the ejection of target micro-regions, by adjusting the scanning angle θ of the galvanometer and focusing the scanned laser light in conjunction with the objective lens.

[0011] The control module is connected to the carrier stage and the laser cutting system signals respectively, and synchronously controls the mechanical slicing and laser cutting;

[0012] The collector is located above the target micro-region on the tissue slice and is used to polarize the bounced target micro-region under the action of the electrostatic field, so as to adsorb the target micro-region into the collector under the action of the dielectrophoretic force.

[0013] Preferably, it further comprises an electrostatic generator, wherein an electrostatic generating head is connected between the electrostatic generator and the collector, and applies static electricity uniformly to the collector.

[0014] Preferably, the area for performing laser scanning cutting is set at the area on the upper surface of the blade between the front end tip of the blade and 10 μm away from the tip.

[0015] Preferably, the beam expander is arranged between the laser and the galvanometer, and includes a first lens close to the laser and a second lens close to the galvanometer.

[0016] Preferably, the objective lens is located obliquely above the blade, and the optical axis of the focused light spot emitted by the objective lens is perpendicular to the upper surface of the blade.

[0017] Preferably, a plurality of collectors are arranged above the target micro-region in a vertical and horizontal array or in a honeycomb pattern. The method for laser cutting and collecting the target micro-region of tissue slices on a blade comprises the following steps:

[0018] S1. placing a sample tissue block on an operating position on a carrier table, imaging the sample tissue block, selecting a target micro-region to be cut, and storing the tissue section position of the sample tissue block and target position information of all target micro-regions;

[0019] S2. Connect the collector to the electrostatic generator and adjust the position of the collector to ensure that the collector is above the target micro-area;

[0020] S3, the laser provides a pulsed laser, which is expanded by a beam expander and scanned by a galvanometer, and then focused by an objective lens into a light spot with energy reaching the set cutting threshold, and waits for the trigger signal to emit light;

[0021] S4. The control module signals the carrier stage to drive the sample tissue block to move horizontally, ensuring that the blade and the sample tissue block move relative to each other to achieve mechanical slicing. When the stage moves to the tissue slicing position stored in step S1, the control module sends a signal to the galvanometer and pulse laser. By adjusting the scanning angle θ and scanning speed v of the galvanometer, a series of target position points on the target micro-area are scanned and ablated one by one. When the last point of a closed curve is ablated, the synchronously generated shock wave pressure is reflected on the blade surface, causing the target micro-area to bounce.

[0022] S5. Turn on the collector, and polarization occurs in the non-uniform electric field generated by the collector, so that the target micro-region is adsorbed into the collector under the action of dielectrophoretic force.

[0023] Preferably, in step S4, the galvanometer adopts a single-axis galvanometer transverse scanning mode, controls the scanning speed by setting the scanning time, and then adjusts the cutting speed v of the pulse laser in the feed direction to ensure that the interval between adjacent ablation points is smaller than the ablation point diameter.

[0024] Preferably, when the diameter of the target micro-region is less than 100 μm, the cutting and ejection of the target micro-region is achieved by focusing the light spot in a direction perpendicular to the upper surface of the blade;

[0025] When the diameter of the target micro-region is greater than 100 μm, a horizontal focused light beam emitted in a horizontal direction is added to increase the ejection force applied to the target micro-region.

[0026] Preferably, when there are multiple target micro-areas and multiple collectors are arranged in an array on the target micro-areas, step S5 further includes adjusting the position of the vacant collector to the top of the next target micro-area for collection after each collector completes collection, until there are no vacant collectors.

[0027] The beneficial effects of the technical solution of the present invention are mainly reflected in:

[0028] 1. Combining laser microdissection with mechanical sectioning ensures the integration of the entire acquisition process of sample morphology from tissue blocks to tissue sections and then to single cells or cell groups (target microregions) in tissue sections. It more accurately retains the three-dimensional position information of target cells or cell groups, which is more conducive to the spatial histological analysis of specific cells. At the same time, compared with traditional laser microdissection methods, there is no need for additional tissue sectioning and patching steps. By tilting the blade and using the blade as the base of the tissue section, and directly performing laser scanning and cutting on the tissue section on the blade surface, it not only improves the cutting efficiency, but also can achieve non-contact cutting to prevent sample contamination.

