Laser microdissection system based on coherent Raman imaging and application thereof

Abstract

The invention belongs to the field of laser microdissection, and relates to a laser microdissection system based on coherent Raman imaging and application thereof.The device comprises a femtosecond laser, a beam splitter, a dichroscope and a laser scanning microscope, and a pump light path and a Stokes light path are arranged between the beam splitter and the dichroscope; an optical parametric amplifier and a movable dispersion broadening module are arranged in the pump light path; the Stokes light path is internally provided with a Martin inner z stretcher; a femtosecond laser beam is split through a beam splitter and then enters a pump light path and a Stokes light path, one beam generates wavelength-tunable first pump light through an optical parametric amplifier, and the other beam generates Stokes light through the Stokes light path; the first pump light and the Stokes light are combined in the dichroscope, and the laser scanning microscope is used for carrying out coherent Raman imaging by using the combined light and is also used for carrying out laser microdissection according to a coherent Raman image. According to the invention, the accuracy, selectivity and real-time performance of the laser microdissection process can be obviously improved.

Description

Technical Field

[0001] The present application belongs to the field of laser microdissection, and more specifically, relates to a laser microdissection system based on coherent Raman imaging and its application. Background Art

[0002] Laser microdissection (LMD) is a technique that uses laser technology to precisely isolate specific cells or tissue regions from tissue sections. Conventional microdissection often uses a widefield microscope to observe tissue sections, identify the specific cells or tissue regions that need to be cut, and then uses methods such as laser cutting to separate the target areas. Finally, the separated tissue or cells are collected. The advantage of laser microdissection technology is its ability to isolate a single type of cell from a complex tissue environment, which is crucial for studying cellular heterogeneity and performing precise molecular analysis. In addition, due to its non-contact operation, it can avoid the cell damage and contamination that may be caused by traditional mechanical cutting.

[0003] Laser microdissection is typically observed using wide-field microscopy, including brightfield microscopy, darkfield microscopy, phase contrast microscopy, and wide-field fluorescence microscopy. For low-contrast situations, staining is required, which can damage the sample. Some stains can also interfere with subsequent molecular analysis, and prolonged staining can even increase the risk of RNA degradation. Therefore, the staining process needs to ensure that the stain does not interfere with subsequent analysis, which increases the complexity of the operation and requires additional time and skills to process the stained sections. Therefore, a new laser microdissection device is needed to address the problem of sample staining required during traditional laser microdissection surgery. Summary of the Invention

[0004] In response to the shortcomings of the existing technology, 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 sample staining, which affects subsequent molecular analysis.

[0005] To achieve the above objectives, the present application provides a laser microdissection system based on coherent Raman imaging, comprising a femtosecond laser, a beam splitter, a dichroic mirror, and a laser scanning microscope, wherein a pump optical path and a Stokes optical path are provided between the beam splitter and the dichroic mirror, an optical parametric amplifier and a movable dispersion broadening module are provided in the pump optical path, and a Martinez stretcher is provided in the Stokes optical path; 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 stretcher are respectively used to adjust the dispersion values ​​of the light beams in the corresponding optical paths to match each other; The dichroic mirror is used to combine the first pump light generated in the pump light path through the optical parametric amplifier and the Stokes light emitted from the Stokes light path; the laser scanning microscope is used to use the combined light emitted from the dichroic mirror to scan the sample and obtain a coherent Raman image; when the dispersion broadening module is removed and the Stokes light path is closed, a beam of split light emitted by 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 uJ, the repetition rate is not less than 200 kHz, and the 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 reflector and a reflector are sequentially arranged after the optical parametric amplifier, the dispersion broadening module is located between the right-angle reflector and the reflector, and the reflector is directly opposite to the output end of the dispersion broadening module; the split light beam enters the optical parametric amplifier, the pump light power control unit, the right-angle reflector, and the dispersion broadening module in sequence, and then enters the reflector, and is reflected by the reflector into the dispersion broadening module, and is reflected by the right-angle reflector into 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 mirror is arranged between the right-angle reflector and the dichroic mirror, and a self-collimating beam position detection module is arranged next to the dichroic mirror. The self-collimating beam position detection module is used to detect the beam combining state of the light beam emitted from the dichroic mirror, and the piezoelectric mirror 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 arranged between the optical parametric amplifier and the pump light power control unit, and an optical power detection unit is arranged 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 arranged between the beam splitter and the optical parametric amplifier, and a beam position detection module is arranged next to the second silver-plated reflector. The beam position detection module is used to detect the position of the light beam emitted from the beam splitter and incident on the second silver-plated reflector.

