Laser microdissection collection system

Abstract

The utility model discloses a laser microdissection collection system which comprises an objective table used for placing tissue slices; the laser cutting system comprises a laser device and a cutting system, the cutting system is arranged below the objective table and converges pulse laser emitted by the laser device into a light spot right opposite to the tissue slice, and the light spot cuts a target micro-area from the tissue slice; the collector is located above the objective table, and the collecting side of the collector directly faces the target micro-area; the light source is arranged above the objective table and is used for irradiating the tissue slice to facilitate imaging; the static electricity generation system comprises a static electricity generation head and a static electricity control system, the static electricity generation head is arranged between the collector and the light source, the static electricity control system controls the static electricity generation head to generate static electricity, and the target micro-area is adsorbed into the collector through the static electricity effect. According to the scheme, the electrostatic generation head is arranged above the collector, the electrostatic generation head is directly controlled to generate static electricity to adsorb and collect the target micro-area, and efficient and pollution-free collection is achieved.

Description

Technical Field

[0001] The utility model relates to the technical field of cell cutting and collection, in particular to a laser microcutting and collection system. Background Art

[0002] In life science research, it is necessary to conduct gene and protein studies on specific cells (groups) within a tissue. Laser micro-cell dissection devices or systems are high-end equipment developed to meet this need. They can isolate specific or characteristic cells (groups) from diverse tissues and collect the separated cells (groups) for research, thus preventing contamination of experimental specimens by other cells, bacteria, or other impurities, making gene and protein analysis more accurate and specific.

[0003] The core technology that distinguishes various laser micro-cell cutting systems is the different collection methods or devices for non-contamination collection of cut cells.

[0004] ① Gravity collection: U.S. patents US7807108B2 and US6907798B2 disclose cell collection devices, which are placed directly below the sample. After laser cutting, the cells (groups) to be collected automatically fall into the cell collector of the device under the action of gravity.

[0005] Disadvantages: The gravity collection method is easily affected by micro-perturbations in the air and is not easy to collect cut samples with a small area, which results in a low collection efficiency and success rate for cut samples with a small area.

[0006] ② Laser pressure ejection collection: WO97 / 29355A discloses a method for collecting cells after excision using laser ejection technology. This method can be used in an inverted laser microcytotomy system. The method defocuses the focused laser beam used for excision, lowering the focal point below the sample surface. A pulsed laser beam is then emitted, using the pressure generated by the laser pulses to eject the excised tissue into the cells for collection.

[0007] Disadvantages: The laser pressure ejection collection method requires adding a high-power laser beam to the cut sample. The inherent characteristics of the laser beam can easily damage the activity of biological tissue, thereby affecting subsequent further research and analysis. In addition, this method is not suitable for the collection of large cells (groups).

[0008] ③ Direct adhesion: U.S. Patent US7318999B2 discloses a laser capture microdissection technology (LCM). This technology uses a laser beam to heat and melt a special film with thermal melting properties. After being heated, the film expands in volume and becomes adhesive, wrapping around the tissue cells to be separated and adhering to the cells (groups), separating the target tissue from the tissue section.

[0009] Disadvantages: This method has low collection accuracy. The collection tube cap will come into direct contact with the uncut sample surface, making it difficult to separate cells with a smaller area and easily causing contamination and damage to the uncut sample.

[0010] ④ Electrostatic capture and collection based on a film that carries a negative charge: Announcement No. CN100526453C discloses a method for collecting cut cells using a polyimide film that is pre-negatively charged and a collector with a polar material.

[0011] Disadvantages: This method requires a thin film that can carry a negative charge for a long time. The film materials that can be used are relatively limited, which limits the types of biological samples that can be cut by the laser microdissection system.

[0012] ⑤ Collection based on plasma edge pressure ejection and dielectrophoretic force: A tissue cutting and collection device and collection method disclosed in announcement number CN113916624B. This technology uses a high voltage (up to 30kV) to polarize the collection tube cover, so that the collection tube cover has a polarized charge, and then the dielectrophoretic force generated by the polarized charge will collect the target micro-area.