[0029] 2. The reflective ejection force generated by the focused laser on the tissue slice during the cutting process is combined with the dielectrophoretic force generated on the target micro-area under the non-uniform electric field of the charged collector to achieve non-contact rapid collection of the target micro-area placed on the inclined blade surface. At the same time, when there are multiple target micro-areas, multiple collectors arranged in a vertical and horizontal array or a honeycomb pattern are set above the target micro-area to collect different target micro-areas in turn, further improving the collection efficiency.

[0030] 3. Different laser cutting methods are used according to the different diameters of the target micro-areas, which has higher compatibility for the collection of target micro-areas of different sizes. For target micro-areas with smaller diameters, the cutting and ejection of the target micro-areas can be achieved through the focused light spot in the direction perpendicular to the upper surface of the blade. For target micro-areas with larger diameters, a focused light beam emitted in the horizontal direction is added to the original focused light spot to increase the ejection force, which is convenient for the collection of target micro-areas with large diameters.

[0031] 4. By adjusting the scanning angle and scanning speed of the galvanometer, the position of the ablation points and the distance between the ablation points of the focused light spot on the slice can be controlled. At the same time, by setting the scanning speed, the interval between adjacent ablation points is ensured to be less than the ablation point diameter, thereby ensuring the continuity of the cutting and the complete cutting of the target micro-area. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] FIG1 is a schematic structural diagram of a target micro-area laser cutting and collection device for tissue sectioning performed on a blade;

[0033] FIG2 is a schematic diagram showing the principle of a method for laser cutting and collecting target micro-areas of tissue sections performed on a blade;

[0034] FIG3 is a flow chart of a method for laser cutting and collecting target micro-regions of tissue sections performed on a blade;

[0035] FIG4 is a schematic diagram showing the principle of laser cutting a target micro-area by synchronously combining scanning angle and scanning speed;

[0036] FIG5 is a schematic diagram of a method for cutting and collecting target micro-regions with larger diameters;

[0037] FIG6 is a schematic diagram of a method for collecting target micro-areas in an embodiment with multiple collectors. DETAILED DESCRIPTION

[0038] To more clearly and in detail illustrate the objectives, advantages, and features of the present invention, the following non-limiting description of preferred embodiments is provided for illustration and explanation. This embodiment is merely a typical example of the application of the technical solution of the present invention. Any technical solution formed by equivalent substitution or equivalent transformation falls within the scope of protection claimed by the present invention.

[0039] It is also stated that in the description of the scheme, it should be noted that the terms "upper", "lower", "inside", "outside", etc. indicate orientations or positional relationships based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of description and simplified description, and do not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as a limitation on the present invention.

[0040] Furthermore, the terms "first" and "second" in this solution are used for descriptive purposes only and should not be construed as indicating or implying a ranking of importance or implicitly specifying the number of technical features shown. Therefore, features defined as "first" or "second" may explicitly or implicitly include one or more of such features. In the present invention, "plurality" means two or more, unless otherwise specifically defined.

[0041] The present invention discloses a device for laser cutting and collecting target micro-regions of tissue slices on a blade 110, wherein a sample tissue block 107 is first sliced, and then a target micro-region 109 is separated from the tissue slice 108 by laser cutting. The target micro-region 109 can be a small-sized target micro-region 109, such as a single cell, or a large-sized target micro-region 109, such as a cell cluster.

[0042] As shown in Figures 1 and 2, the device includes:

[0043] The carrier table is used to place the sample tissue block 107 and control its movement. During the entire cutting process, it is necessary to ensure that the sample tissue block 107 is always fixed at the operating position on the carrier table.

[0044] The blade 110 is tiltedly arranged on one side of the carrier table by a clamp. The blade 110 is stationary, and the carrier table is moved to move the blade 110 relative to the sample tissue block 107 on the carrier table to complete mechanical slicing and obtain the tissue slice 108. After the cutting is completed, the tissue slice 108 is located on the upper surface of the blade 110, and the blade 110 serves as the base of the tissue slice 108. The tilt angle of the blade 110 is φ, so the tilt angle of the tissue slice 108 located on the upper surface of the blade 110 is also φ.

[0045] In some embodiments, the blade 110 can be made of a material selected from diamond, glass knife, steel knife and tungsten carbide knife.

