Photoacoustic microscopy system and method based on gradient needle beam excitation

Abstract

The present invention discloses a photoacoustic microscopy system and method based on gradient needle-shaped beam excitation. The system includes: a water tank for placing samples, an ultrasonic transducer and a three-dimensional displacement platform, both of which are communicatively connected to the host computer, and the three-dimensional displacement platform is fixedly connected to the water tank; a laser output laser beam is adjusted to circular polarization by a polarization adjustment system, and a 4F beam expansion system is used to expand the circularly polarized beam. The expanded beam is reflected by a reflector and transmitted to an ultraviolet optical diffraction element. The expanded beam is modulated by the ultraviolet optical diffraction element to form a gradient needle-shaped beam, which is focused on the sample to generate an ultrasonic signal. An ultrasonic transducer is used to detect the ultrasonic signal and transmit the detected ultrasonic signal to the host computer, so that the host computer can complete image reconstruction based on the received ultrasonic signal. The above-mentioned photoacoustic microscopy system has the ability to image histologically with a large depth of focus and high depth resolution.

Description

Technical Field

[0001] The present invention relates to the technical field of photoacoustic microscopy, and in particular to a photoacoustic microscopy system and method based on gradient needle-shaped light beam excitation. Background Art

[0002] Histopathological examination is the gold standard for disease diagnosis. Its traditional method relies on formalin-fixed, paraffin-embedded (FFPE) tissue samples, which are then micron-sectioned and stained with hematoxylin-eosin (H&E) to observe cells and tissue structures under a brightfield microscope. However, this process requires tedious steps such as fixation, embedding, sectioning, and staining, which takes hours to days and cannot meet real-time diagnostic needs such as rapid judgment of cancer margins during surgery. Although frozen section technology shortens the detection time to tens of minutes through tissue freezing and rapid staining, it is prone to ice crystal artifacts or difficulty in sectioning in edematous tissue or hard tissue (such as cortical bone and calcified tumors), resulting in reduced diagnostic accuracy.

[0003] Microscopic imaging technology based on the photoacoustic effect provides a new path for non-invasive cell nucleus imaging: by utilizing the strong absorption characteristics of DNA / RNA in the ultraviolet band, laser excitation of tissues to generate ultrasonic signals can achieve specific visualization of cell nuclei, avoiding the dependence of traditional staining. However, existing photoacoustic microscopic imaging technology faces two bottlenecks:

[0004] 1. Limitations of focal depth and surface adaptability: Conventional optical focusing systems have a short focal depth. When imaging fresh, thick tissue with irregular surfaces, local defocusing can easily lead to image blur or information loss. Although research has expanded the focal depth range through wavefront manipulation techniques, the depth resolution is still determined by the ultrasound transducer and has not been improved, making it impossible to achieve pathology slice-level depth-resolved imaging (the gold standard pathology slice thickness is approximately 7 µm).

[0005] 2. Physical limitations of ultrasonic transducers: The bandwidth limitation of the transducer limits the slicing capability of photoacoustic imaging to tens of microns, making it difficult to distinguish deep fine structures, which is significantly different from the slice-level resolution required for pathological diagnosis.

[0006] Therefore, existing technologies make it difficult to achieve fast and accurate cell nucleus imaging in unprocessed thick tissue samples. Summary of the Invention

[0007] The purpose of the present invention is to provide a photoacoustic microscopy system and method based on gradient needle beam excitation, aiming to solve the problem that the existing technology is difficult to achieve fast and accurate cell nucleus imaging of untreated thick tissue samples.

