Dual-pulse needle beam laser capture microdissection system and method

Abstract

This invention relates to a dual-pulse needle-beam laser capture micro-cutting system and method. The system includes a laser source module, a beam expander module, a polarization control module, a dual-pulse control module, a spatial light modulation module, an objective lens focusing module, a sample carrier and scanning module, and an image acquisition module. The dual-pulse control module splits a single-pulse laser beam to form a dual-pulse laser that selectively controls energy deposition during the cutting process. The spatial light modulation module modulates the incident laser wavefront to create a light field with an axial range greater than or covering the thickness of the sample to be cut. The sample carrier and scanning module carries the sample to be cut and drives relative motion between the sample and the laser focal zone to complete micro-cutting along a predetermined trajectory. This invention can reduce the effective cutting width and decrease collateral damage induced by side lobes while maintaining the ability to cut thick samples, thereby improving cutting accuracy.

Description

Technical Field

[0001] This invention relates to the field of tissue separation technology, and in particular to a dual-pulse needle-beam laser capture microdissection system and method. Background Technology

[0002] Laser capture microdissection (LCM) is a technique that enables the precise separation of specific cells or localized tissue regions from complex tissue samples. It has been widely applied in pathological analysis, single-cell research, and downstream omics detection such as transcriptomics and proteomics. Compared to mechanical cutting or manual separation methods, LCM offers advantages such as non-contact operation, high spatial resolution, and the ability to manipulate specific regions, thus holding significant value in biomedical research and clinical sample preparation.

[0003] In existing laser-captured microdissection techniques, a Gaussian beam is often focused through a microscope objective to ablate or cut the sample. This approach achieves good cutting results in thinner samples, but for thick biological samples, especially frozen tissue sections tens of micrometers thick, the limited focal domain of the Gaussian beam along the optical axis means the cutting action is mainly concentrated near a narrow focal plane. Therefore, it is difficult to simultaneously achieve a narrow cutting width and a low cutting threshold energy density. When the incident energy is increased to ensure full-thickness cutting of thick samples, thermal effects and mechanical damage can easily occur around the cutting area, leading to a wider incision, blurred tissue boundaries, and consequently affecting the accuracy of subsequent molecular detection results.

[0004] To improve the axial reach of Gaussian beams in thick samples, existing techniques employ non-diffraction beams, such as Bessel beams or other long-depth-of-focus beams, for micromachining or microcutting. These beams have a longer effective reach along their propagation direction, theoretically making them more suitable for cutting thick samples. However, these beams typically have prominent side lobes in addition to the main lobe. The energy of these side lobes also interacts with the sample during cutting, introducing additional damage regions on both sides of the main cut, manifesting as thermomechanical damage, edge structure disruption, or localized non-target ablation. This side-lobe-induced collateral damage reduces cutting precision, affects the integrity and purity of the separated cells or tissue regions, and is detrimental to obtaining high-quality downstream omics samples.

[0005] Therefore, there is an urgent need for a laser capture microdissection technique suitable for thick biological samples, which can reduce the cutting threshold, decrease the cutting width, and suppress collateral damage caused by side lobes while ensuring a longer axial range of action. Summary of the Invention

[0006] The purpose of this invention is to provide a dual-pulse needle-beam laser capture micro-cutting system and method to solve the problems of existing Gaussian beam-based laser capture micro-cutting technology, which is difficult to balance narrow cutting width and low threshold energy density in thick samples, and the side lobe-induced incidental damage in existing non-diffraction beam cutting.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] A dual-pulse needle-beam laser capture micro-cutting system includes a laser source module, a beam expander module, a polarization control module, a dual-pulse control module, a spatial light modulation module, an objective lens focusing module, a sample carrying and scanning module, and an image acquisition module.

[0009] The system comprises the following components: a laser source module for outputting single-pulse laser light; a beam expander module positioned along the output path of the laser source module for expanding the pulsed laser beam to match the effective modulation aperture of the spatial light modulation module; a polarization control module positioned behind the beam expander module for adjusting the polarization state of the laser incident on the spatial light modulation module; a dual-pulse control module positioned behind the polarization control module for splitting the single-pulse laser beam to form a dual-pulse laser beam that selectively modulates energy deposition during the cutting process; a spatial light modulation module positioned behind the dual-pulse control module for wavefront modulation of the incident laser to form a light field with an axial range greater than or covering the thickness of the sample to be cut; an image acquisition module for acquiring an image of the sample before cutting to adjust the position between the sample to be cut and the objective lens to align the imaging plane with the area to be cut; an objective lens focusing module for focusing the modulated laser onto the sample to be cut; and a sample carrying and scanning module for carrying the sample to be cut and driving relative motion between the sample and the laser focal zone to complete microscopic cutting along a predetermined trajectory.

