A laser microdissection system based on coherent raman imaging and its application

Abstract

This application belongs to the field of laser micro-cutting, and relates to a laser micro-cutting system based on coherent Raman imaging and its application. The device includes a femtosecond laser, a beam splitter, a dichroic mirror, and a laser scanning microscope. A pump optical path and a Stokes optical path are provided between the beam splitter and the dichroic mirror. The pump optical path includes an optical parametric amplifier and a movable dispersion broadening module; the Stokes optical path contains a Martinez broadener. The femtosecond laser beam is split in the beam splitter and then enters the pump optical path and the Stokes optical path respectively. One beam is passed through the optical parametric amplifier to generate a wavelength-tunable first pump beam, and the other beam is passed through the Stokes optical path to generate a Stokes beam. The first pump beam and the Stokes beam are combined in the dichroic mirror. The laser scanning microscope is used to perform coherent Raman imaging using the combined beam, and also to perform laser micro-cutting according to the coherent Raman image. This application can significantly improve the accuracy, selectivity, and real-time performance of the laser micro-cutting process.

Description

Technical Field

[0001] This application belongs to the field of laser micro-cutting, and more specifically, relates to a laser micro-cutting system based on coherent Raman imaging and its application. Background Technology

[0002] Laser microdissection (LMD) is a technique that uses laser technology to precisely separate specific cells or tissue regions from tissue sections. Conventional microdissection typically involves using a wide-field microscope to observe tissue sections, identify the specific cells or tissue regions to be cut, and then using methods such as laser cutting to separate the target area. Finally, the separated tissue or cells are collected. The advantage of laser microdissection lies in its ability to separate single-type cells from complex tissue environments, which is crucial for studying cellular heterogeneity and performing precise molecular analysis. Furthermore, its non-contact operation avoids cell damage and contamination that can occur with traditional mechanical cutting.

[0003] Observation methods for laser microdissection typically employ wide-field microscopy, including bright-field microscopy, dark-field microscopy, phase-contrast microscopy, and wide-field fluorescence microscopy. In situations with low contrast, staining is required, which can damage the sample. Some staining agents can interfere with subsequent molecular analysis, and prolonged staining can even increase the risk of RNA degradation. Therefore, the staining process must ensure that the staining agent does not interfere with subsequent analysis, increasing the complexity of the procedure and requiring additional time and skill to handle stained sections. Thus, a new laser microdissection device is needed to address the issue of sample staining required in traditional laser microdissection procedures. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the purpose of this application is to provide a laser microdissection system based on coherent Raman imaging and its application, aiming to solve the problem that traditional laser microdissection surgery requires staining of samples, which affects subsequent molecular analysis.

[0005] To achieve the above objectives, this application provides a laser micro-cutting system based on coherent Raman imaging, including a femtosecond laser, a beam splitter, a dichroic mirror, and a laser scanning microscope. A pump optical path and a Stokes optical path are provided between the beam splitter and the dichroic mirror. The pump optical path includes an optical parametric amplifier and a movable dispersion broadening module, and the Stokes optical path includes a Martinez broadener. The femtosecond laser is used to emit a laser beam; the beam splitter is used to split the laser beam and allow the split beams to enter the pump optical path and the Stokes optical path respectively; the dispersion broadening module and the Martinez broadener are used to adjust the dispersion values ​​of the beams in the corresponding optical paths to match each other; The dichroic mirror is used to combine the first pump light generated by the optical parametric amplifier in the pump light path with the Stokes light emitted from the Stokes light path; the laser scanning microscope is used to scan the sample with the combined light emitted from the dichroic mirror to obtain a coherent Raman image; when the dispersion broadening module is removed and the Stokes light path is turned off, a split beam emitted from the beam splitter enters the optical parametric amplifier to generate a second pump light, the pump light path can adjust the power of the second pump light, and the laser scanning microscope can also use the second pump light with adjusted power to cut the corresponding sample.

[0006] Furthermore, the single-pulse output energy of the femtosecond laser is at least 10 μJ, its repetition rate is not less than 200 kHz, and its pulse width is not greater than 500 femtoseconds.

