Small-size biological sample laser slicing device and laser slicing method

Abstract

The invention discloses a small-size biological sample laser slicing device and a laser slicing method, and belongs to the technical field of biological sample preparation. The laser slicing device comprises an ultrafast laser, a laser beam motion control assembly, a lens and a sample cabin, the sample cabin is a container which is composed of a frame and a replaceable transparent plastic or glass cabin wall and provided with an opening in the upper end, a small-size biological sample is fixed in the sample cabin, a matching medium needs to be added into a living body sample, and the matching medium is matched with the sample. And performing micro-damage cutting on the sample by using Gaussian light or Bessel light. According to the invention, laser is used for cutting the sample, so that the sample is not extruded and pulled during slicing, and the sample is not stuck and curled on the cutting blade, so that the experiment and sample loss are reduced, and the experiment efficiency and success rate are improved; the slicing machine is matched with a stereoscope for use, so that the dependence of a traditional slicing machine on the eyesight of operators can be relieved.

Description

Technical Field

[0001] The invention relates to the technical field of biological sample preparation, and in particular to a laser slicing device and a method for micro-sized microorganism samples. Background Art

[0002] In spatial omics research, biological samples need to be sliced ​​before imaging and omics analysis can be performed. For conventional large-sized biological samples, such as mouse brains, plant rhizomes, etc., they are sliced ​​first and then imaged and analyzed. However, for small-sized samples with high requirements for spatiotemporal characteristics, such as pancreatic islets, they need to be imaged first and then sliced ​​for omics analysis. Because the use of conventional slicing methods such as oscillating slicers or frozen slicers will cause sample extrusion and pulling, resulting in excessive sample loss and difficulty in three-dimensional reconstruction. At the same time, the sample often sticks and curls on the blade, making it difficult to collect the cut sample. In addition, the existing slicer needs to embed the sample before sample preparation, and relies on the operator's human eye observation during cutting, which makes it difficult to slice small samples. The above situations will cause sample loss, especially precious pathological samples. Therefore, it is necessary to invent a device for reliable non-contact slicing of small-sized biological samples with a diameter of 50μm to 500μm. Summary of the invention

[0003] The purpose of the present invention is to provide a device and a laser slicing method for small-sized biological samples, so as to overcome the risks of sample deformation, sample loss, and preparation difficulties that may occur when using existing slicing devices for small-sized biological samples, thereby reducing time, experiments, and sample losses. Through the design and production of a small-scale biological sample slicing device, a sample preparation solution can be provided for laboratories in need, thereby improving the efficiency and success rate of spatial omics experiments.

[0004] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

[0005] A biological sample laser slicing device can perform small-scale biological sample laser slicing operations. The laser slicing device comprises an ultrafast laser, a laser beam motion control component, a lens, and a sample chamber, wherein the ultrafast laser and the laser beam motion control component are connected via a light guide tube, and the laser enters the laser beam motion control component from the ultrafast laser via the light guide tube; the lens is connected to the laser beam motion control component housing via a lens seat, and the laser is emitted into the sample chamber via the lens to slice the sample; the sample chamber is a container with an upper end open and composed of a frame and a replaceable transparent plastic or glass chamber wall.

[0006] The ultrafast laser is a near-infrared laser that can emit 750nm to 1100nm ultrafast laser light. The laser energy range is 50nJ to 3mJ. It acts on the sample in the form of pulses with a repetition frequency of 1kHz to 1GHz, which can achieve cold processing of the sample and protect the out-of-focus sample from damage.