[0012] Furthermore, the dispersion broadening module is composed of two glass rods connected end to end, and the axis thereof is parallel to the propagation direction of the light beam of the pump optical path.

[0013] Furthermore, along the propagation direction of the Stokes optical path, there are also arranged in sequence: a Stokes optical power control module and a delay line, the Stokes optical power control module is arranged 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 the present application, there is also provided a method for applying the aforementioned laser microdissection system, comprising: S1 enables the femtosecond laser to emit a laser beam toward the beam splitter; the beam splitter splits the laser beam and allows the split beams to enter the pump light path and the Stokes light path respectively, generating a first pump light and a Stokes light; S2: causing the dispersion stretching module to adjust the dispersion value of the first pump light and causing the Martinez stretcher to adjust the dispersion value of the Stokes light until the two light beams match each other and then are injected into the dichroic mirror for beam combination; S3: enabling the laser scanning microscope to receive the combined light emitted from the dichroic mirror and scan the sample to obtain a coherent Raman image; S4 removes the dispersion broadening module and closes the Stokes optical path, so that a beam of split light emitted by the beam splitter enters the optical parametric amplifier to generate a second pump light; adjusts the power of the pump light so that the laser scanning microscope uses the second pump light with adjusted power to cut the corresponding sample.

[0015] In general, the above technical solutions conceived by this application have the following beneficial effects compared with the existing technologies: (1) This application combines coherent Raman technology with laser microdissection technology, which can significantly improve the accuracy, selectivity and real-time performance of the cutting 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 labeling, 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, it can monitor the changes in the chemical distribution of sample molecules in real time during the cutting process and dynamically adjust the cutting path.

[0016] (2) In this application, by rationally designing the pump optical path and the Stokes optical path, the acquisition of coherent Raman images and the laser microcutting are directly dependent on the coordinates of the galvanometer itself, thereby avoiding the re-alignment of the imaging space and cutting space coordinates during laser microcutting, 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 will apply different amounts of dispersion values ​​to the femtosecond pulse light of different wavelength ranges generated by the optical parametric amplifier, resulting in different pulse broadening effects. When coherent Raman imaging is performed, this device is controlled by an electric translation stage or other similar displacement device to move the right-angle reflector into the optical path. When laser microdissection is performed, this device is controlled by an electric translation stage to move out. This design scheme can ensure that the coherent Raman imaging has a range of about 10cm. -1 It can also ensure the high peak power of the laser after the module is removed, which is convenient for microscopic laser cutting. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Schematic diagram of a laser microdissection system based on coherent anti-Stokes Raman scattering imaging provided in an embodiment of the present application; Figure 2 Schematic diagram of the light spot outputted by the Stokes light after passing through the Martinez stretcher provided in an embodiment of the present application; Figure 3 Schematic diagram of the corresponding relationship between the moving position of the Martinez stretcher and the output pulse width provided in an embodiment of the present application; Figure 4 The coherent Raman scanning image obtained by the embodiment of the present application; Figure 5 Schematic diagram of an image obtained by coherent Raman scanning provided in an embodiment of the present application after being cut at the upper left and lower right.

[0019] Throughout the 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 optical power control module, 5-D-shaped mirror, 6-Martinez stretcher, 7-grating, 8-plano-convex lens, 9-dispersion stretching module, 10-sample, 11-piezoelectric mirror, 12-right-angle mirror, 13-pump light power control module, 14-beam splitter, 15-beam position detection module, 16-optical power detection module, 17-mirror, 18-first silver-coated mirror, 19-second silver-coated mirror. DETAILED DESCRIPTION

[0020] In order to make the purpose, technical solutions and advantages of this application more clear, the following further describes this application in detail with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain this application and are not intended to limit this application.

[0021] The term "and / or" as used herein describes an association between related objects, indicating that three possible relationships exist. For example, "A and / or B" can represent: A exists alone, A and B exist simultaneously, or B exists alone. The symbol " / " as used herein indicates that the related objects are in an "or" relationship, for example, A / B means either A or B.