[0007] Furthermore, along the propagation direction of the pump light path, a pump light power control unit, a right-angle mirror, and a reflector are sequentially arranged after the optical parametric amplifier. The dispersion broadening module is located between the right-angle mirror and the reflector, with the reflector facing the output end of the dispersion broadening module. The split beam sequentially enters the optical parametric amplifier, the pump light power control unit, the right-angle mirror, and the dispersion broadening module before entering the reflector, being reflected by the reflector to the dispersion broadening module, and then reflected by the right-angle mirror to the dichroic mirror.

[0008] Furthermore, the wavelength of the optical parametric amplifier is 750nm~980nm, its output energy is not less than 1uJ, and its pulse width is not greater than 500fs.

[0009] Furthermore, a piezoelectric reflector is disposed between the right-angle reflector and the dichroic mirror, and a self-collimating beam position detection module is disposed beside the dichroic mirror. The self-collimating beam position detection module is used to detect the beam combining state of the beam emitted from the dichroic mirror, and the piezoelectric reflector is used to adjust the propagation direction of the first pump light using the beam combining state.

[0010] Furthermore, a first silver-plated reflector is disposed between the optical parametric amplifier and the pump optical power control unit, and an optical power detection unit is disposed next to the first silver-plated reflector. The optical power detection unit is used to detect the power of the light beam emitted from the optical parametric amplifier and entering the first silver-plated reflector.

[0011] Furthermore, a second silver-plated reflector is disposed between the beam splitter and the optical parametric amplifier, and a beam position detection module is disposed beside the second silver-plated reflector. The beam position detection module is used to detect the position of the beam emitted from the beam splitter and entering the second silver-plated reflector.

[0012] Furthermore, the dispersion broadening module consists of two glass rods connected end to end, with its central axis parallel to the beam propagation direction of the pump optical path.

[0013] Furthermore, along the propagation direction of the Stokes optical path, a Stokes optical power control module and a delay line are sequentially arranged. The Stokes optical power control module is located between the beam splitter and the Martinez stretcher, and the delay line is located between the Martinez stretcher and the dichroic mirror.

[0014] According to a second aspect of this application, a method for applying the laser micro-cutting system as described above is also provided, comprising: S1 causes the femtosecond laser to emit a laser beam toward the beam splitter; the beam splitter splits the laser beam and causes the split beams to enter the pump optical path and the Stokes optical path respectively, generating a first pump beam and a Stokes beam. S2 causes the dispersion broadening module to adjust the dispersion value of the first pump light, and the Martinez broadener to adjust the dispersion value of the Stokes light, until the two are matched and then enter the dichroic mirror to combine. S3 enables the laser scanning microscope to receive the combined beam of light emitted from the dichroic mirror and scan the sample to obtain a coherent Raman image; S4 removes the dispersion broadening module and shuts down the Stokes optical path, allowing a split beam emitted from the beam splitter to enter the optical parametric amplifier to generate a second pump beam; the power of the pump beam is adjusted so that the laser scanning microscope uses the adjusted second pump beam to cut the corresponding sample.

[0015] Overall, the technical solutions conceived in this application have the following beneficial effects compared with the prior art: (1) This application combines coherent Raman spectroscopy with laser microdissection technology, which can significantly improve the accuracy, selectivity and real-time performance of the dissection process, and bring many advantages in terms of sample purity, imaging depth and operational efficiency. First, coherent Raman imaging can achieve chemically selective imaging based on Raman activity under label-free conditions, providing precise molecular guidance for laser microdissection. Coherent Raman imaging can directly visualize specific chemical components (such as lipids, proteins or metabolites) in the sample without labels, thereby accurately identifying the target area. Second, the combination of coherent Raman and laser microdissection significantly improves the accuracy of laser microdissection. After coherent Raman is combined with the cutting laser, the chemical distribution changes of sample molecules can be monitored in real time during the dissection process, and the dissection path can be dynamically adjusted.

[0016] (2) In this application, by reasonably designing the pump optical path and the Stokes optical path, the acquisition of coherent Raman images and the cutting of laser microscopy directly depend on the coordinates of the galvanometer itself, thereby avoiding the re-registration of the imaging space and cutting space coordinates in laser microscopy cutting, and also avoiding the frequent adjustment and correction of the long-term laser pointing stability after a period of use.