[0010] In the above scheme, by controlling the pulse to form a dual-pulse laser that selectively modulates the energy deposition of the cutting process, the energy deposition can be more concentrated in the main lobe region, reducing damage to the side lobe region and lowering the threshold energy density. By modulating the incident laser wavefront to form a light field with an axial range greater than or covering the thickness of the sample to be cut, it can be adapted to the cutting of thick biological samples. While ensuring the cutting ability of thick samples, it reduces the effective cutting width and reduces collateral damage induced by side lobes, thereby improving the cutting accuracy.

[0011] In a preferred embodiment, the dual-pulse control module provides a reference optical path and a delayed optical path, wherein one pulsed laser beam propagates along the reference optical path and the other pulsed laser beam propagates along the delayed optical path. The delay time between the two pulsed laser beams is adjusted by changing the optical path length of the delayed optical path, so that after one pulsed laser beam acts, a pre-excitation state is formed in the sample to be cut, and the local energy coupling characteristics are changed when the other pulsed laser beam arrives.

[0012] In the above scheme, two laser beams are focused onto the sample to be cut, one after the other. The first arriving laser pulse creates a pre-excitation state in the sample, while the subsequent laser pulse alters the local energy coupling characteristics, resulting in high intensity in the main lobe region and low intensity in the side lobes. This not only reduces damage to the side lobes but also effectively lowers the threshold energy density for micro-cutting of thick samples. The selective control of energy deposition during the cutting process is achieved through a delayed approach, which is simple and easy to implement.

[0013] In a preferred embodiment, the dual-pulse control module includes two beam splitters and at least two mirrors. The two beam splitters form the reference optical path, and the mirrors form the delay optical path. One of the beam splitters splits the single-pulse input light into two paths, and the pulsed lasers output through the reference optical path and the delay optical path, respectively, are coaxially output by the other beam splitter.

[0014] In the above scheme, using a reflector to change the optical path length not only makes it easier to arrange the lens, but also facilitates precise control of the optical path length, which in turn improves cutting accuracy and reduces side lobe damage.

[0015] In a preferred embodiment, a beam transmission and imaging module is also included, disposed between the spatial light modulation module and the objective lens focusing module;

[0016] The spatial light modulation module uses a spatial light modulator, which constructs multiple discrete focal points in the optical axis direction based on spatial multiplexing, and forms a needle-shaped beam with a predetermined axial length through phase superposition.

[0017] The beam transmission and imaging module is used to transmit the light field modulated by the spatial light modulation module to the rear pupil surface of the objective lens and realize conjugate imaging of the light field.

[0018] In the above scheme, a needle-shaped beam is generated using a spatial light modulator. The needle-shaped beam can provide a longer axial range of action, making it more suitable for cutting thick biological samples. This improves the problem that traditional Gaussian beams have insufficient depth of focus in thick samples, making it difficult to cut through or requiring higher energy to cut through.

[0019] The dual-pulse needle-beam laser capture micro-cutting method based on the aforementioned dual-pulse needle-beam laser capture micro-cutting system includes the following steps:

[0020] Step 1: Acquire a sample image before cutting, and adjust the objective lens or sample position according to the sample image to align the imaging plane with the area to be cut.

[0021] Step 2: Output a single-pulse laser, expand the pulsed laser beam, and adjust the polarization state of the expanded laser to adapt to the spatial light modulation module;

[0022] Step 3: Split the single-pulse laser beam to form a dual-pulse laser that controls the selective energy deposition during the cutting process;

[0023] Step 4: Wavefront modulation of the incident laser is performed to form an optical field with an axial range greater than or covering the thickness of the sample to be cut. The modulated laser is then focused on the sample to be cut.

[0024] Step 5: Drive the sample to be cut to move relative to the laser focal zone to complete the micro-cutting along the predetermined trajectory.

[0025] In a preferred embodiment, step 6 is also included: acquiring images of the cut sample and observing the cutting trajectory to evaluate the cutting threshold, cutting width, and morphology of the side lobe-related damage.