[0046] In some embodiments, the position and angle of the blade 110 on the fixture are adjustable. Before mechanical slicing, the position and angle of the blade 110 on the fixture are precisely adjusted to control the uniformity of the overall thickness of the tissue slice 108 during the movement and cutting of the carrier stage. In some embodiments, the inclination angle φ is 30°-45°. When the inclination angle φ of the blade 110 is within this range, the overall thickness of the slice is relatively uniform.

[0047] The laser cutting system includes a laser 101 for providing pulsed laser, a beam expander for expanding the laser emitted by the laser 101, a galvanometer 104 for controlling the scanning angle and scanning speed of the expanded laser, and an objective lens 106 for focusing the scanned laser beam. In one embodiment, a reflector 105 is further provided between the galvanometer 104 and the objective lens 106 for reflecting the beam scanned by the galvanometer 104 to the objective lens 106; wherein the objective lens 106 focuses the scanned laser into a light spot that reaches the cutting threshold, and uses the light spot to complete the cutting of the target micro-region 109. By adjusting the scanning angle θ of the galvanometer 104 and combining with the objective lens 106 to focus the scanned laser, the cutting of the tissue slice 108 and the target micro-region 109 on the tissue slice 108 and the ejection of the target micro-region 109 are achieved.

[0048] The control module 114 is respectively connected to the carrier stage and the laser cutting system signals, and synchronously controls the mechanical slicing and laser cutting, wherein the control module 114 is at least connected to the carrier stage, the laser 101 and the galvanometer 104 signals. During operation, the control module 114 adjusts the moving position of the carrier stage in real time, and synchronously triggers the laser emission of the laser 101 in the laser cutting system, the scanning angle and scanning speed of the galvanometer 104, etc., thereby realizing the cutting and capture of the target micro-area 109.

[0049] The collector 111 is located above the target micro-region 109 on the tissue slice 108 and is used to polarize the bounced target micro-region 109 under the action of the electrostatic field, thereby adsorbing the target micro-region 109 from the bottom of the collector 111 into the collector 111 under the action of the dielectrophoretic force.

[0050] In some embodiments, an electrostatic generator 113 is also provided, and an electrostatic generating head 112 is connected between the electrostatic generator 113 and the collector 111, wherein the electrostatic generator 113 is used to generate static electricity, and the electrostatic generating head 112 is used to uniformly apply static electricity to the collector 111, thereby ensuring that the collector 111 carries static electricity, and polarizes the bounced target micro-region 109 under the action of the electrostatic field, so that it is adsorbed and collected under the action of the dielectrophoretic force; wherein, the optimal distance between the collector 111 and the electrostatic generating head 112 can also be measured through experiments, and the collector 111 and the electrostatic generating head 112 can be adjusted to the optimal distance through a fixing device, so that the collector 111 can store more static electricity, thereby generating a greater electric field strength, and improving the collection efficiency and collection stability. In some embodiments, the collector 111 can be made of a material with a larger dielectric constant, so as to facilitate the storage of more static electricity, generate a stronger electric field, make the target micro-region 109 have a greater polarization intensity, and have a stronger dielectrophoretic adsorption force.

[0051] Among them, after completing the mechanical sectioning, the blade 110 located at the bottom of the tissue slice 108 can be used as a slide to carry the tissue slice 108. In order to ensure the quality of laser cutting, the tissue slice 108 needs to maintain a good fit with the upper surface of the blade 110. The area for implementing laser scanning and cutting is set on the upper surface of the blade between the front tip of the blade and 10 μm away from the tip. Since the front tip of the blade first contacts and cuts the sample tissue block 107, and the blade is set at an angle, after the cutting is completed, the closer the blade is to its front tip, the better the fit with the bottom of the tissue slice 108. Therefore, using the area between the front tip of the blade and 10 μm away from the tip as the area for implementing laser scanning and cutting can better ensure the quality of laser cutting.

[0052] In some embodiments, the laser 101 is an ultraviolet pulse laser 101. The ultraviolet pulse laser 101 has a shorter wavelength, which is convenient for generating a smaller focused spot. When the pulse energy remains unchanged, it can achieve a higher energy density and improve the precision of laser cutting.

[0053] In other embodiments, the laser 101 may also be a picosecond pulse laser 101. Compared with nanosecond pulses, picosecond pulses have nonlinear absorption and can generate stronger shock wave pressure within a short distance at a smaller pulse energy. Therefore, while achieving cutting, the generated shock wave is sufficient to cause the target micro-area 109 to eject the edge at the last point of the laser segmentation, thereby helping to collect the subsequent target micro-area 109.