[0008] In a first aspect, the present invention provides a photoacoustic microscopy system based on gradient needle beam excitation, the system comprising a laser, a polarization adjustment system, a 4F beam expansion system, a reflector, an ultraviolet optical diffraction element, a water tank, an ultrasonic transducer, a host computer, and a three-dimensional displacement platform, wherein the water tank is used to place a sample, the ultrasonic transducer and the three-dimensional displacement platform are both communicatively connected to the host computer, and the three-dimensional displacement platform is fixedly connected to the water tank;

[0009] The laser output laser beam is adjusted to circular polarization through the polarization adjustment system, and the 4F beam expansion system performs a beam expansion operation on the circularly polarized beam. The formed expanded beam is reflected by the reflector and transmitted to the ultraviolet optical diffraction element. The expanded beam is modulated by the ultraviolet optical diffraction element to form a gradient needle beam, and is focused on the sample to generate an ultrasonic signal. The ultrasonic transducer is used to detect the ultrasonic signal and send the detected ultrasonic signal to the host computer, so that the host computer completes image reconstruction based on the received ultrasonic signal.

[0010] Furthermore, the polarization adjustment system includes a first wave plate, a polarization beam splitter and a second wave plate, and the laser beam is adjusted by the first wave plate, the polarization beam splitter and the second wave plate in sequence to form a circularly polarized beam.

[0011] Furthermore, the first wave plate is a half wave plate, and the second wave plate is a quarter wave plate.

[0012] Furthermore, the 4F beam expansion system includes a first lens and a second lens, and the circularly polarized light beam is expanded by the first lens and the second lens in sequence to form an expanded light beam.

[0013] Furthermore, an incident pinhole is provided between the first lens and the second lens for spatial filtering.

[0014] Furthermore, the operating wavelength of the ultraviolet optical diffraction element is selected according to the excitation spectrum peak of the target substance in the sample.

[0015] Furthermore, the sample is located within the focal depth range of the gradient needle-shaped light beam formed by the ultraviolet optical diffraction element.

[0016] Furthermore, the water tank is filled with a coupling medium.

[0017] Furthermore, the coupling medium is deionized water.

[0018] In a second aspect, the present invention further provides a photoacoustic microscopy method based on gradient needle beam excitation, which is applied to the photoacoustic microscopy system based on gradient needle beam excitation in the first aspect, the method comprising:

[0019] The laser output laser beam is adjusted to circular polarization by the polarization adjustment system;

[0020] The 4F beam expansion system performs a beam expansion operation on the circularly polarized light beam, and the formed expanded light beam is reflected by the reflector and transmitted to the ultraviolet optical diffraction element;

[0021] The expanded light beam is modulated by the ultraviolet optical diffraction element to form a gradient needle-shaped light beam, and is focused on the sample to generate an ultrasonic signal;

[0022] The ultrasonic transducer is used to detect ultrasonic signals and send the detected ultrasonic signals to the host computer, so that the host computer completes image reconstruction based on the received ultrasonic signals.

[0023] The present invention discloses a photoacoustic microscopy system and method based on gradient needle-shaped beam excitation. The system includes a laser, a polarization adjustment system, a 4F beam expansion system, a reflector, an ultraviolet optical diffraction element, a water tank, an ultrasonic transducer, a host computer, and a three-dimensional displacement platform. The water tank is used to place a sample. The ultrasonic transducer and the three-dimensional displacement platform are both communicatively connected to the host computer, and the three-dimensional displacement platform is fixedly connected to the water tank. The laser outputs a laser beam, and the polarization state of the beam is adjusted to circular polarization by the polarization adjustment system. The 4F beam expansion system expands the circularly polarized beam. The formed expanded beam is reflected by the reflector and transmitted to the ultraviolet optical diffraction element. The expanded beam is modulated by the ultraviolet optical diffraction element to form a gradient needle-shaped beam, which is focused on the sample to generate an ultrasonic signal. The ultrasonic transducer is used to detect the ultrasonic signal and send the detected ultrasonic signal to the host computer, so that the host computer completes image reconstruction based on the received ultrasonic signal. The above-mentioned photoacoustic microscopy system combines photoacoustic microscopy technology and light field control technology, breaking through the poor depth resolution caused by the bandwidth limitation of the ultrasonic transducer and possessing the ability of histological imaging with large focal depth and high depth resolution. Specifically, the light beam wavefront is manipulated by ultraviolet optical diffraction elements, and the amplitude of the photoacoustic signal excited by the cell nucleus is associated with the depth position under the premise of large focal depth. The photoacoustic signals of different amplitudes generated by the gradient needle focus are decoded. While the focal depth is significantly extended by more than ten times, the deep slicing capability is improved to ~8 µm. It can directly observe untreated thick tissue samples with uneven surfaces, and obtain slice-level depth-resolved histological images that are benchmarked against the gold standard without slicing or labeling, providing new technical support for intraoperative pathological diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0025] Figure 1 A schematic diagram of a photoacoustic microscopy system provided in an embodiment of the present invention;