[0026] In the above scheme, by acquiring sample images after cutting, the quality of this cutting is evaluated, which is beneficial to adjust relevant parameters based on the evaluation results, thereby achieving more accurate cutting.

[0027] Compared with the prior art, the present invention has at least the following beneficial effects:

[0028] (1) The present invention uses a needle beam for laser capture micro-cutting, which can provide a longer axial range of action, making it more suitable for cutting thick biological samples. It improves the problem that traditional Gaussian beams have insufficient depth of focus in thick samples, making it difficult to cut through or requiring higher energy to cut through.

[0029] (2) By setting a dual-pulse delay control, the first pulse forms a pre-excitation state in the sample, and the second pulse further enhances the energy coupling of the main cutting area, which helps to reduce the threshold energy density required to achieve continuous cutting and improve energy utilization efficiency.

[0030] (3) This invention combines dual-pulse temporal modulation with needle-beam spatial modulation, so that energy deposition is more concentrated in the main lobe region, while ensuring the cutting ability of thick samples, reducing the effective cutting width and reducing the collateral damage induced by side lobes, thereby improving the cutting accuracy.

[0031] (4) The present invention can achieve more precise separation of target areas (the target area is the tissue or cell area that is to be precisely cut, separated and collected) in thick biological samples, reduce the thermomechanical damage of non-target areas (the non-target area is the tissue area around the target area that is not to be affected by the laser), and help maintain the integrity and purity of the separated cells or tissue areas, thereby improving the quality of sample preparation for subsequent omics analysis.

[0032] (5) The system structure of the present invention is clear and the cutting parameters are adjustable. It can be used for laser capture micro-cutting of biological tissue sections, and can also be extended to other micro-processing scenarios with high requirements for fine processing accuracy and low damage of thick samples. Attached Figure Description

[0033] Figure 1 This is a schematic diagram of a dual-pulse needle-beam laser capture micro-cutting system as an example in the embodiments.

[0034] Figure 2 This is a schematic diagram of the structure of the dual-pulse control module exemplified in the embodiment.

[0035] Figure 3 This is a schematic diagram illustrating the principle of needle-shaped beam generation.

[0036] Figure 4 This is a schematic diagram illustrating the working principle of a dual-pulse needle-shaped light beam acting on a sample.

[0037] Figure 5 The following is a flowchart illustrating a dual-pulse needle-beam laser capture micro-cutting method as an example in the embodiments.

[0038] In the diagram, the following modules are labeled: 10-Laser source module; 20-Beam expander module; 30-Polarization control module; 40-Dual pulse control module; 50-Spatial light modulation module; 60-Beam transmission and imaging module; 70-Sample carrying and scanning module; 80-Objective focusing module; 90-Image acquisition module; 401-First beam splitter; 402-Second beam splitter; 403-First reflecting mirror; 404-Second reflecting mirror. Detailed Implementation

[0039] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0040] Please see Figure 1 The dual-pulse needle-beam laser capture micro-cutting system provided in this embodiment includes a laser source module 10, a beam expander module 20, a polarization control module 30, a dual-pulse control module 40, a spatial light modulation module 50, a beam transmission and imaging module 60, an objective lens focusing module 80, a sample carrying and scanning module 70, and an image acquisition module 90.

[0041] The laser source module 10 is used to output pulsed laser light; the beam expander module 20 is disposed on the output path of the laser source module 10 and is used to expand the laser beam to match the effective modulation aperture of the subsequent spatial light modulation module 50; the polarization control module 30 is disposed behind the beam expander module 20 and is used to adjust the polarization state of the laser incident on the spatial light modulation module 50; the dual-pulse control module 40 is disposed behind the polarization control module 30 and is used to split the single-path laser into two paths and recombin them to form a dual-pulse laser with an adjustable time interval; the spatial light modulation module... The beam beam transmission and imaging module 60 is located behind the dual-pulse control module 40 and is used to load a preset phase distribution to modulate the incident laser wavefront to generate a needle-shaped beam. The beam transmission and imaging module 60 is used to transmit the laser beam modulated by the spatial light modulation module 50 to the rear pupil plane of the objective lens. The objective lens focusing module 80 is used to focus the modulated laser beam onto the sample. The sample carrying and scanning module 70 is used to carry the sample to be cut and drive the sample to move relative to the laser focal zone to complete the microscopic cutting along a predetermined trajectory. The image acquisition module 90 is used to illuminate the sample to acquire sample images.