[0022] The terms "first" and "second" in this specification and claims are used to distinguish different objects rather than to describe a specific order of objects. For example, "first response message" and "second response message" are used to distinguish different response messages rather than to describe a specific order of response messages.

[0023] In the embodiments of this application, words such as "exemplary" or "for example" are used to indicate examples, illustrations, or descriptions. Any embodiment or design described as "exemplary" or "for example" in the embodiments of this application should not be interpreted as being preferred or advantageous over other embodiments or designs. Rather, the use of words such as "exemplary" or "for example" is intended to present the relevant concepts in a concrete manner.

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

[0025] Traditional methods for targeting microdissection rely primarily on brightfield or fluorescence imaging. However, these methods can only distinguish samples based on morphology or marker signals and lack molecular selectivity. Traditional microdissection often uses widefield imaging before cutting, lacking real-time chemical guidance and prone to cutting errors due to sample drift or positioning errors.

[0026] Specifically, in order to achieve accurate and clean cutting in conventional laser microdissection, the single pulse energy of the pulsed laser used is often in the microjoule level, and the repetition rate is around tens of kHz. This type of laser has too much energy and too low a repetition rate for coherent Raman imaging. In order to avoid sample damage while taking into account high scanning imaging speed, conventional coherent Raman imaging uses a single pulse energy of the laser in the nanojoule level, and the repetition rate is above Mhz. In order to achieve higher spectral resolution, coherent Raman imaging requires the use of picosecond pulse light or the use of dispersion spectrum focusing to broaden the femtosecond pulse light for imaging. For laser microdissection, shorter laser pulses mean higher peak power. The broadened pulse reduces the peak power of the pulse, which is not conducive to laser microdissection. Therefore, there are essential differences in the lasers used for scanning imaging and laser cutting. Due to the aforementioned difficulties and issues, there are currently no systems that combine coherent Raman with laser microdissection. The following table lists some commercially available laser microdissection systems and how they generate images for marking the cut.

[0027] Table 1 Conventional microdissection systems and corresponding methods for marking and cutting images

[0028] Microdissection based on wide-field imaging requires the alignment of the cutting coordinate system with the observation coordinate system. In actual application, due to long-term operation, the laser beam pointing may shift, resulting in a mismatch between the cutting position and the area of ​​interest, which may also lead to inaccurate cutting position and contamination of the cells of interest.

[0029] Therefore, the present application proposes a laser microdissection system based on coherent anti-Stokes Raman scattering imaging. By utilizing the advantages of coherent Raman imaging for dye-free labeling and high-sensitivity chemical-specific imaging, the problem of needing to stain samples during traditional laser microdissection surgery can be avoided. The images obtained based on coherent Raman imaging can directly provide guidance for laser microdissection. At the same time, the problem of needing to position and align the CCD captured image with the laser microdissection path coordinates during laser microdissection is eliminated. The sample image obtained based on coherent Raman is naturally dependent on the scanning path of the galvanometer. Therefore, there will be no misalignment between the cutting position and the observation position during laser microdissection, and the need for daily correction and maintenance of the laser light path direction during microdissection is avoided.

[0030] The embodiments of the present application are described below in conjunction with the drawings in the embodiments of the present application.

[0031] This application provides a laser microdissection 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 light path and a Stokes light 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 light path; a Martinez stretcher 6 is provided in the Stokes light path; the femtosecond laser is used to emit a laser beam, and the yellow laser beam enters the beam splitter 14 and is split into two beams. The blue beam of the two beams enters the pump light path and emits the first pump light after passing through the optical parametric amplifier, and the green beam enters the Stokes light path to generate Stokes light; dispersion broadening Module 9 and Martinez stretcher 6 are used to adjust the dispersion values ​​of the light beams in the corresponding optical paths to match each other; the dichroic mirror 3 is used to combine the first pump light emitted from the pump optical path and the Stokes light emitted from the Stokes optical path; the laser scanning microscope is used to use the yellow combined light emitted from the dichroic mirror 3 to scan the sample 10 to obtain a coherent Raman image; when the dispersion stretching module 9 is removed and the Stokes optical path is closed, the laser beam enters the optical parametric amplifier to generate a second pump light, the pump optical 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 femtosecond laser in the aforementioned amplification stage 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 needs of high-speed scanning imaging.