[0026] Figure 2 This is a comparison chart of the depth-resolved image produced by a traditional objective lens and the depth-resolved image produced by a large focal depth.

[0027] Figure 3 A schematic diagram of a process for photoacoustic microscopy provided by an embodiment of the present invention;

[0028] Among them, the reference numerals in the figures are as follows:

[0029] 1. Laser; 2. First wave plate; 3. Polarization beam splitter; 4. Second wave plate; 5. First lens; 6. Second lens; 7. Reflector; 8. Ultraviolet optical diffraction element; 9. Water tank; 10. Three-dimensional displacement platform; 11. Ultrasonic transducer; 12. Host computer; 13. Incident pinhole. DETAILED DESCRIPTION

[0030] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0031] It will be understood that when used in this specification and the appended claims, the terms “comprises” and “comprising” indicate the presence of described features, integers, steps, operations, elements and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components and / or groups thereof.

[0032] It should also be understood that the terms used in the present specification are only for the purpose of describing particular embodiments and are not intended to limit the present invention. As used in the present specification and the appended claims, the singular forms "a", "an", and "the" are intended to include the plural forms unless the context clearly indicates otherwise.

[0033] It should be further understood that the term "and / or" used in the present description and appended claims refers to and includes any and all possible combinations of one or more of the associated listed items.

[0034] See also Figure 1 , Figure 1 Schematic diagram of a photoacoustic microscopy system provided in an embodiment of the present invention. Figure 1 As shown, the present invention proposes a photoacoustic microscopy imaging system based on gradient needle beam excitation, the system includes a laser 1, a polarization adjustment system, a 4F beam expansion system, a reflector 7, an ultraviolet optical diffraction element 8, a water tank 9, an ultrasonic transducer 11, a host computer 12 and a three-dimensional displacement platform 10, the water tank 9 is used to place the sample, the ultrasonic transducer 11 and the three-dimensional displacement platform 10 are both communicated with the host computer 12, and the three-dimensional displacement platform 10 is fixedly connected to the water tank 9; the laser beam output by the laser 1 is polarized and adjusted. The system adjusts the polarization state of the light beam to circular polarization, and the 4F beam expansion system performs a beam expansion operation on the circularly polarized light beam. The formed expanded light beam is reflected by the reflector 7 and transmitted to the ultraviolet optical diffraction element 8. The expanded light beam is modulated by the ultraviolet optical diffraction element 8 to form a gradient needle-shaped beam, and is focused on the sample to generate an ultrasonic signal. The ultrasonic transducer 11 is used to detect the ultrasonic signal and send the detected ultrasonic signal to the host computer 12, so that the host computer 12 completes image reconstruction based on the received ultrasonic signal.

[0035] In this embodiment, the water tank 9 is used to place the sample, the ultrasonic transducer 11 and the three-dimensional displacement platform 10 are both connected to the host computer 12 for communication, the three-dimensional displacement platform 10 is fixedly connected to the water tank 9, and the three-dimensional displacement platform 10 is used to drive the water tank 9 to move, thereby driving the sample to move; the present invention adopts a laser 1 with a wavelength of 266nm as an excitation light source, and its output laser beam is adjusted to a circular polarization state by the polarization adjustment system, and the 4F beam expansion system expands the circularly polarized beam, and the formed expanded beam is reflected by the reflector 7 and transmitted to the The ultraviolet optical diffraction element 8 is used to adjust the wavefront of the expanded light beam. The expanded light beam is modulated by the ultraviolet optical diffraction element 8 to form a gradient needle beam and focused on the sample. Under the irradiation of the gradient needle beam, due to the transient thermoelastic effect and photothermal effect, the sample generates ultrasonic waves at the focus. The ultrasonic transducer 11 detects the generated ultrasonic signal and sends the detected ultrasonic signal to the host computer 12, so that the host computer 12 completes image reconstruction based on the received ultrasonic signal. The reconstructed image can reflect the specific morphology of the cell nucleus.