[0017] (3) The dispersion broadening module in this application applies different dispersion values ​​to femtosecond pulses of different wavelength ranges generated by the optical parametric amplifier, producing different pulse broadening effects. When performing coherent Raman imaging, this device is controlled by an electric stage or other similar displacement device to move the right-angle mirror into the optical path. When performing laser micro-cutting, this device is controlled by an electric stage to move out. This design scheme can ensure that approximately 10cm is available for coherent Raman imaging. -1 It has excellent spectral line resolution and can ensure that the laser has a high peak power after being removed from the module, which is convenient for micro laser cutting. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of a laser micro-cutting system based on coherent anti-Stokes Raman scattering imaging provided in an embodiment of this application; Figure 2 This is a schematic diagram of the light spot output by the Stokes beam after passing through the Martinez stretcher, as provided in the embodiments of this application. Figure 3 This is a schematic diagram showing the correspondence between the moving position of the Martinez stretcher and the output pulse width provided in the embodiments of this application; Figure 4 The coherent Raman scan images obtained for the embodiments of this application; Figure 5 This is a schematic diagram of the image obtained by coherent Raman scanning after being cut off at the upper left and lower right, as provided in an embodiment of this application.

[0019] In all the accompanying drawings, the same reference numerals are used to denote the same elements or structures, wherein: 1-Delay line, 2-Beam position detection module, 3-Dichroic mirror, 4-Stokes power control module, 5-D-shaped mirror, 6-Martinez broadener, 7-Grate, 8-Plan-convex lens, 9-Dispersion broadening module, 10-Sample, 11-Piezoelectric mirror, 12-Right-angle mirror, 13-Pump power control module, 14-Beam splitter, 15-Beam position detection module, 16-Power detection module, 17-Mirror, 18-First silver-plated mirror, 19-Second silver-plated mirror. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0021] In this article, the term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. The symbol " / " in this article indicates that the related objects are in an "or" relationship; for example, A / B means A or B.

[0022] The terms "first" and "second," etc., used in the specification and claims herein are used to distinguish different objects, not to describe a specific order of objects. For example, "first response message" and "second response message," etc., are used to distinguish different response messages, not to describe a specific order of response messages.

[0023] In the embodiments of this application, the terms "exemplary" or "for example" are used to indicate that something is an example, illustration, or description. Any embodiment or design that is described as "exemplary" or "for example" in the embodiments of this application should not be construed as being more preferred or advantageous than other embodiments or design. Specifically, the use of the terms "exemplary" or "for example" is intended to present the relevant concepts in a specific manner.

[0024] In the description of the embodiments of this application, unless otherwise stated, "multiple" means two or more, for example, multiple processing units means two or more processing units, multiple elements means two or more elements, etc.

[0025] Traditional target localization methods in microdissection mainly rely on bright-field or fluorescence imaging. However, these methods can only distinguish samples based on morphological or labeling signals and lack molecular selectivity. Traditional microdissection often uses wide-field imaging before cutting, lacking real-time chemical guidance, and is prone to cutting deviations due to sample drift or localization errors.

[0026] Specifically, conventional laser micro-cutting, to achieve accurate and clean cutting, often uses pulsed lasers with single-pulse energies in the microjoule range and repetition rates around tens of kHz. Such lasers are too powerful and have too low a repetition rate for coherent Raman imaging. Conversely, conventional coherent Raman imaging, to avoid sample damage while maintaining high scanning speed, typically uses lasers with single-pulse energies in the nanojoule range and repetition rates above MHz. To achieve high spectral resolution, coherent Raman imaging requires picosecond pulses or dispersive spectral focusing to broaden femtosecond pulses for imaging. However, for laser micro-cutting, shorter laser pulses mean higher peak power; broadened pulses reduce peak power, which is detrimental to laser micro-cutting. Therefore, the lasers used for scanning imaging and laser cutting are fundamentally different. Due to the aforementioned difficulties and problems, there is currently no system that combines coherent Raman spectroscopy with laser micro-cutting. The table below lists some commercially available laser micro-cutting systems and the methods used to generate images for marking the cuts.

[0027] Table 1. Conventional microdissection systems and corresponding marked dissection images.

[0028] Microsurgical cutting techniques based on wide-field imaging require registration of the cutting coordinate system with the observation coordinate system. In practical applications, due to long-term operation, the laser beam direction may shift, causing a mismatch between the cutting position and the region of interest. This can also lead to inaccurate cutting positions and contamination of cells of interest.