[0042] In this embodiment, the laser source module 10 uses an ultraviolet picosecond laser as the cutting source. Of course, other pulsed laser sources can also be used, such as ultraviolet lasers of other wavelengths, visible light pulsed lasers, near-ultraviolet pulsed lasers, femtosecond lasers, or other ultrafast lasers with different pulse widths.

[0043] The beam expander module 20 is used to adjust the diameter of the incident laser beam to match the effective modulation region of the spatial light modulation module 50. In this embodiment, the beam expander module 20 can be composed of a Galilean beam expander system, a Keplerian beam expander system, or other optical components capable of beam expanding, such as a beam expander system formed by combining two or more lenses.

[0044] The polarization control module 30 is used to adjust the laser polarization state to meet the operating requirements of the spatial light modulation module 50. In this embodiment, the polarization control module 30 can be composed of one or more combinations of polarizers, half-wave plates, quarter-wave plates, polarization beam splitters, and liquid crystal polarization controllers, or other devices capable of polarization control can be used.

[0045] In this embodiment, the dual-pulse control module 40 includes a beam splitter and a reflector. The beam splitter splits the single-pulse input light into two paths. One beam propagates along a reference optical path, and the other beam propagates along a delayed optical path formed by the reflector. The time delay between the two pulses is adjusted by changing the optical path length of the delayed optical path. After beam merging (meaning they coincide in spatial position but are sequential in time), a coaxially propagating dual-pulse laser is output. The time delay is preferably set on the order of nanoseconds so that the first pulse laser forms a pre-excitation state in the sample. When the second pulse laser arrives, it can change the local energy coupling characteristics, that is, the second pulse laser further enhances the energy coupling of the main lobe region and relatively weakens the side lobe correlation response, thereby achieving a better cutting effect.

[0046] Please see Figure 2 In a preferred embodiment, the dual-pulse control module 40 includes a first beam splitter 401, a second beam splitter 402, a first reflector 403, and a second reflector 404. The first beam splitter 401 and the second beam splitter 402 form a reference optical path, and the first reflector 403 and the second reflector 404 form a delay optical path. The single-pulse input light is split into two paths by the first beam splitter 401; one beam is incident on the second beam splitter 402 and then transmitted and output by the second beam splitter 402; the other beam is incident on the first reflector 403, then reflected by the first reflector 403 to the second reflector 404, then reflected by the second reflector 404 to the second beam splitter 402, and finally reflected and output by the second beam splitter 402.

[0047] In this embodiment, a beam splitting, delaying, and combining method is used to form a double pulse, but other implementation methods can also be used, such as free space delay lines, fiber delay lines, pulse shaping modules, double pulse output structures inside the laser, or equivalent double pulse schemes formed by pulse gating.

[0048] In this embodiment, the spatial light modulation module 50 employs a spatial light modulator, constructing multiple discrete focal points along the optical axis based on spatial multiplexing, and forming a needle-shaped beam with a predetermined axial length through phase superposition, such as... Figure 3 As shown, the axial length of the needle-shaped beam is greater than the thickness of the thick biological sample to be cut, so that the laser's effective range can cover the entire sample thickness. A blazed grating can also be superimposed on the phase diagram loaded by the spatial light modulation module 50 to separate the zero-order light and improve the quality of the effectively modulated beam.

[0049] In this embodiment, a spatial light modulator is used to generate a needle-shaped beam, but it is not limited to this. Any scheme that can form a light field distribution with an extended range of action along the optical axis that is greater than or covers the thickness of the sample to be cut can be used, such as Bessel-type beams, needle-shaped beams, or multifocal axially superimposed beams generated by diffractive optical elements, axial conical lenses, phase plates, digital micromirror devices, or other wavefront modulation elements.

[0050] The beam transmission and imaging module 60 is used to transmit the light field modulated by the spatial light modulation module 50 to the rear pupil plane of the objective lens and to achieve conjugate imaging of the light field. The beam transmission and imaging module 60 can be a 4f system composed of one or more lenses, or it can be constructed using a relay lens group, a telescope system, a scanning lens and tube lens combination system, or other optical systems capable of beam transmission, magnification / reduction, and conjugate imaging. Preferably, the beam transmission and imaging module 60 can be constructed as a 4f imaging system composed of a first lens and a second lens, used to conjugate image the phase modulation surface of the spatial light modulation module 50 onto the rear focal plane of the objective lens.