[0036] Preferably, the host computer 12 uses LABVIEW to control the movement of the three-dimensional displacement platform 10, thereby driving the movement of the sample. For thin tissue samples (~7um), a single XY axis scan can fully image the histological information of the sample. Moreover, since the light beam is distributed in a gradient needle shape, for thick tissue samples, the depth information of the cell nucleus can be accurately obtained by capturing the difference in the amplitude of the photoacoustic signal of the cell nucleus at different depths.

[0037] The present invention combines photoacoustic microscopy technology and light field control technology to design a photoacoustic microscopy system based on gradient needle beam excitation. By using a customized ultraviolet optical diffraction element 8 to manipulate the excitation beam wavefront, label-free, section-free histological imaging is performed. By decoding the amplitude difference of the photoacoustic signal generated by the gradient needle focus, the focal depth is significantly extended by more than ten times, while the sectioning capability in the depth direction is improved to ~8 µm. Therefore, by controlling the light field distribution of the photoacoustic excitation beam, the present invention can obtain histological images with slice-level depth resolution within a large focal depth range with a single scan. This method can image fresh tissue samples and achieve section-free, label-free, and precise acquisition of cell nuclear information, avoiding cumbersome sample preparation procedures. Tissue pathology images with subcellular resolution and slice-level depth resolution can be obtained within 20 minutes, providing rapid and accurate pathological diagnostic information for intraoperative resection margin assessment.

[0038] The present invention has been experimentally verified that the developed photoacoustic microscopy imaging system based on gradient needle beam excitation has achieved large depth of focus and depth resolution imaging in untreated mouse thick brain slice samples (~3mm thickness). The hematoxylin and eosin (H&E) stained images of the samples have verified that the system can obtain good information on the nuclei and cytoplasm of brain slices, and the information is reliable. Figure 2 As shown, conventional objective images suffer from defocus due to insufficient depth of focus. However, images obtained with a wide depth of focus, mediated by light field manipulation, capture complete histological images of uneven sample surfaces, highly similar to the gold standard H&E staining image, demonstrating imaging accuracy. However, depth-resolved images obtained with conventional objectives lack depth resolution, preventing accurate separation of cell nuclei at different locations, resulting in nearly identical images. In contrast, deep-resolved images obtained with a wide depth of focus, using gradient needle beam excitation, achieve improved depth resolution because the depth position of the nuclei is accurately decoded from the photoacoustic signal amplitude. This allows for precise separation of nuclei at different depths, enabling non-destructive acquisition of pathological information at the slice level. (In H&E staining, hematoxylin binds to the DNA of the cell nucleus as a nucleophilic dye, giving the nucleus a dark blue or purple color; eosin stains the cytoplasm and extracellular matrix as an acidic dye, giving them a pink color.)

[0039] Preferably, the ultrasonic transducer 11 is arranged directly above the water tank 9 to reduce energy scattering in the sound beam propagation path.

[0040] Preferably, the present invention can also achieve simultaneous image acquisition and depth-resolved imaging of the cell nucleus and cytoplasm by capturing synchronously excited fluorescence signals and based on confocal fluorescence imaging technology.

[0041] In one embodiment, if Figure 1 As shown, the polarization adjustment system includes a first wave plate 2, a polarization beam splitter 3 and a second wave plate 4. The laser beam is adjusted by the first wave plate 2, the polarization beam splitter 3 and the second wave plate 4 in sequence to form a circularly polarized beam.