[0029] Therefore, this application proposes a laser microdissection system based on coherent anti-Stokes Raman scattering imaging. Utilizing the advantages of coherent Raman imaging, such as stain-free labeling and high-sensitivity chemically specific imaging, it avoids the need for sample staining in traditional laser microdissection procedures. Images obtained from coherent Raman imaging can directly guide laser microdissection. Simultaneously, it eliminates the need for positioning and registration of CCD-acquired images with the laser microdissection path coordinates during laser microdissection. Since the sample images obtained from coherent Raman imaging are inherently dependent on the galvanometer scanning path, misalignment between the cutting and observation positions during laser microsurgery is avoided, and routine correction and maintenance of the laser beam direction during microdissection are also unnecessary.

[0030] The embodiments of this application are described below with reference to the accompanying drawings.

[0031] This application provides a laser micro-cutting system based on coherent Raman imaging, such as... Figure 1As shown, it includes a femtosecond laser, a beam splitter 14, a dichroic mirror 3, and a laser scanning microscope. A pump optical path and a Stokes optical path are provided between the beam splitter 14 and the dichroic mirror 3. The pump optical path includes an optical parametric amplifier and a movable dispersion broadening module 9. The Stokes optical path contains a Martinez broadener 6. The femtosecond laser emits a laser beam. The yellow laser beam enters the beam splitter 14 and is split into two beams. The blue beam enters the pump optical path and, after passing through the optical parametric amplifier, is emitted as the first pump beam. The green beam enters the Stokes optical path, generating Stokes light. Dispersion broadening is then performed. Module 9 and Martinez broadener 6 are used to adjust the dispersion value of the beam in the corresponding optical path to match each other; dichroic mirror 3 is used to combine the first pump light emitted from the pump light path and the Stokes light emitted from the Stokes light path; the laser scanning microscope is used to scan the sample 10 with the yellow combined light emitted from the dichroic mirror 3 to obtain a coherent Raman image; when the dispersion broadening module 9 is removed and the Stokes light path is turned off, the laser beam enters the optical parametric amplifier to generate a second pump light, the pump light path can adjust the power of the second pump light, and the laser scanning microscope can use the second pump light with adjusted power to cut the corresponding sample 10.

[0032] Specifically, the aforementioned amplification stage femtosecond laser is used to generate high repetition rate and high energy femtosecond pulses. Its energy can be used to pump the optical parametric amplifier at the back end, while also leaving a margin for use as Stokes light in coherent Raman imaging. At the same time, its repetition rate should be as high as possible to meet the requirements of high-speed scanning imaging.

[0033] In this embodiment, the single-pulse output energy of the femtosecond laser is at least 10 μJ, its repetition rate is not less than 200 kHz, and its pulse width is not greater than 500 femtoseconds. Specifically, the amplification stage femtosecond laser is used to generate high-repetition-rate, high-energy near-infrared laser with a repetition rate of 1 MHz, a single-pulse energy of about 20 μJ, and a pulse width of about 250 fs. Of this, 19 μJ of energy is used to pump the optical parametric amplifier, and the remaining 1 μJ is used as Stokes light in coherent Raman imaging.

[0034] The spectral focusing module consists of two parts: a dispersion broadening module 9 placed in the Stokes beam path and a Martinez broadener 6 placed in the pump beam path. It is used to apply equal amounts of dispersion chirp to the pump beam and the Stokes beam while ensuring that the spatial pointing of the two beams is aligned, thereby achieving high spectral line resolution scanning imaging in coherent Raman scanning imaging.

[0035] Specifically, for Stokes light wavelengths, the Martinez broadener 6 uses a grating 7 with high dispersion efficiency and transmittance, paired with a D-shaped mirror 5, a plano-convex lens 8 with a certain focal length, and a mirror with a suitable coating (a general silver-plated mirror is sufficient) and a climbing mirror assembly. The plano-convex lens 8, grating 7, and a mirror are placed on a one-dimensional motion platform. The D-shaped mirror 5 is placed on the propagation path of the Stokes light transmitted from the Stokes light power control module 4 to the delay line 1. The movement distance of the one-dimensional motion platform can be controlled according to the wavelength of different lights used to generate different levels of dispersion, matching the dispersion generated by the dispersion broadening module 9 placed in the pump optical path, thereby realizing the function of high spectral line resolution scanning imaging in coherent Raman scanning imaging. Figure 2 As shown, the beam output from the Stokes beam after passing through the Martinez stretcher 6 still possesses good beam quality. Figure 3 The figure shows the correspondence between the output pulse width and the partial structural movement in the Martinez stretcher 6.