[0033] In this embodiment, the single-pulse output energy of the femtosecond laser is at least 10uJ, its repetition rate is not less than 200khz, and the pulse width is not greater than 500 femtoseconds; specifically, the amplifier-stage femtosecond laser is used to generate high-repetition-rate, high-energy near-infrared laser, with a repetition rate of 1MHz, a single-pulse energy of approximately 20uJ, and a pulse width of approximately 250fs, of which 19uJ of energy is used to pump the optical parametric amplifier, and the remaining 1uJ 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 light path and a Martinez stretcher 6 placed in the pump light path. It is used to apply equal amounts of dispersion chirp to the pump light and Stokes light while ensuring that the two beams are spatially aligned, thereby achieving high spectral line resolution scanning imaging in coherent Raman scanning imaging.

[0035] Specifically, for the wavelength of Stokes light, the Martinez stretcher 6 uses a grating 7 with high dispersion efficiency and transmittance, paired with a D-shaped reflector 5, a plano-convex lens 8 of a certain focal length, a reflector with a suitable coating (a general silver-coated reflector can be used), and a climbing mirror assembly. The plano-convex lens 8, the grating 7, and a reflector are placed on a one-dimensional motion platform, and the D-shaped reflector 5 is placed on the propagation optical 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 the different light used to generate dispersion of different sizes, which matches the dispersion generated by the dispersion stretching module 9 placed in the pump light path, thereby realizing the function of high spectral line resolution scanning imaging in coherent Raman scanning imaging. Figure 2 As shown in FIG, the light spot outputted by the Stokes light after passing through the Martinez stretcher 6 still has good beam quality. Figure 3 FIG. 1 shows the corresponding relationship between the output pulse width and the movement of a part of the structure in the Martinez stretcher 6 .

[0036] In this embodiment, the aforementioned pump light path is specifically provided with an optical parametric amplifier, a pump light power control unit 13, a right-angle reflector 12 and a reflector 17 in sequence. The dispersion broadening module 9 is located between the right-angle reflector 12 and the reflector 17, and the reflector 17 is directly opposite to the output end of the dispersion broadening module 9. The split light beam enters the optical parametric amplifier, the pump light power control unit 13, the right-angle reflector 12, and the dispersion broadening module 9 in sequence, and then enters the reflector 17, and is reflected by the reflector 17 into the dispersion broadening module 9, and then reflected by the right-angle reflector 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, 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 pumping 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 (i.e., the first pump light) in coherent Raman imaging, and also as the laser used for cutting sample tissue in microscopic laser 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 mirror 11 is further provided between the aforementioned right-angle reflector 12 and the dichroic mirror 3. A self-collimating beam position detection module 2 is provided next to the dichroic mirror 3. The self-collimating beam position detection module 2 is used to detect the beam combining state of the light beams 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. That is, when a deviation is found in the overlap of the light beams, the piezoelectric mirror can be fine-tuned to make the two light beams overlap.

[0039] The aforementioned self-collimating beam position detection module 2 can detect the overlap and pointing stability of the two beams in the optical path, ensuring that the optical path has no deviation after the beam combination and that the coherent Raman signal has a high signal-to-noise ratio.

[0040] In this embodiment, a first silver-plated reflector 18 is further provided between the optical parametric amplifier and the pump light 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 light power control unit 13. At the same time, part of the light will be transmitted from 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-coated reflector 19 is further provided between the beam splitter 14 and the optical parametric amplifier. A beam position detection module 15 is provided next to the second silver-coated reflector 19. The beam position detection module 15 can be a laser position tester or a beam position detector, and is used to detect the position of the light beam emitted from the beam splitter 14 and entering the second silver-coated reflector 19. Specifically, a beam of light split by the beam splitter 14 enters the second silver-coated reflector 19 and is reflected by the second silver-coated reflector 19 into the optical parametric amplifier. Part of the light will pass through the second silver-coated reflector 19, and the beam position detection module 15 captures this part of the transmitted light to detect the beam position.

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

[0043] Specifically, the dispersion broadening module 9 consists of two glass rods connected end to end, with their axis parallel to the propagation direction of the pump light path. Specifically, the glass rods can be high-dispersion glass rods. Anti-reflection coatings are applied to both ends of the glass rods for the pump light's wavelength band to improve light transmission efficiency. The length of the glass rods is selected based on the second-order dispersion coefficient of the glass material and the target chirp amount. A length of less than 10 cm can be achieved. -1The spectral line resolution capability of coherent Raman imaging based on wave number.