[0042] In this embodiment, the polarization adjustment system includes a first wave plate 2, a polarization beam splitter 3 and a second wave plate 4. The first wave plate 2 and the second wave plate 4 are both used to adjust the polarization state of the laser beam, so that the laser beam is adjusted by the first wave plate 2, the polarization beam splitter 3 and the second wave plate 4 in sequence to form a circularly polarized beam.

[0043] In one embodiment, if Figure 1 As shown, the first wave plate 2 is a half wave plate, and the second wave plate 4 is a quarter wave plate.

[0044] In this embodiment, the first wave plate 2 is a half wave plate, the second wave plate 4 is a quarter wave plate, and the laser beam is adjusted by the first wave plate 2, the polarization beam splitter 3 and the second wave plate 4 in sequence to form a circularly polarized beam.

[0045] In one embodiment, if Figure 1 As shown, the 4F beam expansion system includes a first lens 5 and a second lens 6. The circularly polarized light beam is expanded by the first lens 5 and the second lens 6 in sequence to form an expanded light beam.

[0046] In this embodiment, the 4F beam expansion system includes a first lens 5 and a second lens 6. The circularly polarized light beam is expanded sequentially by the first lens 5 and the second lens 6 to form an expanded light beam. By providing the first lens 5 and the second lens 6, the present invention reduces the beam divergence angle and improves the collimation.

[0047] In one embodiment, if Figure 1 As shown, an incident pinhole 13 is provided between the first lens 5 and the second lens 6 .

[0048] In this embodiment, an incident pinhole 13 is provided between the first lens 5 and the second lens 6 for spatial filtering.

[0049] In one embodiment, if Figure 1As shown, the working wavelength of the ultraviolet optical diffraction element 8 is selected according to the excitation spectrum peak of the target substance in the sample.

[0050] In this embodiment, the ultraviolet optical diffraction element 8 is used to adjust the wavefront of the expanded light beam. The expanded light beam is modulated by the ultraviolet optical diffraction element 8 to form a gradient needle-shaped beam. The working wavelength of the ultraviolet optical diffraction element 8 is selected according to the excitation spectrum peak of the target substance in the sample. The present invention manipulates the light beam wavefront by using a customized ultraviolet optical diffraction element 8, stretching the light beam (ultraviolet photoacoustic excitation beam) in the axial direction, so that the spot size at different axial positions remains almost consistent and has a gradient intensity distribution along the optical axis. Under the irradiation of this gradient needle-shaped light beam, because the cell nucleus is rich in DNA / RNA and has a strong light absorption coefficient, the amplitude of the excited photoacoustic signal is directly related to the light intensity, that is, the amplitude of the photoacoustic signal excited by the cell nucleus at different depths is different; since the light intensity of the gradient needle-shaped light beam is gradient-distributed and the light intensity at each depth is known, the light intensity is associated with the specific depth position, and the precise depth position information of the cell nucleus can be obtained by quantitatively measuring the size of the photoacoustic signal. After collection and reconstruction, it can accurately reflect the morphology and spatial distribution of the cell nucleus in the tissue, providing a fast and reliable technical means for intraoperative pathological diagnosis.

[0051] In one embodiment, if Figure 1 As shown, the sample is located within the focal depth range of the gradient needle-shaped beam formed by the ultraviolet optical diffraction element 8 .

[0052] In this embodiment, the sample is placed at the bottom of the water tank 9 so that it is exactly at the focus of the ultraviolet optical diffraction element 8 .

[0053] In one embodiment, if Figure 1 As shown, the water tank 9 is filled with coupling medium.

[0054] In this embodiment, the water tank 9 is filled with a coupling medium, which is deionized water. The system can be applied to both transmission detection scenarios and reflection detection scenarios; in transmission detection scenarios (such as Figure 1 As shown), the sample is completely immersed in the coupling medium in the water tank, the gradient needle beam penetrates the sample, and the ultrasonic transducer 11 detects the ultrasonic signal. At this time, the sound wave transmission direction is the same as the light transmission direction.