[0036] In this embodiment, the aforementioned pump optical path is specifically provided with an optical parametric amplifier, a pump optical power control unit 13, a right-angle mirror 12, and a mirror 17 in sequence. The dispersion broadening module 9 is located between the right-angle mirror 12 and the mirror 17, with the mirror 17 facing the output end of the dispersion broadening module 9. The split beam enters the optical parametric amplifier, the pump optical power control unit 13, the right-angle mirror 12, and the dispersion broadening module 9 in sequence, and then enters the mirror 17. It is reflected by the mirror 17 into the dispersion broadening module 9, and then reflected by the right-angle mirror 12 into the dichroic mirror 3 to participate in beam combining.

[0037] In this embodiment, the wavelength of the optical parametric amplifier is 750nm~980nm, such as 750nm, 780nm, 800nm, 850nm, 900nm or 950nm, etc., and its output energy is not less than 1uJ and its pulse width is not greater than 500fs. Specifically, when the aforementioned femtosecond laser is selected, the optical parametric amplifier will generate signal light and idler light under the pump of 19uJ femtosecond pulse light. The generated light is split by a dichroic mirror, and the signal light portion is used as the pump light in coherent Raman imaging (i.e., the first pump light), and also as the laser used to cut sample tissue in microlaser surgery (i.e., the second pump light). The conversion efficiency of the optical parametric amplifier is generally about 7%. In this embodiment, about 1μJ of signal light will be output through the optical parametric amplifier, and its pulse width is about 300fs.

[0038] In this embodiment, a piezoelectric reflector 11 is also provided between the aforementioned right-angle reflector 12 and dichroic mirror 3. A self-collimating beam position detection module 2 is provided on the side of the dichroic mirror 3. The self-collimating beam position detection module 2 is used to detect the beam combining state of the beam emitted from the dichroic mirror 3. The piezoelectric reflector 11 is used to adjust the propagation direction of the first pump light by utilizing the beam combining state. That is, when a deviation is found in the beam combining state, the piezoelectric reflector can be finely adjusted to make the two beams combine.

[0039] The aforementioned self-collimating beam position detection module 2 can detect the overlap and pointing stability of two beams in the optical path, ensuring that the optical path does not shift after beam merging, while ensuring that the coherent Raman signal has a high signal-to-noise ratio.

[0040] In this embodiment, a first silver-plated reflector 18 is also provided between the optical parametric amplifier and the pump power control unit 13. An optical power detection unit 16 is provided next to the first silver-plated reflector 18. The optical power detection unit 16 is used to detect the power of the light beam emitted from the optical parametric amplifier and entering the first silver-plated reflector 18. Specifically, the light beam emitted from the optical parametric amplifier is reflected by the first silver-plated reflector 18 and enters the pump power control unit 13. At the same time, some light will pass through the silver-plated reflector 18. The optical power detection unit 16 can capture this part of the transmitted light for power detection, which is used as a reference for adjusting the laser beam.

[0041] In a more preferred embodiment, a second silver-plated reflector 19 is further disposed between the beam splitter 14 and the optical parametric amplifier. A beam position detection module 15 is disposed beside the second silver-plated reflector 19. The beam position detection module 15 can be a laser position tester or a beam position detector, used to detect the position of the beam emitted from the beam splitter 14 and entering the second silver-plated reflector 19. Specifically, one beam of light after being split by the beam splitter 14 enters the second silver-plated reflector 19 and is reflected into the optical parametric amplifier by the second silver-plated reflector 19. A portion of the light will pass through the second silver-plated reflector 19, and the beam position detection module 15 captures this portion of the passed-through light to detect the beam position.

[0042] In this embodiment, the aforementioned dispersion broadening module 9 is disposed on an electric displacement platform, the moving stroke of the electric displacement platform is no more than 300mm, and the stepping accuracy is less than 2μm.