[0054] The beam expander is arranged between the laser 101 and the galvanometer 104, and includes a first lens 102 close to the laser 101 and a second lens 103 close to the galvanometer 104. In some embodiments, the first lens 102 and the second lens 103 can be made of high-purity fused quartz material and coated with an ultraviolet anti-reflection film to ensure high transmittance and reduce energy loss. The beam expansion factor of the beam expander can be adjusted in real time as needed, which will not be elaborated here.

[0055] The objective lens 106 is located obliquely above the blade 110. In a preferred embodiment, the optical axis of the focused light spot emitted by the objective lens 106 is perpendicular to the upper surface of the blade 110, thereby ensuring the accuracy of laser cutting.

[0056] A plurality of collectors 111 may be arranged on the target micro-area 109 to improve the collection efficiency and flux. In some embodiments, a plurality of collectors 111 are arranged above the target micro-area 109 in a vertical and horizontal array, and the number of collectors 111 arranged in the vertical and horizontal rows is, for example, M×N (1≤M≤10, 1≤N≤10, M and N are both positive integers). During the collection process, a group of target micro-areas 109 can be continuously cut on the slice in advance by laser cutting, and then a plurality of collectors 111 above a group of target micro-areas 109 are synchronously opened for collection. After the collection is completed, the position of the next group of collectors 111 is adjusted, and the cutting and collection of the next group of target micro-areas 109 are performed. In other embodiments, a plurality of collectors 111 may be arranged in a honeycomb shape above the target micro-area 109.

[0057] In some embodiments, a variety of imaging modules coupled to the target micro-area laser cutting and collection device for tissue slicing performed on the blade 110 can also be provided. The imaging module is used to image the surface of the sample tissue block 107 and locate the cutting position of the target micro-area 109. Before the carrier stage drives the sample tissue block 107 to move to the bottom of the blade 110, it first moves to the imaging module for imaging, selects the target micro-area 109 to be cut, and records the position information to facilitate subsequent simultaneous mechanical cutting and laser cutting.

[0058] As shown in FIG3 , the method for laser cutting and collecting the target micro-region 109 of the tissue slice 108 performed on the blade 110 includes the following steps:

[0059] S1. Place the sample tissue block 107 at the operating position on the carrier table, image the sample tissue block 107, select the target micro-region 109 to be cut, and store the position of the tissue slice 108 of the sample tissue block 107 and the target position information of all target micro-regions 109.

[0060] S2 . The collector 111 is connected to the electrostatic generator 113 , and the position of the collector 111 is adjusted to ensure that the collector 111 is located above the target micro-area 109 .

[0061] S3. The laser 101 provides a pulsed laser, which is expanded by a beam expander and scanned by a galvanometer 104 and then focused by an objective lens 106 into a light spot with energy reaching a set cutting threshold, and waits for a trigger signal to emit light.

[0062] S4: The control module 114 signals the carrier stage to drive the sample tissue block 107 to move horizontally, ensuring that the blade 110 and the sample tissue block 107 move relative to each other to achieve mechanical slicing. When the blade 110 moves to the tissue slice 108 position stored in step S1, the control module 114 sends a signal to the galvanometer 104 and the pulsed laser. By adjusting the scanning angle θ and scanning speed v of the galvanometer 104, a series of target position points on the target micro-area 109 are scanned and ablated one by one. When the last point of a closed curve is ablated, the synchronously generated shock wave pressure is reflected on the blade surface, causing the target micro-area 109 to bounce.

[0063] In some embodiments, in step S4, the galvanometer 104 adopts a single-axis galvanometer 104 transverse scanning mode, controls the ablation point position of the pulsed laser by adjusting the scanning angle θ of the galvanometer 104, controls the scanning speed by setting the scanning time, and then adjusts the cutting speed v of the pulsed laser in the feed direction to ensure that the interval between adjacent ablation points is less than the ablation point diameter.