[0055] The embodiment of the present invention further provides a method for photoacoustic microscopy based on gradient needle beam excitation, which is applied to the above-mentioned photoacoustic microscopy system based on gradient needle beam excitation, such as Figure 3 As shown, the method includes steps S110-S140:

[0056] S110, the laser output laser beam is adjusted to circular polarization by the polarization adjustment system;

[0057] S120, the 4F beam expansion system performs a beam expansion operation on the circularly polarized light beam, and the formed expanded light beam is reflected by the reflector and transmitted to the ultraviolet optical diffraction element;

[0058] S130, the expanded light beam is modulated by the ultraviolet optical diffraction element to form a gradient needle beam, and is focused on the sample to generate an ultrasonic signal;

[0059] S140: The ultrasonic transducer is used to detect ultrasonic signals and send the detected ultrasonic signals to the host computer, so that the host computer completes image reconstruction based on the received ultrasonic signals.

[0060] In this embodiment, the laser output laser beam is adjusted to a circular polarization state by the polarization adjustment system, and the 4F beam expansion system performs a beam expansion operation on the circularly polarized beam. The formed expanded beam is reflected by the reflector and transmitted to the ultraviolet optical diffraction element. The ultraviolet optical diffraction element is used to adjust the wavefront of the expanded beam. The expanded beam is modulated by the ultraviolet optical diffraction element to form a gradient needle beam and focused on the sample. Under the irradiation of the gradient needle beam, due to the transient thermoelastic effect and the photothermal effect, the sample generates ultrasonic waves at the focus. The ultrasonic transducer detects the generated ultrasonic signal and sends the detected ultrasonic signal to the host computer, so that the host computer completes image reconstruction based on the received ultrasonic signal. The reconstructed image can reflect the specific morphology of the cell nucleus. The present invention manipulates the light beam wavefront through ultraviolet optical diffraction elements, associates the amplitude of the photoacoustic signal excited by the cell nucleus with the depth position under the premise of large focal depth, decodes the photoacoustic signals of different amplitudes generated by the gradient needle focus, and while significantly extending the focal depth by more than ten times, improves the deep slicing capability to ~8 µm. This allows direct observation of untreated thick tissue samples with uneven surfaces, and obtains slice-level depth-resolved histological images that are comparable to the gold standard without slicing or labeling, providing new technical support for intraoperative pathological diagnosis.

[0061] The present invention discloses a photoacoustic microscopy system and method based on gradient needle-shaped beam excitation. The system includes a laser, a polarization adjustment system, a 4F beam expansion system, a reflector, an ultraviolet optical diffraction element, a water tank, an ultrasonic transducer, a host computer, and a three-dimensional displacement platform. The water tank is used to place a sample. The ultrasonic transducer and the three-dimensional displacement platform are both communicatively connected to the host computer, and the three-dimensional displacement platform is fixedly connected to the water tank. The laser outputs a laser beam, and the polarization state of the beam is adjusted to circular polarization by the polarization adjustment system. The 4F beam expansion system expands the circularly polarized beam. The formed expanded beam is reflected by the reflector and transmitted to the ultraviolet optical diffraction element. The expanded beam is modulated by the ultraviolet optical diffraction element to form a gradient needle-shaped beam, which is focused on the sample to generate an ultrasonic signal. The ultrasonic transducer is used to detect the ultrasonic signal and send the detected ultrasonic signal to the host computer, so that the host computer completes image reconstruction based on the received ultrasonic signal. The above-mentioned photoacoustic microscopy system combines photoacoustic microscopy technology and light field control technology, breaking through the poor depth resolution caused by the bandwidth limitation of the ultrasonic transducer and possessing the ability of histological imaging with large focal depth and high depth resolution. Specifically, the light beam wavefront is manipulated by ultraviolet optical diffraction elements, and the amplitude of the photoacoustic signal excited by the cell nucleus is associated with the depth position under the premise of large focal depth. The photoacoustic signals of different amplitudes generated by the gradient needle focus are decoded. While the focal depth is significantly extended by more than ten times, the deep slicing capability is improved to ~8 µm. It can directly observe untreated thick tissue samples with uneven surfaces, and obtain slice-level depth-resolved histological images that are benchmarked against the gold standard without slicing or labeling, providing new technical support for intraoperative pathological diagnosis.