[0051] It should be noted that in this embodiment, the spatial light modulation module 50 loads the phase distribution used to generate the needle-shaped beam, not the final light field itself. The spatially modulated beam undergoes diffraction evolution during free propagation, causing the light field distribution reaching the rear pupil of the objective lens to differ from the designed phase distribution of the output surface of the spatial light modulation module 50. Therefore, a beam transmission and imaging module 60 (such as a 4f imaging system or other relay imaging system) is needed to conjugate the spatial light modulation surface onto the rear pupil of the objective lens to ensure that the phase modulation result accurately acts on the objective lens and forms the expected needle-shaped beam. However, theoretically, when the distance between the spatial light modulation module 50 and the objective lens is extremely short, or when other methods are used to ensure that the phase distribution after spatial light modulation can be effectively transmitted to the rear pupil of the objective lens, a separate beam transmission and imaging module 60 may not be necessary.

[0052] In this embodiment, the objective focusing module 80 includes a low numerical aperture microscope objective for generating and focusing a needle-shaped beam. By selecting a lower numerical aperture objective, the influence of defocusing aberration on the axial extension characteristics of the needle-shaped beam can be reduced, thereby obtaining a needle-shaped beam that meets the requirements for cutting thick samples.

[0053] In the experimental example, the objective focusing module 80 may also include a high numerical aperture microscope objective. This high numerical aperture objective can be used to generate a Gaussian beam, which is then compared with the needle-like beam to analyze the effectiveness of the needle-like beam. Generally, ≥0.6 is considered a high numerical aperture, and <0.4 is considered a low numerical aperture.

[0054] The sample carrying and scanning module 70 includes an inverted microscope platform and a controllable displacement stage. The sample to be cut is placed on the displacement stage. The displacement stage drives the sample to move according to a preset speed and trajectory, so that the needle-shaped laser beam completes a single scan and cut along a predetermined cutting line. It is easy to understand that the inverted microscope platform is simply based on... Figure 1 The structural layout shown allows for different mounting methods for the microscope platform depending on the specific local conditions.

[0055] The sample to be cut is a thick biological sample, preferably a tissue sample that has been fixed, dehydrated, protected, and frozen into sections. The sample can be mounted on a glass slide with a polyethylene naphthalate (PEN) membrane. Of course, the system described in this embodiment is not only suitable for frozen tissue sections, but also for paraffin tissue sections, cell sheet samples, thin-layer samples of living tissue, and other thick samples that require low-damage, high-precision separation.

[0056] In this embodiment, a displacement stage is used to drive the sample to generate relative motion with the laser focal zone to complete the cutting, but it is not limited to this. Beam scanning or a method of coordinated movement between the beam and the sample can also be used to achieve cutting along a predetermined trajectory, such as galvanometer scanning, piezoelectric platform scanning, and motorized displacement stage scanning.

[0057] The image acquisition module 90 includes an illumination source and an image sensor, used to acquire bright-field images before and after cutting under fixed imaging conditions (i.e., the displacement stage is stationary). Sample images are acquired before cutting to align the imaging plane with the area to be cut, and sample images are acquired after cutting to observe and record the cutting results for subsequent evaluation of cutting width and damage morphology. The delay time parameter and incident energy density between the dual-pulse lasers can also be adjusted based on the cutting effect.

[0058] It is easy to understand that, Figure 1 The structure shown is merely an example of one possible implementation of the above system; the specific composition of each module can be implemented in other different ways.

[0059] See also Figure 4 This diagram illustrates the working principle of a dual-pulse needle-shaped light beam applied to a sample. Compared to a traditional Gaussian focused beam, the needle-shaped beam has a longer axial range of action, capable of covering the thickness of thicker biological samples. This avoids the problem of insufficient depth of focus in thick samples caused by Gaussian beams, which sometimes result in incomplete cutting or require higher energy for cutting. Simultaneously, the dual-pulse irradiation method induces free electron generation and local pre-excitation in the sample with the first pulse. When the second pulse arrives, the main lobe region, due to its higher initial light intensity, exhibits stronger subsequent energy coupling, while the side lobes, with their lower initial light intensity, show relatively limited enhancement in subsequent response. Therefore, the dual-pulse needle-shaped beam allows energy deposition to be more spatially concentrated in the main lobe region, thus maintaining the cutability of thick samples while reducing collateral damage induced by side lobes.