[0043] Specifically, the dispersion broadening module 9 consists of two glass rods connected end-to-end, with their central axes parallel to the beam propagation direction of the pump light path. High-dispersion glass rods can be used, and anti-reflection coatings are deposited on both ends of the glass rods to improve light transmission efficiency in the waveband used for the pump light. The length of the glass rods is selected based on the second-order dispersion coefficient of the glass material and the target chirp, achieving a length of less than 10cm. -1Wavenumber coherent Raman imaging spectral line resolution.

[0044] More specifically, the high-dispersion glass rods are placed end to end, so that the pump light passes back and forth in the glass rods four times and is then broadened into a pulse of about 3 ps. The Martinez stretcher is adaptively designed according to the center wavelength and pulse width of the aforementioned femtosecond laser. It should be understood that those skilled in the art can make adaptive designs according to femtosecond lasers with different center wavelengths and pulse widths, which will not be elaborated here.

[0045] The aforementioned Stokes optical path is sequentially configured with: Stokes optical power control module 4, D-shaped reflector 5, and delay line 1, with Martinez stretcher 6 located between D-shaped reflector 5 and delay line 1.

[0046] The Stokes light power control module 4 is placed in the pump light path to adjust the optical power of the pump beam. Its working principle is as follows: a stepper motor drives a half-wave plate to rotate, which, in conjunction with a polarization beam splitter, controls the optical power. During coherent Raman imaging, the optical power of both the pump light and the Stokes light is attenuated to approximately 30mW to obtain a clear image while avoiding damage to the laser power of the sample 10. During microsurgical laser cutting, the pump light power control module adjusts the second pump light, increasing its laser power to approximately 300mW, and adjusts its wavelength to match the absorption of the sample to be cut, thus efficiently removing the corresponding sample 10.

[0047] The aforementioned delay line 1 is used to control the time synchronization of the pump light and the Stokes light. Specifically, it consists of a hollow retroreflector and a one-dimensional motion platform. Since its structure is an existing design, it will not be described in detail here. Using a hollow retroreflector in the delay line helps ensure that the directivity of the Stokes light remains unchanged after passing through the delay line, and that the Stokes light and pump light achieve good spatial collinearity at any position of the one-dimensional motion platform. The aforementioned hollow retroreflector has a 60mm aperture for easy adjustment within the optical path, and its surface is coated with a gold film to ensure high reflectivity throughout the tunable range of the pump light.

[0048] The aforementioned laser scanning microscope consists of conventional units such as a digital galvanometer, a scanning relay optical path, a non-decoherent acquisition module, and a high numerical aperture objective lens. It has high transmittance for the laser band and can achieve scanning of the excitation light on the sample surface in a tightly focused state and acquire signals generated nonlinearly.

[0049] The operation method of the laser micro-cutting system based on coherent Raman imaging provided in the foregoing embodiments is as follows: (1) Power on and preheat the femtosecond laser and optical parametric amplifier, and then ensure that the femtosecond laser is in normal working condition according to the reading of the optical power meter and the beam position detection module 15.

[0050] (2) Prepare for coherent Raman scanning imaging of the sample. First, adjust the power of the two lasers to a low level, generally around tens of milliwatts, depending on the sample properties and the signal strength in the laser scanning microscope. Then, move the dispersion broadening module at the pump light into the optical path, and move the Martinez broadening stage to a suitable position according to the wavelength of the pump light, so that the dispersion magnitude of the Stokes light matches that of the pump light, meeting the requirements of high spectral line resolution during coherent Raman imaging. Finally, use the beam position detection module 15 to confirm the overlap of the pump light and the Stokes light beams. If the overlap is good, the subsequent coherent Raman scanning imaging experiment can be carried out. If there is a deviation in the overlap, the piezoelectric reflector can be finely adjusted to make the two beams overlap.

[0051] It should be noted that this optical path module layout design can ensure that the beam direction remains unchanged when moving the displacement stage of the Martinez expander and after moving the dispersion broadening module into and out of the optical path. Therefore, it is generally not necessary to adjust the piezoelectric mirror. However, the beam direction may inevitably deviate during long-term operation of the system. At this time, the piezoelectric mirror can be used for adjustment to ensure that the beam direction position remains unchanged.

[0052] After completing the above steps, coherent Raman scanning imaging can be performed. As an example, the images acquired during this process can be as follows: Figure 4 As shown in the figure, the acquired images can be used as a navigation basis for subsequent laser micro-cutting.