[0062] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in the present invention, and such modifications or substitutions are intended to be within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be subject to the scope of protection of the claims.

Claims

1. A photoacoustic microscopy system based on gradient needle beam excitation, characterized in that: The system includes a laser, a polarization adjustment system, a 4F beam expansion system, a reflector, an ultraviolet optical diffraction element, a water tank, an ultrasonic transducer, a host computer and a three-dimensional displacement platform. The water tank is used to place the sample. The ultrasonic transducer and the three-dimensional displacement platform are both communicatively connected to the host computer, and the three-dimensional displacement platform is fixedly connected to the water tank. The laser output laser beam is adjusted to a circular polarization state by the polarization adjustment system, the 4F beam expansion system performs a beam expansion operation on the circularly polarized beam, and the formed expanded beam is reflected by the reflector and transmitted to the ultraviolet optical diffraction element, the expanded beam is modulated by the ultraviolet optical diffraction element to form a gradient needle beam, and is focused on the sample to generate an ultrasonic signal, the ultrasonic transducer is used to detect the ultrasonic signal and send the detected ultrasonic signal to the host computer, so that the host computer completes image reconstruction based on the received ultrasonic signal; The polarization adjustment system includes a first wave plate, a polarization beam splitter, and a second wave plate. The laser beam is adjusted by the first wave plate, the polarization beam splitter, and the second wave plate in sequence to form a circularly polarized beam. The first wave plate is a half wave plate, and the second wave plate is a quarter wave plate; the laser beam is adjusted by the first wave plate, the polarization beam splitter, and the second wave plate in sequence to form a circularly polarized beam; The spot size of the gradient needle beam at different axial positions is consistent, the light intensity is distributed in a gradient along the optical axis, and the light intensity at each depth is known. The light intensity is associated with the specific depth position to obtain the precise depth position information of the cell nucleus.

2. The photoacoustic microscopy system based on gradient needle beam excitation according to claim 1, characterized in that: The 4F beam expansion system includes a first lens and a second lens. The circularly polarized light beam is expanded by the first lens and the second lens in sequence to form an expanded light beam.

3. The photoacoustic microscopy system based on gradient needle beam excitation according to claim 2, characterized in that: An incident pinhole is provided between the first lens and the second lens for spatial filtering.

4. The photoacoustic microscopy system based on gradient needle beam excitation according to claim 1, characterized in that: The operating wavelength of the ultraviolet optical diffraction element is selected according to the excitation spectrum peak of the target substance in the sample.

5. The photoacoustic microscopy system based on gradient needle beam excitation according to claim 1, characterized in that: The sample is located within the focal depth range of the gradient needle-shaped beam formed by the ultraviolet optical diffraction element.

6. The photoacoustic microscopy system based on gradient needle beam excitation according to claim 1, characterized in that: The water tank is filled with a coupling medium.

7. The photoacoustic microscopy system based on gradient needle beam excitation according to claim 6, characterized in that: The coupling medium is deionized water.

8. A method for photoacoustic microscopy based on gradient needle beam excitation, the method being applied to the photoacoustic microscopy system based on gradient needle beam excitation according to any one of claims 1 to 7, characterized in that: The method comprises: The laser output laser beam is adjusted to circular polarization by the polarization adjustment system; The 4F beam expansion system performs a beam expansion operation on the circularly polarized light beam, and the formed expanded light beam is reflected by the reflector and transmitted to the ultraviolet optical diffraction element; The expanded light beam is modulated by the ultraviolet optical diffraction element to form a gradient needle-shaped light beam, and is focused on the sample to generate an ultrasonic signal; The ultrasonic transducer is used to detect ultrasonic signals and send the detected ultrasonic signals to the host computer, so that the host computer completes image reconstruction based on the received ultrasonic signals.

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