[0060] Please see Figure 5 This embodiment also provides a dual-pulse needle-beam laser capture micro-cutting method based on the above system, including the following steps:

[0061] Step 1: Sample positioning and focusing. Acquire an image of the sample before cutting using the image acquisition module. Adjust the objective lens or sample position based on the image to align the imaging plane with the area to be cut.

[0062] Step 2: Output pulsed laser and perform beam expansion and polarization adjustment. The pulsed laser output from the laser source module is expanded by the beam expansion module and then enters the polarization control module to obtain a polarization state suitable for the operation of the spatial light modulator.

[0063] Step 3: Generate a dual-pulse laser. The single-pulse laser is split into two beams to form a dual-pulse laser with selective energy deposition for controlling the cutting process. For example, the polarization-adjusted laser enters the dual-pulse control module, is split into two paths, and then propagates along the reference optical path and the delay optical path, respectively. They are then recombined in space to form a dual-pulse laser with a predetermined time interval. By adjusting the optical path length of the delay optical path, different dual-pulse time delay parameters can be obtained.

[0064] Step 4, Laser Modulation. The incident laser is wavefront modulated to create an optical field with an axial effective range greater than or covering the thickness of the sample to be cut. For example, a phase distribution for generating a needle-like beam is loaded onto a spatial light modulator. After a dual-pulse laser is incident on the spatial light modulator, multiple axially discrete focal points are distributed along the propagation direction and superimposed to form a needle-like beam with an extended focal range. The needle-like beam is transmitted to the rear pupil plane of the objective lens via a beam transmission and imaging module, and then focused onto the sample area by the objective lens. To compensate for thick samples and potential deviations between the cutting focal plane and the imaging focal plane in the actual optical path, the longer axial effective range of the needle-like beam is used, ensuring that the effective laser area covers the target tissue layer.

[0065] Step 5: Perform dual-pulse needle beam cutting. Control the sample carrying and scanning module to move the sample relative to the needle beam along a preset path, completing the target trajectory cutting under single-scan conditions. By adjusting the dual-pulse delay parameters and incident energy density, the first pulse preferentially induces pre-excitation in the main lobe region, and the second pulse further enhances the energy coupling in the main lobe region and relatively weakens the sidelobe correlation response, thereby reducing the cutting threshold, narrowing the effective cutting width, and mitigating sidelobe collateral damage.

[0066] After cutting, the cutting effect can be evaluated so that parameters can be adjusted to achieve a better cutting result. Therefore, the above method may also include:

[0067] Step 6: Acquire and evaluate the cutting results. Use the image acquisition module to acquire images of the cut samples and observe the cutting trajectory under fixed imaging conditions. The cutting threshold, cutting width, and morphology of sidelobe-related damage can be evaluated. The cutting threshold can be defined as the minimum incident energy density required to form a continuous slit along a preset cutting trajectory in a single scan. The cutting width can be defined as the lateral range of the main cutting area. Sidelobe-related damage can be characterized by the morphology of the non-target affected areas on both sides of the main slit.

[0068] The evaluation method is not limited to manual observation. Image processing, automatic recognition, or closed-loop feedback control can also be used to optimize and adjust the double pulse delay, incident energy density, cutting trajectory, or cutting quality.

[0069] The above specific embodiments are merely several optional embodiments of the present invention. Based on the technical solutions of the present invention and the relevant teachings of the above embodiments, those skilled in the art can make various alternative improvements and combinations to the above specific embodiments.