[0053] (3) Adjust the optical path module for laser micro-cutting. First, turn off the Stokes beam in the optical path, which can be achieved by removing the dispersion broadening module; then adjust the pump light power to the level of hundreds of milliwatts. Since the repetition rate of the femtosecond laser used is 1 MHz, the single pulse energy of the pump light is about 100 nanojoules and the pulse width is about 200 femtoseconds. These pulse parameters are sufficient for laser micro-cutting; depending on the different absorption properties of the sample at different wavelengths, the pump light wavelength can be adjusted to the point where the sample absorption efficiency is the highest, so as to improve the cutting efficiency and ensure the cutting quality.

[0054] After setup, cutting begins. The cutting content is selected and outlined on the computer based on the previously acquired coherent Raman imaging data. Then, an adjusted pump beam (i.e., the second pump beam) is used to perform laser cutting along the outlined position. Since both imaging and cutting in this application use the same scanning imaging optical path from the microscope, the problem of mismatch between the cutting position and the observed image, common in typical laser micro-cutting systems, is avoided.

[0055] Through the above cutting, such as Figure 5 As shown, a square area and an elliptical area were accurately cut out from the upper left and lower right of the sample, respectively.

[0056] It should be understood that expressions such as “comprising” and “may include” used in this application indicate the existence of the disclosed functions, operations, or constituent elements, and do not limit one or more additional functions, operations, and constituent elements. In this application, terms such as “comprising” and / or “having” are to be interpreted as indicating a particular characteristic, number, operation, constituent element, component, or combination thereof, but not to exclude the existence or possibility of adding one or more other characteristics, numbers, operations, constituent elements, components, or combinations thereof.

[0057] Furthermore, in this application, the expression "and / or" includes any and all combinations of the associated listed words. For example, the expression "A and / or B" may include A, may include B, or may include both A and B.

[0058] In the description of the embodiments of this application, it should be noted that, unless otherwise explicitly specified and limited, the term "connection" should be interpreted broadly. For example, "connection" can be a detachable connection or a non-detachable connection; it can be a direct connection or an indirect connection through an intermediate medium. "Fixed connection" refers to a connection where the relative positional relationship remains unchanged after connection. "Rotary connection" refers to a connection where the components can rotate relative to each other after connection. "Sliding connection" refers to a connection where the components can slide relative to each other after connection. The directional terms mentioned in the embodiments of this application, such as "top," "bottom," "inner," "outer," "left," and "right," are only for reference to the directions in the accompanying drawings. Therefore, the directional terms used are for better and clearer explanation and understanding of the embodiments of this application, and are not intended to indicate or imply that the device or component referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on the embodiments of this application.

[0059] Furthermore, the mathematical concepts mentioned in the embodiments of this application, such as symmetry, equality, parallelism, and perpendicularity, are limitations specific to the current technological level, rather than absolute and strict mathematical definitions. Slight deviations are permissible; approximations of symmetry, equality, parallelism, and perpendicularity are all acceptable. For example, "A and B are parallel" means that A and B are parallel or approximately parallel, and the angle between A and B can be between 0 and 10 degrees. "A and B are perpendicular" means that A and B are perpendicular or approximately perpendicular, and the angle between A and B can be between 80 and 100 degrees.

[0060] The above description is merely a specific embodiment of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A laser micro-cutting system based on coherent Raman imaging, characterized in that, It includes a femtosecond laser, a beam splitter (14), a dichroic mirror (3) and a laser scanning microscope. A pump optical path and a Stokes optical path are provided between the beam splitter (14) and the dichroic mirror (3). An optical parametric amplifier and a movable dispersion broadening module (9) are provided in the pump optical path. A Martinez broadening device (6) is provided in the Stokes optical path. The femtosecond laser is used to emit a laser beam; the beam splitter (14) is used to split the laser beam and allow the split beams to enter the pump optical path and the Stokes optical path respectively; the dispersion broadening module (9) and the Martinez broadener (6) are used to adjust the dispersion values ​​of the corresponding beams to match each other; The dichroic mirror (3) is used to combine the first pump light generated by the optical parametric amplifier in the pump light path with the Stokes light emitted from the Stokes light path; the laser scanning microscope is used to scan the sample with the combined light emitted from the dichroic mirror (3) to obtain a coherent Raman image; when the dispersion broadening module (9) is removed and the Stokes light path is closed, a split beam emitted from the beam splitter (14) enters the optical parametric amplifier to generate a second pump light, the pump light path can adjust the power of the second pump light, and the laser scanning microscope can also use the second pump light with adjusted power to cut the corresponding sample.