[0007] The laser beam motion control component can be an optical device group that can control the Gaussian light motion of the ultrafast laser emission and scanning direction, including a light-guiding optical path, which specifically contains a reflector, a beam expander, a three-dimensional scanner, etc. The reflector guides the laser beam emitted by the ultrafast laser to the processing device, and guides the laser beam into the beam expander to achieve the required spot size, and then the three-dimensional scanner drives the laser beam to perform three-dimensional scanning. The selection of the reflector needs to reduce the laser energy loss, and a dielectric film high-reflectivity reflector or a metal reflector can be selected, which can be in the form of a discrete device or an integrated light pipe. The beam expander matches the light beam emitted by the laser with the tail aperture of the microscope objective lens selected subsequently to ensure that the designed spot size can be achieved, so it is necessary to select a suitable magnification, and at the same time, it is necessary to ensure that it is completely coaxial with the optical path during use. The 3D scanner drives the light beam to perform the designed 3D scanning on the target sample to achieve the cutting action. The 3D scanner includes a 2D scanner such as a 2D galvanometer. The 2D scanner controls the light beam to scan in its orthogonal plane (YZ plane). Its selection needs to match the field of view of the selected objective lens. The 3D scanner also needs to include a longitudinal scanner, which can be a precision electric translation stage or a piezoelectric actuation platform, installed on the microscope objective lens, driving the lens to perform short-distance high-precision longitudinal scanning. When using Gaussian light as the cutting light source, the size of the light spot in the XY direction can theoretically reach less than 1μm.

[0008] The laser beam motion control component can also be an optical device group that can modulate the laser emitted by the ultrafast laser into a non-diffraction Bessel light sheet and control its movement, specifically including the aforementioned reflector, beam expander, two-dimensional scanner, and phase modulation device. Specifically, the phase modulation device includes but is not limited to a corner cube prism (Axicon), a liquid crystal spatial light modulator (LC-SLM), and a digital micromirror modulator (DMD). The reflector allows the laser emitted by the ultrafast laser to enter the beam expander to achieve the required spot size; the phase modulation device modulates the original Gaussian light wave into a conical phase light wave. After passing through the lens, the light beam is transformed into a slender Bessel light beam, which can be combined with the scanning action of the two-dimensional scanner to form a Bessel light sheet to achieve a large depth of cutting action. When Bessel light is used as a cutting light source, the thickness of the non-diffraction Bessel light sheet formed can theoretically reach less than 2μm. Using both Gaussian light and Bessel light as cutting light sources can achieve micro-damage cutting of samples (cutting thickness is less than 20μm, i.e., typical cell thickness).

[0009] The lens has a working distance of 0.5 cm to 1.5 cm and a numerical aperture of 0.6 to 1.

[0010] Preferably, the frame of the sample chamber is composed of a bottom plate and a plurality of columns located on the bottom plate, wherein the bottom plate can be made of plastic material, the columns are plastic columns covered with silicone, and each column is provided with two slideways; the frame cooperates with a cabin wall composed of a replaceable transparent plastic film (such as an FEP film with a refractive index close to that of water), a plastic sheet (such as an FEP sheet) or a glass sheet to form a container with an open top, wherein the transparent plastic film can be wrapped around the column, and the gap is sealed with an adhesive (such as a tissue adhesive or dental cement); the plastic sheet or the glass sheet can be inserted into the slideway of the column, and the elastic force of the slideway material itself and / or the adhesive (such as a tissue adhesive or dental cement) is used to achieve sealing. In some embodiments of the present invention, the bottom plate of the sample chamber frame is rectangular, and four columns are provided at the four corners. Four plastic sheets or glass sheets are inserted into the slideways of the columns to form a rectangular container with an open top.

[0011] During the laser sectioning operation, for samples with biological activity, a matching medium needs to be added to the sample chamber. The matching medium is a liquid or colloid that does no damage to the sample and has a refractive index difference of less than ±10% with the sample.

[0012] Compared with existing slicing devices, the biological sample laser slicing device of the present invention uses laser to cut samples, which will not cause squeezing and pulling of the samples during slicing, and will not cause the samples to stick to and curl on the cutting blade, thereby ensuring the integrity of the samples. It is particularly suitable for laser slicing operations of small-sized biological samples.