[0013] Disadvantages: This method involves high voltage, which may pose a safety hazard to the experimental operator. In addition, the dielectrophoretic force generated by the polarization charge on the collection tube cover is relatively weak, and tiny impurities will affect the collection of the target micro-area, and the collection success rate will be low. During the collection process, the polarization charge disappears in about a few minutes. Therefore, it is generally necessary to replace a single collection tube after completing a collection and then reuse the high-voltage polarization collection tube. The steps of replacing the collection tube and polarizing the collection tube hinder the possibility of continuous collection by the laser microdissection system and reduce the collection efficiency. The collection method based on plasma edge pressure ejection and dielectrophoretic force has a short existence time of dielectrophoretic force, which is not enough to support the multi-hole collection tube to complete the collection under the condition of a single polarization collection tube. Therefore, only a single-hole collection tube can be used for collection, which greatly increases the cost of using the system.

[0014] In summary, how to safely collect target microareas without damaging them, improve the collection success rate, improve the collection efficiency, reduce the cost of system use, simplify the experimental operation process, and increase the types of samples that can be cut are problems that need to be solved urgently. Utility Model Content

[0015] The purpose of the utility model is to overcome the deficiencies in the prior art and provide a laser microdissection collection system.

[0016] The purpose of this utility model is achieved through the following technical solutions:

[0017] The laser microdissection collection system includes a stage for placing tissue slices; a laser cutting system including a laser and a cutting system, wherein the cutting system is arranged below the stage and converges the pulsed laser emitted by the laser into a light spot facing the tissue slice, and the light spot cuts out a target micro-region from the tissue slice; a collector is located above the stage with its collection side facing the target micro-region; and a light source is also included, which is arranged above the stage and is used to irradiate the tissue slice for imaging; an electrostatic generating system includes an electrostatic generating head and an electrostatic control system, wherein the electrostatic generating head is arranged between the collector and the light source, and the electrostatic control system controls the electrostatic generating head to generate static electricity and utilizes the electrostatic effect to adsorb the target micro-region into the collector.

[0018] Preferably, the thickness of the electrostatic generating head is 1 mm to 2 mm, and the distance between the electrostatic generating head and the collector is 5±1 mm.

[0019] Preferably, the electrostatic control system includes an electrostatic generator, a programmable logic controller, and a control module. The electrostatic generator is connected to the electrostatic generating head, and the control module is connected to the electrostatic generator through the programmable logic controller to control the opening and closing of the electrostatic generator by controlling the output voltage.

[0020] Preferably, the electrostatic generator is the IONFIX Compact electrostatic generator of FRASER, and the electrostatic generating head is a combination of a 7700 junction box, a 7700 electrode, a 7701 shunt, and a 7097 handheld discharge head that matches the IONFIX Compact electrostatic generator of FRASER.

[0021] Preferably, the control module is connected to the laser and the stage respectively to control the laser and the stage to start synchronously.

[0022] Preferably, the stage is movably disposed above the cutting system, and the stage moves so that the light spot cuts out the target micro-region.

[0023] Preferably, the cutting system includes a first beam expander, a second beam expander, a reflector, and a microscope objective lens. The laser emitted by the laser passes through the first beam expander, the second beam expander, the reflector, and the microscope objective lens in sequence, and the reflector is tilted at an angle of °. The microscope objective lens is perpendicular to the stage, so that the light spot formed by the microscope objective lens is directly opposite to the tissue slice.

[0024] Preferably, the bottom of the collector has an array of collecting tubes, and the collecting tubes are arranged in a horizontal and vertical array or in a honeycomb shape.

[0025] The beneficial effects of the present invention are mainly reflected in:

[0026] 1. Maximize and optimize the tissue section cutting and collection process: Effectively combine the electrostatic generation system with the entire laser cutting system, set up a control module to connect and control the laser, stage and electrostatic generation head respectively, and fix the electrostatic generation head in the electrostatic generation system above the collector. This allows the control module to directly control the electrostatic generation head to generate static electricity to adsorb and collect the target micro-area after the target micro-area is segmented, achieving contactless collection and achieving high efficiency and pollution-free.

[0027] 2. Improved experimental safety: The control module directly controls the electrostatic generator through an editable logic controller to control the electrostatic generating head to turn on or off the electrostatic charge. Compared with the electrostatic capture collection method based on a thin film that carries its own negative charge, this solution reduces the experimental process of charging the thin film. Compared with the collection methods based on plasma edge pressure ejection and dielectrophoresis force, this solution does not require the experimenter to specifically disassemble the polarization collection tube, avoiding direct contact between the user and the electrostatic generator, which is safer.