2. The laser micro-cutting system as described in claim 1, characterized in that, The single-pulse output energy of the femtosecond laser is at least 10 μJ, its repetition rate is not less than 200 kHz, and its pulse width is not greater than 500 femtoseconds.

3. The laser micro-cutting system as described in claim 1, characterized in that, Along the propagation direction of the pump light path, a pump light power control unit (13), a right-angle mirror (12), and a mirror (17) are arranged sequentially after the optical parametric amplifier. The dispersion broadening module (9) is located between the right-angle mirror (12) and the mirror (17). The mirror (17) is directly opposite the output end of the dispersion broadening module (9). The split beam enters the optical parametric amplifier, the pump light power control unit (13), the right-angle mirror (12), and the dispersion broadening module (9) in sequence and then enters the mirror (17). It is reflected by the mirror (17) to the dispersion broadening module (9) and then reflected by the right-angle mirror (12) to the dichroic mirror (3).

4. The laser micro-cutting system as described in claim 3, characterized in that, The wavelength of the optical parametric amplifier is 750nm~980nm, its output energy is not less than 1uJ, and its pulse width is not greater than 500fs.

5. The laser micro-cutting system as described in claim 3, characterized in that, A piezoelectric mirror (11) is provided between the right-angle mirror (12) and the dichroic mirror (3). A self-collimating beam position detection module (2) is provided on the side of the dichroic mirror (3). The self-collimating beam position detection module (2) is used to detect the beam combining state of the beam emitted from the dichroic mirror (3). The piezoelectric mirror (11) is used to adjust the propagation direction of the first pump light using the beam combining state.

6. The laser micro-cutting system as described in claim 3, characterized in that, A first silver-plated reflector (18) is provided between the optical parametric amplifier and the pump optical power control unit (13). An optical power detection unit (16) is provided next to the first silver-plated reflector (18). The optical power detection unit (16) is used to detect the power of the light beam emitted from the optical parametric amplifier and entering the first silver-plated reflector (18).

7. The laser micro-cutting system as described in claim 3, characterized in that, A second silver-plated reflector (19) is provided between the beam splitter (14) and the optical parametric amplifier. A beam position detection module (15) is provided on the side of the second silver-plated reflector (19). The beam position detection module (15) is used to detect the position of the beam emitted from the beam splitter (14) and into the second silver-plated reflector (19).

8. The laser micro-cutting system as described in claim 1, characterized in that, The dispersion broadening module (9) consists of two glass rods connected end to end, with its central axis parallel to the beam propagation direction of the pump optical path.

9. The laser micro-cutting system as described in claim 1, characterized in that, Along the propagation direction of the Stokes optical path, a Stokes optical power control module (4) and a delay line (1) are also arranged in sequence. The Stokes optical power control module (4) is located between the beam splitter (14) and the Martinez stretcher (6), and the delay line (1) is located between the Martinez stretcher (6) and the dichroic mirror (3).

10. A method of applying the laser micro-cutting system as described in any one of claims 1-9, comprising: S1 causes the femtosecond laser to emit a laser beam toward the beam splitter (14); The beam splitter (14) splits the laser beam and allows the split beams to enter the pump optical path and the Stokes optical path respectively, generating the first pump light and the Stokes light; S2 causes the dispersion broadening module (9) to adjust the dispersion value of the first pump light, and the Martinez broadener (6) to adjust the dispersion value of the Stokes light until the two are matched and then enter the dichroic mirror (3) to combine. S3 enables the laser scanning microscope to receive the combined beam of light emitted from the dichroic mirror (3) and scan the sample to obtain a coherent Raman image; S4 removes the dispersion broadening module (9) and closes the Stokes optical path, allowing a beam of split light emitted from the beam splitter (14) to enter the optical parametric amplifier to generate a second pump light; adjusts the power of the pump light so that the laser scanning microscope can use the adjusted power second pump light to cut the corresponding sample.

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