[0013] The present invention also provides a method for laser slicing a small-sized biological sample using the biological sample laser slicing device, comprising the following steps:

[0014] For biologically active samples:

[0015] 1. Prepare the matching medium solution. Prepare the isotonic solution for culturing samples. After the preparation is completed, pump 95% O 2 With 5% CO 2 The mixed gas composition makes the isotonic solution contain O 2 Fully saturated. Use Abbe refractometer to measure the refractive index of the isotonic solution at this time. The refractive index of the isotonic solution is generally between 1.30 and 1.40. At this time, if the material of the sample cabin wall is a cover glass insert, add an appropriate amount of sucrose solution to the isotonic solution to adjust its refractive index to 1.41±0.01, and the resulting liquid is the matching medium solution; if the material of the sample cabin wall is FEP film, the isotonic solution can be directly used as the matching medium.

[0016] 2. Make a sample chamber. Figure 2As shown, if the sample cabin wall is made of a cover glass insert, it can be directly inserted between the sample cabin frame columns, and the gap can be sealed with tissue adhesive or dental cement; if the sample cabin wall is made of FEP film, the FEP film can be wrapped around the sample cabin frame columns, and the gap can be sealed with tissue adhesive or dental cement.

[0017] 3. Sample placement. According to the experimental purpose, pre-cool the oxygen-saturated matching medium with ice-water mixture or adjust the temperature to room temperature or adjust the temperature to the biological body temperature. Take out the sample to be sliced ​​from the living organism by surgical means, or take out the sample pre-cultured in the incubator. If necessary, use a scalpel to trim the sample, and then fix the sample to the slide at the bottom of the sample chamber with biological glue or 502 (if the experimental method permits). Quickly pour in the oxygen-saturated matching medium.

[0018] 4. Sample slicing and post-processing. Place the sample chamber into the laser slicing device of the present invention, and adjust the distance between the sample and the lens to the calibrated working distance of the lens. According to the experimental requirements, the supporting program of the laser beam motion control component sets the laser beam route, power and working time, turns on the machine, and slices the sample. The cut samples can be quickly transferred to a 35°C isotonic solution through an incubation frame for recovery according to experimental requirements, and can be used for subsequent experiments after half an hour; or directly used in subsequent experiments.

[0019] For non-biologically active / fixed samples:

[0020] 1. Fix the sample on the slide by tissue adhesive or 502 (if the experimental method permits), and place the slide in the sample chamber. There is no need to insert a cover glass or FEP film between the columns of the sample chamber frame.

[0021] 2. Place the sample chamber into the laser slicing device of the present invention, and adjust the distance between the sample and the lens to the working distance calibrated by the lens. According to the experimental requirements, the laser beam motion control component supporting program sets the laser beam travel route, power and working time, turns on the machine, and slices the sample. The cut sample can be directly used for subsequent experiments.

[0022] Technical effects of the present invention:

[0023] The laser slicing device of the present invention is used to prepare small-sized biological slices required for spatial omics and other research. Compared with existing slicing devices, the use of laser for sample cutting will not cause squeezing and pulling of the sample during slicing, and will not cause the sample to stick and curl on the cutting blade, thereby avoiding sample deformation during slicing, reducing sample loss in the depth direction, and reducing the requirements for the experimental personnel's operating skills during the sample preparation process, thereby ensuring the integrity of the sample and improving the experimental efficiency and success rate. The laser slicing device can be used in conjunction with a stereoscope, thereby reducing the dependence of traditional slicers on the operator's eyesight. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is a schematic structural diagram of a small-size biological sample laser slicing device according to an embodiment;

[0025] Figure 2 is a schematic diagram of the sample chamber described in the embodiment, wherein the upper left is a side sectional view without a sample and a matching medium, the upper right is a top view without a sample and a matching medium, the lower left is a side sectional view with a sample and a matching medium, and the lower right is a top view with a sample and a matching medium;

[0026] Figure 3 is a schematic diagram of the small-size biological sample laser slicing device in operation according to the embodiment;

[0027] Figure 4 It is a schematic top view of the laser forward path when the laser used in the small-size biological sample laser slicing device described in the embodiment is Gaussian light.