[0044] More specifically, high-dispersion glass rods are placed end-to-end so that the pump light passes back and forth through the glass rods four times before being broadened into pulsed light of approximately 3 ps. The Martinez stretcher is adaptively designed based on the central wavelength and pulse width of the aforementioned femtosecond laser. It should be understood that those skilled in the art can adaptively design it based on femtosecond lasers with different central wavelengths and pulse widths, and this will not be described in detail here.

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

[0046] The Stokes optical power control module 4, placed in the pump optical path, adjusts the optical power of the pump beam. Its specific operating principle is as follows: a stepper motor drives the rotation of the half-wave plate, which cooperates with the polarization beam splitter cube to achieve optical power control. During coherent Raman imaging, the optical power of both the pump and Stokes beams is attenuated to approximately 30mW to obtain a clear image while avoiding laser power damage to sample 10. During microscopic laser surgery, the pump light power control module adjusts the second pump beam to approximately 300mW, and adjusts its wavelength to match the absorption of the sample to be cut, allowing for efficient removal of 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 is composed of a hollow reflector and a one-dimensional motion platform. Since its structure is an existing design, it will not be described in detail here. The use of a hollow reflector in the delay line is conducive to ensuring that the directivity of the Stokes light after passing through the delay line does not change, and the Stokes light and the pump light can be well spatially collinear at any position where the one-dimensional motion platform stays. The aforementioned hollow reflector has a light aperture of 60mm, which is convenient for debugging in the optical path. Its surface is gold-plated to ensure high reflectivity within 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 a high transmittance for the laser band, and can scan the excitation light on the sample surface in a tightly focused state and collect the signals generated by nonlinearity.

[0049] The operating method of the laser microdissection system based on coherent Raman imaging provided in the above embodiment is: (1) Turn on the femtosecond laser and optical parametric amplifier to preheat, 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 to perform coherent Raman scanning imaging on the sample. First, adjust the power of the two lasers to a low level, generally around tens of milliwatts, depending on the properties of the sample and the signal size in the laser scanning microscope. Then, move the dispersion broadening module at the pump light into the optical path, and move the Martinez broadener stage to a suitable position according to the wavelength setting of the pump light, so that the dispersion size of the Stokes light matches the dispersion size 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 state of the pump light and the Stokes light beam. If the overlap state is good, the subsequent coherent Raman scanning imaging test can be carried out. If the overlap state deviates, the piezoelectric reflector can be fine-tuned to make the two beams overlap.

[0051] It should be noted that this optical path module layout design ensures that the beam pointing direction remains unchanged when moving the Martinez stretcher's translation stage and after moving the dispersion stretching module in and out of the optical path. Therefore, piezoelectric mirror adjustment is generally not required. However, long-term operation of the system will inevitably lead to deviations in the beam pointing direction. In this case, the piezoelectric mirror can be used to adjust the beam pointing position to ensure that it remains unchanged.

[0052] After completing the above work, coherent Raman scanning imaging can be performed. As an example, the image collected by the scanning process can be as follows: Figure 4 As shown in , the acquired images can be used as the navigation basis for subsequent laser microdissection.

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

[0054] Once the setup is complete, cutting begins. The computer then outlines the selected area to be cut based on the previously collected coherent Raman imaging data. Laser cutting is then performed along the outlined location using the adjusted pump light (i.e., the second pump beam). Because both imaging and cutting in this application utilize the same scanning imaging optical path within the microscope, the mismatch between the cutting position and the observed image, as is common with laser microdissection systems, is eliminated.

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

[0056] It should be understood that expressions such as "include" 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 "include" and / or "have" may be interpreted as indicating specific characteristics, numbers, operations, constituent elements, components, or combinations thereof, but may not be interpreted as excluding the existence or possibility of adding one or more other characteristics, numbers, operations, constituent elements, components, or combinations thereof.

[0057] In addition, 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 the present application, it should be noted that, unless otherwise clearly specified and limited, the term "connection" should be understood in a broad sense. 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. Among them, "fixed connection" means that the two are connected to each other and the relative position relationship after the connection remains unchanged. "Rotational connection" means that the two are connected to each other and can rotate relative to each other after the connection. "Sliding connection" means that the two are connected to each other and can slide relative to each other after the connection. The directional terms mentioned in the embodiments of the present application, such as "top", "bottom", "inside", "outside", "left", "right", etc., are only reference to the directions of the accompanying drawings. Therefore, the directional terms used are for better and clearer explanation and understanding of the embodiments of the present application, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as a limitation on the embodiments of the present application.