[0064] As shown in FIG4 , a coordinate system is established with the upper left corner of the sample tissue block 107 as the coordinate origin o. Before slicing, its surface is imaged and the position information of the target micro-region 109 is stored. Afterwards, the sample tissue block 107 is cut into a tissue slice 108 by a blade 110, and an inclination angle φ is generated along with the blade 110. At this time, the upper left corner of the tissue slice 108 is used as the coordinate system origin O', and the target micro-region 109 is laser cut on the tissue slice 108. In order to reduce the complexity of transforming the target position information, a single-axis galvanometer 104 is used for horizontal scanning. Only the scanning angle along the x-direction needs to be set. As shown in FIG4 , for two ablation points at the same height in the x-direction, a horizontal scan is performed, scanning from ablation point 1 to 2, and then from ablation point 3 to 4, and then accurately determine the target position of each ablation point for cutting on the target micro-area 109. For the y direction, that is, the feed direction, the scanning speed of the galvanometer 104 is determined by controlling the scanning time, and then the cutting speed v is accurately controlled to ensure that the interval Δn between two adjacent ablation points at different heights (such as ablation point 1 and ablation point 3) is less than the diameter of the ablation point, avoiding the existence of uncut parts between adjacent ablation points, thereby achieving cutting continuity and ensuring the complete separation of the target micro-area 109; in addition, considering that the fitting area between the tissue slice 108 and the surface of the blade 110 is extremely small, this method of lateral scanning combined with mechanical cutting can not only achieve precise synchronization, but also ensure that the laser beam used for ablation is always located on the focal plane of the target micro-area 109, thereby ensuring the cutting quality.

[0065] S5 , turning on the collector 111 , causing polarization in the non-uniform electric field generated by the collector 111 , so that the target micro-region 109 is adsorbed into the collector 111 under the action of dielectrophoretic force.

[0066] When the diameter of the target micro-region 109 is less than 100 μm, the diameter of the target micro-region 109 is small, and the area gravity is also small. The cutting and ejection of the target micro-region 109 can be achieved by focusing the light spot in a direction perpendicular to the upper surface of the blade 110.

[0067] As shown in FIG5 , when the diameter of the target micro-region 109 is greater than 100 μm, the area and gravity of the target micro-region 109 also increase due to the larger diameter of the target micro-region 109. At this time, the shock wave generated by the focused laser beam at the edge of the target micro-region 109 is insufficient to cause the target micro-region 109 to be ejected, and cannot reach the electrostatic field generated by the upper collector 111. Therefore, the vertical focused spot cannot achieve the cutting and ejection of the target micro-region 109. To ensure the smooth collection of the target micro-region 109, a horizontal focused light beam is added to increase the ejection force applied to the target micro-region 109 in the horizontal direction to increase the ejection force. The shock wave pressure generated in the direction perpendicular to the light beam when interacting with the tissue is used to lift the target micro-region 109 with a larger diameter. At this time, due to the large area of ​​the target micro-region 109, the local damage of the horizontal laser beam 116 to the target micro-region 109 in the horizontal direction can be ignored.

[0068] As shown in FIG6 , when there are multiple target micro-areas 109 and multiple collectors 111 are arranged in an array on the target micro-areas 109, step S5 further includes adjusting the position of the vacant collectors 111 to the top of the next target micro-area 109 for collection after each collector 111 completes collection, until there are no vacant collectors 111. After a row of target micro-areas 109 is laser cut, the same row of collectors 111 collects the corresponding target micro-areas 109 in sequence (as shown in FIG6 , 109a, 109b, 109c, etc. are collected in sequence), and the position of the mobile collectors 111 is gradually adjusted during the collection process. After the target micro-areas 109 in the same row are collected, the next row of target micro-areas 109 is cut and collected, and before the next row of target micro-areas 109 is collected, the corresponding row of collectors 111 is moved to the top of the target micro-areas 109 to be collected. In FIG6 , a standard eight-tube collector 117 is used to be compatible with the centrifuge and facilitate subsequent RNA and protein sequencing.

[0069] There are many implementation methods of the present invention, and all technical solutions formed by equivalent transformation or equivalent transformation fall within the protection scope of the present invention.

Claims

1. A device for laser cutting and collecting target micro-regions of tissue sections performed on a blade, characterized in that: Comprising: A carrier stage for placing a sample tissue block and controlling its movement; A blade, which is obliquely arranged on one side of the carrier stage through a fixture. The inclination angle of the blade is φ. The blade is stationary, and the blade moves relative to the sample tissue block on the carrier stage to complete mechanical sectioning and obtain tissue sections. After cutting, the tissue section is located on the upper surface of the blade, and the blade serves as the base of the tissue section; A laser cutting system, including a laser for providing pulsed laser, a beam expander for expanding the laser emitted by the laser, a galvanometer for controlling the scanning angle and scanning speed of the expanded laser, and an objective lens for focusing the scanned laser beam. By adjusting the scanning angle θ of the galvanometer and combining with the objective lens, the scanned laser is focused to achieve cutting of the tissue section and the target micro-region on the tissue section and the bouncing of the target micro-region; A control module, which is signal-connected to the carrier stage and the laser cutting system respectively, and synchronously controls mechanical sectioning and laser cutting; A collector, which is located above the target micro-region on the tissue section, and is used to polarize the bounced target micro-region under the action of an electrostatic field, so that the target micro-region is adsorbed into the collector under the action of dielectrophoresis force.