[0028] Figure 5 The diagram is a top view of the laser's advancing path during operation when the laser used in the small-size biological sample laser slicing device described in the embodiment is Bessel light.

[0029] In the figure: 1-ultrafast laser, 2-laser beam motion control assembly, 3-lens, 4-sample chamber, 5-matching medium, 6-frame, 7-FEP film or glass, 8-sample, 9-glass slide, 10-ultrafast laser, 11-Gaussian light focusing position, 12-Bessel light sheet, 13-laser movement path. DETAILED DESCRIPTION

[0030] The small-size biological sample laser slicing device and its implementation method of the present invention are further described below through specific embodiments in conjunction with the accompanying drawings.

[0031] like Figure 1 As shown, the small-size biological sample laser slicing device for preparing small-size biological sample slices includes an ultrafast laser 1, a laser beam motion control component 2, a lens 3, a sample chamber 4, and a matching medium 5 that matches the refractive index of the biological sample and is safe and harmless to the sample. Among them, the laser outlet of the ultrafast laser 1 needs to be aligned with the laser inlet of the laser beam motion control component 2, and the laser outlet of the laser beam motion control component 2 needs to be on the same straight line as the optical axis of the lens 3.

[0032] like Figure 2As shown in the above figure, the sample chamber 4 is composed of a frame 6 and a FEP film or glass 7. The frame 6 is composed of a square bottom plate and four columns at the four corners of the bottom plate, wherein the bottom plate is made of plastic material, the columns are plastic columns covered with silicone, each column has two slides, and the distance between adjacent columns is 2.4 cm; the FEP film or glass 7 is a detachable and replaceable component, and four pieces of FEP film or glass 7 are inserted into the column slides of the frame 6, and the elastic force of the silicone material itself and the tissue adhesive or dental cement are used to seal the FEP film or glass 7 and the space in the slide, and the combination forms a rectangular parallelepiped with an open top.

[0033] like Figure 2 As shown in the following figure, when the sample chamber 4 is in use, a sample 8 fixed on a glass slide 9 is placed in the chamber, and a matching medium 5 is added to ensure the alignment of the laser light path during cutting.

[0034] like Figure 3 As shown, when the small-size biological sample laser slicing device is working, the laser emitted by the ultrafast laser 1 enters the laser beam motion control component 2, is modulated to form a single beam scanning light or Bessel light sheet, is emitted through the lens 3 and enters the sample chamber 4, and completes the slicing operation of the sample 8 in the matching medium 5 which has the same refractive index as the sample 8 and is friendly to the sample 8.

[0035] like Figure 4 As shown, when Gaussian light is used as the cutting light source, the Gaussian light emitted by the laser reaches the scanning module through the beam control component and the laser beam matched with the beam width. The cutting method is: the cutting point (i.e., the light spot) is focused on the energy in the X direction by changing the focal depth through the depth scanning device to cut the sample; after passing through the entire sample, the light beam is translated in the Y direction through the two-dimensional scanning device, and then cut again in the X direction; this process is repeated until the entire sample is cut.

[0036] like Figure 5 As shown, when Bessel light is used as the cutting light source, the Gaussian light emitted by the laser is guided by the beam control component and the beam width matched laser beam reaches the beam shaping module. After the beam is phase modulated, the Gaussian beam becomes a diffraction-free Bessel light sheet. The cutting method is: after the Bessel light sheet is emitted, it passes through the entire sample to complete a cutting. After that, the light sheet is translated by the two-dimensional scanner and pushed in the Y direction along the cutting line direction to achieve cutting of the sample.