[0028] 3. Improve the collection effect of the collector: The electrostatic generator in this scheme controls the electrostatic generating head to directly generate static electricity to form a stable electrostatic force directly above the target micro-area, ensuring a stable and reliable adsorption force on the target micro-area. Compared with the collection methods based on plasma edge pressure ejection and dielectrophoresis force, the electrostatic force of this scheme will not be weakened by the use time of the collector and the number of collections. The electrostatic force can also be adjusted at any time by the electrostatic generator, thereby ensuring a stable and controllable adsorption force on the target micro-area, thereby improving the collector's collection success rate, efficiency and reliability for the target micro-area.

[0029] 4. Improve the collection efficiency of the collector and reduce the cost of use: The collector is an array collector, which can be moved for continuous collection, realizing the continuous collection of the laser microdissection system and improving the collection efficiency. Compared with the single-hole collection tube, the cost of collecting the same number of target micro-areas is greatly reduced by using the array collection tube;

[0030] 5. Enriched the types of cutting samples: Compared with the electrostatic capture collection method that carries a negatively charged film, it does not require a film that can maintain the charge for a long time, is not selective for the type of cutting samples, and can break the limitation of sample types. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The technical solution of the utility model is further described below with reference to the accompanying drawings:

[0032] Figure 1 : A schematic structural diagram of an embodiment of the present utility model;

[0033] Figure 2: A partial structural diagram of an embodiment of the present utility model. DETAILED DESCRIPTION

[0034] The present invention will be described in detail below with reference to the specific embodiments shown in the accompanying drawings. However, these embodiments are not limited to the present invention, and any structural, methodological, or functional modifications made by a person skilled in the art based on these embodiments are included within the scope of protection of the present invention.

[0035] In the description of the scheme, it should be noted that the terms "center", "up", "down", "left", "right", "front", "back", "vertical", "horizontal", "inside", "outside", etc., indicating the orientation or positional relationship, are based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience and simplification of 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. Therefore, they should not be understood as limitations on the present invention. In addition, the terms "first", "second", and "third" are used for descriptive purposes only and should not be understood as indicating or implying relative importance. Moreover, in the description of the scheme, with the operator as a reference, the direction close to the operator is the proximal end, and the direction away from the operator is the distal end.

[0036] like Figures 1 to 2 As shown, the present invention discloses a laser microdissection collection system, including a stage 106 for placing tissue slices; a laser cutting system, including a laser 101 and a cutting system, wherein the cutting system is arranged below the stage 106 and converges the pulsed laser emitted by the laser 101 into a light spot facing the tissue slice, and the light spot cuts out a target micro-region 107 from the tissue slice; a collector 108, located above the stage 106 and with its collecting side facing the target micro-region 107; and also including a light source 113, arranged above the stage 106, for irradiating the tissue slice for imaging; an electrostatic generating system, including an electrostatic generating head 109 and an electrostatic control system, wherein the electrostatic generating head 109 is arranged between the collector 108 and the light source 113, and the electrostatic control system controls the electrostatic generating head 109 to generate static electricity, and utilizes the electrostatic effect to adsorb the target micro-region 107 into the collector 108.

[0037] Specifically, the electrostatic control system includes an electrostatic generator 110, a programmable logic controller 111, and a control module 112. The electrostatic generator 110 is connected to the electrostatic generating head 109. The control module 112 is connected to the electrostatic generator 110 through the programmable logic controller 111 to control the opening and closing of the electrostatic generator 110 by controlling the output voltage.

[0038] This solution effectively combines the electrostatic generation system with the entire laser cutting system to maximize the optimization of the tissue slice cutting and collection process. Specifically, a control module 112 is set in the electrostatic generation system to respectively connect and control the laser 101, the stage 106 and the electrostatic generation head 109, and the electrostatic generation head 109 in the electrostatic generation system is set above the collector 108, so that after the target micro-area 107 is segmented, the control module 112 can directly control the electrostatic generation head 109 to generate static electricity to adsorb and collect the target micro-area 107, thereby achieving contactless collection of the target micro-area 107 and achieving pollution-free collection while efficiently collecting the target micro-area 107.

[0039] Furthermore, in the electrostatic generation system, the control module 112 directly controls the output level of the electrostatic generator 110 through the programmable logic controller 111, and thus controls the electrostatic generating head 109 to turn on or off the electrostatics based on the level of the electrostatic generator 110. Specifically, in one feasible embodiment, the control module 112 is programmed using the Siemens official analog library, setting the high level of the output to 10V and the low level to 0V, and setting the programmable logic controller 111 to output any voltage between 0V and 10V. The external trigger switch of the electrostatic generator 110 is set to turn on when receiving a 10V level, and control the electrostatic generating head 109 to turn on the electrostatics to attract the target micro-area 107; and to turn off when receiving a 0V level, not attracting the target micro-area 107. This collection method enables the electrostatic generator 110 to control the electrostatic generating head 109 to directly generate static electricity. Compared with the electrostatic capture collection method based on the film that carries negative charge itself, this solution reduces the experimental process of charging on the film; compared with the collection method based on plasma edge pressure ejection and dielectrophoresis force, this solution does not require the experimenter to specially disassemble the polarization collection tube, avoiding direct contact between the user and the electrostatic generator, thereby improving the safety of the experiment.