2. The laser cutting and collection device for the target micro-region of tissue sections performed on the blade according to claim 1, characterized in that: It further includes an electrostatic generator. An electrostatic generating head is connected between the electrostatic generator and the collector, and an electrostatic is uniformly applied to the collector.

3. The target micro-region laser cutting and collection device for tissue sections performed on a blade according to claim 1, characterized in that: The area where laser scanning cutting is implemented is set in the area between the tip of the front end of the blade and 10 μm away from the tip on the upper surface of the blade.

4. The laser cutting and collection device for the target micro-region of tissue sections performed on the blade according to claim 1, characterized in that: The beam expander is arranged between the laser and the galvanometer, and includes a first lens close to the laser and a second lens close to the galvanometer.

5. The laser cutting and collection device for the target micro-region of tissue sections performed on the blade according to claim 1, characterized in that: The objective lens is located obliquely above the blade, and the optical axis of the focused light spot emitted by the objective lens is perpendicular to the upper surface of the blade.

6. The laser cutting and collection device for the target micro-region of tissue sections performed on the blade according to claim 1, characterized in that: Above the target micro-region, a plurality of collectors are arranged in a vertical and horizontal array or honeycomb arrangement.

7. Method for laser cutting and collecting target microregions of tissue sections performed on a blade, characterized in that: Including the following steps: S1. Place the sample tissue block at the operation position on the carrier stage, image the sample tissue block, select the target micro-region to be cut, and store the tissue section position of the sample tissue block and the target position information of all target micro-regions; S2. Connect the collector to the electrostatic generator, and adjust the position of the collector to ensure that the collector is located above the target micro-region; S3. The laser provides pulsed laser. The pulsed laser is expanded by the beam expander and scanned by the galvanometer, and then focused by the objective lens into a light spot with energy reaching the set cutting threshold, and waits for the trigger signal to emit light; S4. The control module signals to control the carrier stage to drive the sample tissue block to move horizontally, ensuring that the blade moves relative to the sample tissue block to achieve mechanical sectioning. When moving to the tissue section position stored in step S1, the control module sends signals to the galvanometer and the pulsed laser. By adjusting the scanning angle θ and scanning speed v of the galvanometer, a series of target position points on the target micro-region are scanned and ablated one by one. When the last point of a closed curve is ablated, the shock wave pressure generated synchronously is reflected on the blade surface to make the target micro-region bounce; S5. Turn on the collector, and polarization occurs in the non-uniform electric field generated by the collector, so that the target micro-region is adsorbed into the collector under the action of dielectrophoresis force.

8. The method for laser cutting and collecting of the target micro-region of the tissue section performed on the blade according to claim 7, characterized in that: In step S4, the galvanometer uses a single-axis galvanometer for lateral scanning. The scanning speed is controlled by setting the scanning time, and then the cutting speed v of the pulsed laser in the feed direction is adjusted to ensure that the interval between adjacent ablation points is less than the ablation point diameter.

9. The method for laser cutting and collecting of the target micro-region of the tissue section on the blade according to claim 7, characterized in that: When the diameter of the target micro-region is less than 100 μm, the cutting and ejection of the target micro-region are realized by the focused light spot in the direction perpendicular to the upper surface of the blade. When the diameter of the target micro-region is greater than 100 μm, a horizontal focused beam is added that is emitted in the horizontal direction to increase the ejection force applied to the target micro-region.

10. The method for laser cutting and collecting the target micro-region of the tissue section performed on the blade according to any one of claims 7-9, characterized in that: When there are multiple target micro-regions and multiple collectors are arranged in an array on the target micro-region, step S5 further includes, after each collector completes collection, sequentially adjusting the position of the vacant collector above the next target micro-region for collection until there is no vacant collector.

Images