[0037] Example 1. Preparation of pancreatic islet slices

[0038] 1. Isolation and Culture of Pancreatic Islets

[0039] The purpose of this step is to isolate and culture islets from the mouse pancreas to a state suitable for slicing. The specific method is: prepare a collagenase P solution with a concentration of 0.5 mg / mL, select mice aged 8-20 weeks for euthanasia, perfuse the prepared collagenase P solution into the mouse pancreas, digest the pancreas for 15-17 minutes, and then separate the islets under a dissecting microscope / stereoscope or other type of object image magnification device. The isolated islets are placed in islet culture medium and cultured in a cell culture incubator at 37°C and 5% carbon dioxide. The islet culture medium is RPMI1640 medium containing 10% fetal bovine serum, 5.5 mL glucose, 100 units / mL penicillin, and 100 mg / mL streptomycin. After 20 to 50 hours of culture, it can be used for slicing experiments.

[0040] 2. Sample Chamber Assembly and Islet Placement

[0041] The purpose of this step is to assemble the sample chamber and add the appropriate matching medium according to the experimental requirements, and then fix the islets to be sliced ​​in the sample chamber so that they can remain stable during the slicing process. The specific method is: remove the sample chamber bracket, insert a 0.17mm thick cover glass or wrap FEP film in the bracket slide, seal the lower edge with tissue adhesive or dental cement, and add matching medium. The matching medium is an aqueous solution containing chemicals (such as 10% sucrose) that cause the islets to produce biological reactions. Low-melting point agar is prepared into a 15% to 20% solution, and after it cools and solidifies, it is processed into small pieces that can be placed in the sample chamber to provide support for the islets. Remove the islets from the cell culture incubator, use a pipette equipped with a 10μL pipette tip to transfer the islets to the low-melting point agar block, and use tissue adhesive to bond the islets to the low-melting point agar block. Use tweezers and other instruments to transfer the low-melting point agar block with the islets to the sample chamber, and also use tissue adhesive to bond the low-melting point agar block to the bottom of the sample chamber. Since pancreatic islets are spherical in shape, there is no need to distinguish between the long and short axes.

[0042] 3. Islet Slicing

[0043] The purpose of this step is to slice the islets using a small-size biological sample laser slicer so that they are segmented into a thickness suitable for single-cell spatial omics studies. Figure 4 , Figure 5As shown, the movement of the laser cutting line varies depending on the nature of the light source. When slicing, a metal bath is used at the bottom of the sample chamber to keep the liquid temperature in the sample chamber at 37°C. The specific method is: after the pancreatic islet sample is placed in the sample chamber and fixed by tissue adhesive, a matching medium preheated to 37°C is quickly added. The sample is cut using a laser with a wavelength of 880±20nm. The laser power at the lens end is adjusted according to the rated power of the laser and the state of the sample, and is generally set to 50% to 80% of the rated power of the laser. The laser beam motion range is set by the software supporting the laser beam motion control component to include the entire space where the sample is located. Set the Z-axis step to 10μm, and the laser beam action to wait after the XY plane movement is completed. After the experimenter enters the start command through the software, the laser beam executes the Z-axis movement of 10μm and the next movement along the XY plane. Start the machine to slice the sample. After each sample is cut, it is immediately transferred to the incubation frame for incubation and arranged in sequence according to the slicing order. After the entire sample slicing work is completed, all slices will be transferred sequentially to the sample chip of the single-cell spatial omics device to ensure the integrity of the spatial information when reconstructing the single-cell spatial omics data.

[0044] 4. Islet Fixation

[0045] The above steps 1-3 describe the experimental process of live islet slices. The purpose of this step is to fix the islets to preserve the biological characteristics under specific time and conditions. The islets are fixed and become biologically inactive / fixed samples. The specific method is: prepare PBS buffer, 30% sucrose solution, and PFA fixative. Take out the islets separated and cultured in step 1, rinse them with PBS buffer 3 times, and immerse them in PFA fixative for fixation. The fixation time is generally 30 to 60 minutes depending on the size of the islet sample. Then soak in 30% sucrose solution for 60 to 90 minutes to achieve the purpose of dehydration. The fixed islet samples can continue with the operations in steps 2 and 3. When performing step 2, there is no need to add matching medium. When performing step 3, there is no need to use a metal bath, and there is no need to incubate the sliced ​​samples.