[0040] To ensure effective integration between the static generator head 109 and the collector 108, the preferred static generator 110 in this solution is the FRASER IONFIX Compact, specifically model E73020N-AC. The static generator head 109 is preferably compatible with the FRASER IONFIX Compact, specifically the FRASER IONFIX IML in-mold labeling accessory, including the 7700 junction box, 7700 electrodes, 7701 splitter, and 7097 handheld discharge head.

[0041] In a preferred embodiment, the thickness of the electrostatic generating head 109 is 1 mm to 2 mm, and the distance between the electrostatic generating head 109 and the collector 108 is 5 ± 1 mm. This structural arrangement ensures that the electrostatic generating head 109, under the control of the electrostatic generator 110, generates a stable electrostatic force directly above the target micro-region 107, thereby ensuring a stable and reliable adsorption force on the target micro-region 107. Compared with the collection methods based on plasma edge pressure ejection and dielectrophoresis force, the electrostatic force of this solution will not be weakened by the use time of the collector 108 or the number of collections. Moreover, the electrostatic force can be adjusted at any time by adjusting the output level of the electrostatic generator 110, thereby ensuring a stable and controllable adsorption force on the target micro-region 107, thereby improving the success rate, efficiency, and reliability of the collector's collection of the target micro-region.

[0042] Furthermore, the control module 112 is connected to the laser 101 and the stage 106 respectively to control the laser 101 and the stage 106 to start synchronously.

[0043] The stage 106 is movably positioned above the cutting system. The stage 106 moves to cause the laser spot to cut the target micro-region 107. The stage 106 is equipped with a motion device (not shown) that drives its movement. This motion device is conventional and will not be described in detail here. The tissue section on the stage 106 is first imaged to determine the edge position of the target micro-region 107. The control module 112 then determines the cutting trajectory of the target micro-region 107 and, through the motion device, controls the stage 106 to move along the desired cutting trajectory, allowing the laser spot formed by the laser cutting system to cut the target micro-region 107 into any desired shape.

[0044] In some feasible embodiments, to facilitate separation of the target micro-region 107 from the tissue slice, the stage 106 is covered with a transparent film. The transparent film is preferably made of polyethylene naphthalate or polyethylene terephthalate and has a thickness between 1 and 8 μm. Such a transparent film has a higher UV absorption rate than the tissue slice, resulting in a lower cutting threshold for the transparent film than for the tissue slice. When the light spot is cut, the transparent film is cut synchronously with the target micro-region 107, facilitating separation of the target micro-region 107 from the stage 106.

[0045] Specifically, the cutting system includes a first beam expander 102, a second beam expander 103, a reflector 104, and a microscope objective 105. The laser light emitted by the laser 101 passes through the first beam expander 102, the second beam expander 103, the reflector 104, and the microscope objective 105 in sequence. The reflector 104 is tilted at a 45° angle. The microscope objective 105 is perpendicular to the stage 106, so that the light spot formed by the microscope objective 105 is aligned with the tissue slice. This is prior art and is not the focus of this solution, so it will not be described in detail here.

[0046] like Figure 1 As shown, the bottom of the collector 108 has an array of collection tubes arranged in a horizontal and vertical array or a honeycomb pattern. When the collection tubes are arranged in a horizontal and vertical array, the number of collection holes in the vertical and horizontal rows is, for example, M×N (1≤M≤10, 1≤N≤10, M and N are both positive integers). The collector 108 is an array-type collector that can be moved for continuous collection, enabling continuous collection for the laser microdissection system, improving collection efficiency. Compared to single-hole collection tubes, the array-type collection tube significantly reduces the cost of collecting the same number of target micro-regions 107.