Claims

1. A dual-pulse needle-beam laser capture micro-cutting system, characterized in that, The laser light source module, the beam expansion module, the polarization control module, the double-pulse control module, the spatial light modulation module, the objective focusing module, the sample support and scanning module, and the image acquisition module are included. The laser light source module is configured to output a single-pulse laser light. The beam expansion module is arranged on the light path of the laser light source module and configured to expand the pulse laser beam to match the effective modulation aperture of the spatial light modulation module. The polarization control module is arranged behind the beam expansion module and configured to adjust the polarization state of the laser light incident on the spatial light modulation module. The double-pulse control module is arranged behind the polarization control module and configured to split the single-pulse laser light into two beams and form a double-pulse laser light with adjustable energy deposition selectivity during the cutting process. The spatial light modulation module is arranged behind the double-pulse control module and configured to modulate the wavefront of the incident laser light to form a light field with an axial action range greater than or covering the thickness of the sample to be cut. The image acquisition module is configured to acquire the image of the sample before cutting to adjust the position between the sample to be cut and the objective lens to align the imaging plane with the cutting area. The objective focusing module is configured to focus the modulated laser light on the sample to be cut. The sample support and scanning module is configured to support the sample to be cut and drive the relative movement between the sample to be cut and the laser focal domain to complete the micro-cutting along the predetermined trajectory.

2. The dual-pulsed needle beam laser capture microdissection system of claim 1, wherein, The double-pulse control module provides a reference light path and a delay light path. One of the pulse laser lights propagates along the reference light path, and the other pulse laser light propagates along the delay light path. The delay time between the two pulse laser lights is adjusted by changing the optical path length of the delay light path, so that one pulse laser light forms a pre-excitation state in the sample to be cut after acting, and the other pulse laser light changes the local energy coupling characteristics when it arrives.

3. The dual-pulsed needle beam laser capture microdissection system of claim 2, wherein, The double-pulse control module includes two beam splitters and at least two mirrors. The two beam splitters form the reference light path, and the mirrors form the delay light path. One of the beam splitters splits the single-pulse input light into two beams, and the pulse laser lights output from the reference light path and the delay light path are coaxially output by the other beam splitter.

4. The dual-pulsed needle beam laser capture microdissection system of claim 1, wherein, The light beam transmission and imaging module is arranged between the spatial light modulation module and the objective focusing module. The spatial light modulation module uses a spatial light modulator to construct multiple discrete focal points in the optical axis direction based on spatial multiplexing, and forms a needle-shaped light beam with a predetermined axial length through phase superposition. The light beam transmission and imaging module is configured to transmit the light field modulated by the spatial light modulation module to the objective back pupil plane and realize the conjugate imaging of the light field.

5. The dual-pulsed needle beam laser capture microdissection system of claim 4, wherein, The light beam transmission and imaging module is a 4f imaging system composed of a first lens and a second lens.

6. The dual-pulsed needle beam laser capture microdissection system of claim 1, wherein, The sample support and scanning module includes a microscope platform and a displacement table. The sample to be cut is arranged on the displacement table, and the displacement table is configured to drive the sample to be cut to move at a predetermined speed and trajectory, so that the laser completes single-scan cutting along the predetermined cutting line.

7. A method for double-pulsed needle beam laser capture microdissection realized by the double-pulsed needle beam laser capture microdissection system according to claim 1, characterized in that, The method includes the following steps: Step 1: Acquire the image of the sample before cutting, and adjust the objective lens or the sample position according to the sample image to align the imaging plane with the cutting area. Step 2, output single pulse laser, expand the pulse laser beam, and adjust the polarization state of the expanded laser to adapt to the spatial light modulation module; Step 3, split the single pulse laser, and form a double pulse laser with selective energy deposition for regulating the cutting process; Step 4, wavefront modulation is performed on the incident laser to form a light field with an axial action range greater than or covering the thickness of the sample to be cut, and the modulated laser is focused on the sample to be cut; Step 5, drive the sample to be cut to move relative to the laser focal domain to complete the micro-cutting of the predetermined track.

8. The dual-pulsed needle beam laser capture microdissection method of claim 7, wherein, Step 6, collect the image of the cut sample, observe the cutting track, and evaluate the cutting threshold, cutting width, and sidelobe damage morphology.

9. The dual-pulsed needle beam laser capture microdissection method of claim 7, wherein, In step 2, after splitting the single pulse laser into two beams, one of the pulse lasers propagates along a reference light path, and the other pulse laser propagates along a delay light path. By changing the optical path length of the delay light path, the delay time between the two pulse lasers is adjusted to form a pre-excitation state in the sample to be cut after the action of one pulse laser, and the other pulse laser changes the local energy coupling characteristics when it arrives.

10. The dual-pulsed needle beam laser capture microdissection method of claim 7, wherein, In step 3, a spatial light modulator is used to construct multiple discrete focal points in the optical axis direction based on spatial multiplexing, and a needle-shaped beam with a predetermined axial length is formed by phase superposition.

Images