[0046] Example 2. Zebrafish heart slice preparation

[0047] 1. Zebrafish Heart Isolation

[0048] The purpose of this step is to isolate the heart from the zebrafish and make it suitable for slicing. The specific method is: select zebrafish between the ages of 4 months and 1 year old and at least 3 cm in length, and put them in an ice-water mixture at 0℃ to 4℃ for 20 minutes to kill them. If the experimental design requires trauma to the heart, the trauma experiment needs to be performed before the zebrafish is killed. After the zebrafish is killed, dissect and remove the heart and place it in Hanks' balanced salt solution (HBSS) at 4℃. Use a syringe needle to puncture the heart cavity. During this process, it is necessary to control the diameter of the puncture to less than 0.5mm. Rinse the zebrafish heart with a large amount of 4℃ HBSS to completely remove most of the red blood cells.

[0049] 2. Sample Chamber Assembly and Heart Placement

[0050] The purpose of this step is to assemble the sample chamber and add the appropriate matching medium according to the experimental requirements, and then fix the zebrafish heart to be sliced ​​in the sample chamber so that it can remain stable during the slicing process. The specific method is: take out the sample chamber bracket, insert a 0.17mm thick cover glass or wrap FEP film in the bracket slide, seal the lower edge with tissue adhesive or dental cement, and add matching medium. The matching medium is HBSS. Low melting point agar is prepared as a 15% to 20% solution, and after it cools and solidifies, it is processed into small blocks that can be placed in the sample chamber to provide support for the zebrafish heart. Use tweezers to transfer the zebrafish heart to the low melting point agar block, and use tissue adhesive to bond the heart to the low melting point agar block. Then transfer the low melting point agar block with the heart to the sample chamber, and also use tissue adhesive to bond the low melting point agar block to the bottom of the sample chamber.

[0051] 3. Heart Slicing

[0052] The purpose of this step is to use a small-scale biological sample laser sectioning device to slice the zebrafish heart into sections with a thickness suitable for single-cell spatial omics studies. Figure 4 , Figure 5As shown, the movement of the laser cutting line is different depending on the nature of the light source. After each sample is cut, it is immediately transferred to the sample chip of the single-cell spatial omics device and arranged in sequence in the order of slicing to ensure the spatial integrity of the single-cell spatial omics data reconstruction. The specific method is: after the zebrafish sample is placed in the sample chamber and fixed with a tissue adhesive, a matching medium preheated to 22°C is quickly added. The sample is cut using a laser with a wavelength of 880±20nm. The laser power at the lens end is adjusted according to the rated power of the laser and the state of the sample, and is generally set to 70% to 100% of the rated power of the laser. The laser beam motion range is set by the software supporting the laser beam motion control component to include the entire space where the sample is located. Set the Z-axis step to 15μm, and the laser beam action to wait after the XY plane movement is completed. After the experimenter enters the start command through the software, the laser beam executes the Z-axis movement of 15μm and the next movement along the XY plane. After completing an XY plane sample cut, it is immediately transferred to the incubation frame for incubation and arranged in sequence in the order of slicing. After the entire sample slicing work is completed, all slices will be transferred sequentially to the sample chip of the single-cell spatial omics device to ensure the integrity of the spatial information when reconstructing the single-cell spatial omics data.

[0053] Finally, it should be noted that the purpose of publishing the embodiments is to help further understand the present invention, but those skilled in the art can understand that various substitutions and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. Therefore, the present invention should not be limited to the contents disclosed in the embodiments, and the scope of protection claimed by the present invention shall be subject to the scope defined in the claims.