[0059] In addition, in the embodiments of the present application, the mathematical concepts mentioned include symmetry, equality, parallelism, and perpendicularity. These limitations are all for the current state of the art, rather than being absolutely strict definitions in a mathematical sense. A small amount of deviation is allowed, and it is possible to be approximately symmetric, approximately equal, approximately parallel, or approximately perpendicular. For example, A and B are parallel, which 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, which 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 the present application, but the scope of protection of the present application is not limited thereto. Any changes or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in this application should be included in the scope of protection of the present application. Therefore, the scope of protection of the present application should be based on the scope of protection of the claims.

Claims

1. A laser microdissection system based on coherent Raman imaging, characterized in that: The invention comprises a femtosecond laser, a beam splitter (14), a dichroic mirror (3) and a laser scanning microscope, wherein 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, and a Martinez broadener (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 respectively used to adjust the dispersion values ​​of the corresponding light 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 and the Stokes light emitted by the Stokes light path; the laser scanning microscope is used to scan the sample using 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 beam of split light emitted by 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 microdissection system according to claim 1, wherein: The single pulse output energy of the femtosecond laser is at least 10 uJ, the repetition rate is not less than 200 khz, and the pulse width is not greater than 500 femtoseconds.

3. The laser microdissection system according to claim 1, wherein: Along the propagation direction of the pump light path, a pump light power control unit (13), a right-angle reflector (12) and a reflector (17) are sequentially arranged after the optical parametric amplifier, the dispersion broadening module (9) is located between the right-angle reflector (12) and the reflector (17), and the reflector (17) faces the output end of the dispersion broadening module (9); the split light beam sequentially enters the optical parametric amplifier, the pump light power control unit (13), the right-angle reflector (12), and the dispersion broadening module (9) and then enters the reflector (17), and is reflected by the reflector (17) into the dispersion broadening module (9), and then reflected by the right-angle reflector (12) into the dichroic mirror (3).

4. The laser microdissection system according to claim 3, wherein: 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 microdissection system according to claim 3, wherein: A piezoelectric reflector (11) is provided between the right-angle reflector (12) and the dichroic mirror (3), and a self-collimating light beam position detection module (2) is provided beside the dichroic mirror (3). The self-collimating light beam position detection module (2) is used to detect the beam combining state of the light beam emitted from the dichroic mirror (3), and the piezoelectric reflector (11) is used to adjust the propagation direction of the first pump light using the beam combining state.

6. The laser microdissection system according to claim 3, wherein: A first silver-plated reflector (18) is provided between the optical parametric amplifier and the pump light power control unit (13), and 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 a light beam emitted from the optical parametric amplifier and entering the first silver-plated reflector (18).

7. The laser microdissection system according to claim 3, wherein: A second silver-plated reflector (19) is provided between the beam splitter (14) and the optical parametric amplifier, and a light beam position detection module (15) is provided next to the second silver-plated reflector (19). The light beam position detection module (15) is used to detect the position of a light beam emitted from the beam splitter (14) and incident on the second silver-plated reflector (19).

8. The laser microdissection system according to claim 1, wherein: The dispersion broadening module (9) is composed of two glass rods connected end to end, and the axis thereof is parallel to the light beam propagation direction of the pump light path.

9. The laser microdissection system according to claim 1, wherein: Along the propagation direction of the Stokes optical path, there are also arranged in sequence: a Stokes optical power control module (4) and a delay line (1), wherein the Stokes optical power control module (4) is arranged 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 for using the laser microdissection system according to any one of claims 1 to 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 light path and the Stokes light path respectively, generating a first pump light and a Stokes light; S2 causes the dispersion broadening module (9) to adjust the dispersion value of the first pump light, and causes the Martinez broadener (6) to adjust the dispersion value of the Stokes light, until the two light match each other and then are injected into the dichroic mirror (3) for beam combination; S3 enables the laser scanning microscope to receive the combined 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, so that a beam of split light emitted by the beam splitter (14) enters the optical parametric amplifier to generate a second pump light; and adjusts the power of the pump light so that the laser scanning microscope uses the second pump light with adjusted power to cut the corresponding sample.

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