[0047] The collection method of the laser microdissection collection system disclosed in the present invention includes the following steps:

[0048] S1. Place the tissue slice on the stage 106, start the light source 113, image the sample, and find the target micro-region 107 to be cut;

[0049] S2. Adjust the position of the collector 108 so that the collection tube at the bottom of the collector 108 is located directly above the target micro-region 107;

[0050] S3, adjust the static electricity generating head 109 to be just above the collector 108;

[0051] S4, the control module 112 controls the laser 101 to provide a pulsed laser, which is expanded by the first beam expander 102 and the second beam expander 103 in sequence, and then refracted by the reflector 104 into the microscope objective 105. The microscope objective 105 converges the pulsed laser to form a light spot that reaches the set cutting threshold; at the same time, the control module 112 controls the stage 106 in step S1 to drive the tissue slice to move horizontally, so that the light spot cuts out the target micro-region 107 from the tissue slice and separates from the tissue slice; at the same time, the control module 112 turns on the electrostatic generator 110 through the programmable logic controller 111, so that the electrostatic generating head 109 generates static electricity and adsorbs the target micro-region 107 into the collection tube at the bottom of the collector 108.

[0052] When there are multiple target micro-areas 107 that need to be cut, the process further includes step S5 of sequentially adjusting the positions of the collectors 108 so that the remaining collecting tubes are located directly above the next target micro-area 107 for cutting and collection, until there are no remaining collecting tubes.

[0053] It should be understood that although this specification is described in terms of implementation methods, not every implementation method contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each implementation method can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

[0054] The series of detailed descriptions listed above are only specific descriptions of feasible implementation methods of the present invention. They are not intended to limit the scope of protection of the present invention. Any equivalent implementation methods or changes that do not deviate from the technical spirit of the present invention should be included in the scope of protection of the present invention.

Claims

1. Laser microdissection collection system, including a stage (106) for placing tissue slices; A laser cutting system, comprising a laser (101) and a cutting system, wherein the cutting system is arranged below the stage (106) and converges the pulsed laser light emitted by the laser (101) into a light spot facing the tissue slice, and the light spot cuts a target micro-region (107) from the tissue slice; A collector (108), located above the stage (106) and with its collecting side facing the target micro-region (107); Its characteristics are: It also includes a light source (113), which is arranged above the stage (106) and is used to illuminate the tissue slice to facilitate imaging; The electrostatic generation system comprises an electrostatic generation head (109) and an electrostatic control system. The electrostatic generation head (109) is arranged between the collector (108) and the light source (113). The electrostatic control system controls the electrostatic generation head (109) to generate static electricity and utilizes the electrostatic effect to adsorb the target micro-region (107) into the collector (108).

2. The laser microdissection collection system according to claim 1, wherein: The thickness of the electrostatic generating head (109) is 1 mm to 2 mm, and the distance between the electrostatic generating head (109) and the collector (108) is 5±1 mm.

3. The laser microdissection collection system according to claim 1, wherein: The electrostatic control system comprises an electrostatic generator (110), a programmable logic controller (111), and a control module (112). The electrostatic generator (110) is connected to the electrostatic generating head (109). The control module (112) is connected to the electrostatic generator (110) via the programmable logic controller (111) to control the opening and closing of the electrostatic generator (110) by controlling the output voltage.

4. The laser microdissection collection system according to claim 3, wherein: The electrostatic generator (110) is an IONFIX Compact electrostatic generator of FRASER, and the electrostatic generating head (109) is a combination of a 7700 junction box, a 7700 electrode, a 7701 shunt, and a 7097 handheld discharge head that matches the IONFIX Compact electrostatic generator of FRASER.

5. The laser microdissection collection system according to claim 4, characterized in that: The control module (112) is connected to the laser (101) and the stage (106) respectively to control the laser (101) and the stage (106) to start synchronously.

6. The laser microdissection collection system according to any one of claims 1 to 5, characterized in that: The object stage (106) is movably arranged above the cutting system, and the object stage (106) moves so that the light spot cuts out the target micro-region (107).

7. The laser microdissection collection system according to any one of claims 1 to 5, characterized in that: The cutting system comprises a first beam expander (102), a second beam expander (103), a reflector (104), and a microscope objective lens (105). The laser light emitted by the laser (101) passes through the first beam expander (102), the second beam expander (103), the reflector (104), and the microscope objective lens (105) in sequence, and the reflector (104) is tilted at an angle of 45°. The microscope objective lens (105) and the stage (106) are vertically arranged so that the light spot formed by the microscope objective lens (105) is directly opposite to the tissue slice.

8. The laser microdissection collection system according to any one of claims 1 to 5, characterized in that: The bottom of the collector (108) is provided with an array of collecting tubes, and the collecting tubes are arranged in a horizontal and vertical array or in a honeycomb shape.

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