Claims

1. A biological sample laser slicing device, used for performing small-scale biological sample laser slicing operations, the laser slicing device comprises an ultrafast laser, a laser beam motion control component, a lens and a sample chamber, wherein: The ultrafast laser is connected to the laser beam motion control component through a light guide, and the laser enters the laser beam motion control component from the ultrafast laser through the light guide; the lens is connected to the laser beam motion control component housing through a lens seat, and the laser is emitted into the sample chamber through the lens to slice the sample; the sample chamber is a container with an upper end opening composed of a frame and a replaceable transparent plastic or glass chamber wall.

2. The biological sample laser slicing device according to claim 1, characterized in that: The frame of the sample cabin is composed of a base plate and a plurality of columns located on the base plate, and each column is provided with two slides; the replaceable transparent plastic or glass cabin wall is an FEP film, an FEP sheet or a glass sheet, and the FEP sheet or the glass sheet is inserted into the slide of the column, and the sealing is achieved by utilizing the elastic force and / or adhesive of the slide material itself, or the FEP film is wrapped around the column, and the gap is sealed by utilizing adhesive.

3. The biological sample laser slicing device according to claim 2, characterized in that: The frame of the sample chamber comprises a rectangular plastic bottom plate and four upright posts on its four corners, and the upright posts are plastic posts wrapped with silica gel; the adhesive is a tissue adhesive or dental cement.

4. The biological sample laser slicing device according to claim 1, characterized in that: The ultrafast laser is a near-infrared laser that emits ultrafast laser light of 750nm to 1100nm.

5. The biological sample laser slicing device according to claim 1, characterized in that: The laser beam motion control assembly is an optical device group that controls the emission of ultrafast lasers and the motion of Gaussian light in the scanning direction, or an optical device group that modulates the laser light emitted by ultrafast lasers into a non-diffraction Bessel light sheet and controls its motion.

6. The biological sample laser slicing device according to claim 5, characterized in that: The laser beam motion control component includes a reflector, a beam expander, and a three-dimensional scanner, wherein the reflector guides the laser beam emitted by the ultrafast laser into the beam expander to achieve the required spot size, and then the three-dimensional scanner drives the laser beam to perform three-dimensional scanning; the three-dimensional scanner includes a two-dimensional scanner and a longitudinal scanner, the two-dimensional scanner controls the light beam to scan within its orthogonal plane, and the longitudinal scanner is a precision electrically controlled translation stage or a piezoelectric actuation platform, which drives the lens to perform short-distance and high-precision longitudinal scanning.

7. The biological sample laser slicing device according to claim 5, characterized in that: The laser beam motion control component includes a reflector, a beam expander, a phase modulation device and a two-dimensional scanner, wherein the reflector allows the laser emitted by the ultrafast laser to enter the beam expander to achieve the required spot size; the phase modulation device modulates the original Gaussian light wave into a conical phase light wave, and after passing through the lens, the light beam is transformed into a slender Bessel light beam, and the two-dimensional scanner controls the Bessel light beam to scan in a plane.

8. The biological sample laser slicing device according to claim 1, characterized in that: The lens has a working distance of 0.5 cm to 1.5 cm and a numerical aperture of 0.6 to 1.

9. A method for laser slicing a small-sized biological sample, implemented by using the biological sample laser slicing device according to any one of claims 1 to 8, wherein: For samples with biological activity, the samples are placed in a sample chamber with replaceable transparent plastic or glass chamber walls, and then a matching medium is added after being fixed. Then the sample chamber is placed in a laser slicing device for laser slicing operations. For samples without biological activity, the samples are directly fixed in a sample chamber with only a frame, and then the sample chamber is placed in a laser slicing device for laser slicing operations.

10. The method according to claim 9, characterized in that The matching medium is a liquid or colloid that does no harm to the sample and has a refractive index difference of less than ±10% with the sample. Preferably, the matching medium is an oxygen-saturated isotonic solution, which is precooled with an ice-water mixture or adjusted to room temperature or biological body temperature before use.

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