Two-dimensional multi-focus optical resolution photoacoustic microscopic imaging system and imaging method

Abstract

The invention is applied to the technical field of optical microscopic imaging, and discloses a two-dimensional multi-focus optical resolution photoacoustic microscopic imaging system and an imaging method. The system comprises a laser, a lens assembly, a filtering pinhole, a reflector assembly, an optical diffraction element, a focusing lens, a self-made 3D scanner, an acoustic coding mask, a plane focusing ultrasonic transducer, an amplifier, a data acquisition card, a signal generator and an imaging processing terminal. According to the two-dimensional multi-focus optical resolution photoacoustic microscopic imaging system, a laser device outputs a laser beam, and the laser beam is projected to a subsequent optical diffraction element after being subjected to beam expanding by a beam expanding unit composed of a pair of plano-convex lenses and spatial filtering processing of a pinhole in sequence; and the imaging processing terminal obtains the digital photoacoustic signal and processes the digital photoacoustic signal to obtain an imaging image of the sample, so that the purpose of providing a solution for rapid OR-PAM in an economical and efficient manner is achieved, and the speed limitation caused by the laser repetition rate is broken.

Description

Technical Field

[0001] This invention relates to the field of optical microscopy imaging technology, specifically to a two-dimensional multifocal optical resolution photoacoustic microscopy imaging system and imaging method. Background Technology

[0002] With the continuous advancement and development of modern science and technology, photoacoustic imaging is an emerging and highly promising non-destructive testing method. It is a functional imaging method using ultrasound as a medium, combining the advantages of high penetration depth and high resolution of ultrasound imaging with the high contrast of optical imaging. In photoacoustic imaging, an ultrashort pulse laser is typically used to irradiate the object under test. The object absorbs laser energy and is excited. Then, through a non-radiative excitation process, the absorbed light energy is converted into heat energy, causing the material under test to expand and contract, resulting in periodic pressure changes. This generates an ultrasonic signal, known as a photoacoustic signal. After being received by an ultrasonic transducer, this signal is used to obtain a photoacoustic image, i.e., the internal structure of the object under test, using image reconstruction algorithms. Photoacoustic imaging is a hybrid, non-invasive imaging method with the advantages of being radiation-free, non-invasive, and multi-scale. It has developed rapidly in recent decades and has played an important role in biomedical monitoring and diagnosis, and industrial non-destructive testing. Optical resolution photoacoustic microscopy is a widely used imaging technique in clinical applications. It is a non-invasive biomedical imaging technique that can provide high-resolution and high-sensitivity visualization of wavelength-dependent light absorption at the cellular level. However, the imaging speed of high-resolution OR-PAM is limited because it requires point-by-point scanning with a focused laser beam. JiangboChen et al. achieved an increase in scanning speed by using a laser with a higher pulse repetition frequency and rapid control of a mirror mounted on a piezoelectric scanner or microelectromechanical system. However, the scanning speed is still limited by the characteristics of the laser crystal material and the microelectromechanical structure and control system. Liang Song et al. used a one-dimensional microlens array and a one-dimensional ultrasonic array to achieve an imaging speed that is tens of times faster than the traditional point-by-point scanning method without increasing the pulse repetition frequency of the laser. The multi-channel detection and data acquisition system in this method is both complex and expensive, and a compromise needs to be made between system cost and imaging speed. Yang Li et al. developed a spatiotemporal coding method using ergodic repeaters or chaotic cavities. These techniques can generate images with fewer ultrasonic transducers. However, they require recalibration for different samples, which leads to excessively long imaging times. In addition, due to their sensitivity to boundary conditions, they are difficult to achieve long-term stability in various environments. Yide Zhang et al. introduced an improved solution that enables single-shot 3D imaging without object-related calibration, called photoacoustic computed tomography via ER. However, due to the long acoustic delay line in PACTER, this method results in significant acoustic signal attenuation, especially for high-frequency signals. Furthermore, the imaging resolution of PACTER is limited by the frequency and bandwidth of the ultrasonic transducer. In summary, existing photoacoustic microscopy still suffers from problems such as system complexity and high cost, as well as limitations in imaging speed, highlighting the need for new low-cost and rapid imaging methods. Summary of the Invention

[0003] The purpose of this invention is to provide a two-dimensional multifocal optical resolution photoacoustic microscopy imaging system and imaging method to solve the problems of existing photoacoustic microscopy imaging techniques mentioned in the background art, such as system complexity and high cost and limited imaging speed.

[0004] To achieve the above objectives, the present invention provides the following technical solution: a two-dimensional multifocal optical resolution photoacoustic microscopy imaging system, the system comprising: a laser, a lens assembly, a filter pinhole, a reflector assembly, an optical diffraction element, a focusing lens, a self-made 3D scanner, an acoustic coding mask, a planar focusing ultrasonic transducer, an amplifier and a data acquisition card, a signal generator, and an imaging processing terminal, among other key components. The laser is used to generate a laser beam that illuminates the imaged object, and the laser beam is sequentially passed through a beam expander lens group and a spatial filter pinhole before illuminating subsequent optical elements. The optical diffraction element can convert a single incident beam of light into multiple focal points in a two-dimensional direction, and the focusing lens is used to focus the beam so that the focal point achieves a predetermined resolution on the original focal plane. The acoustic coding mask is a stepped acoustic coding mask located on the ultrasonic path between the cylindrical planar focused ultrasonic transducer and the sample. It spatially codes the photoacoustic signals at different focal points by introducing a position-related delay. The planar focused ultrasonic transducer is used to receive the encoded photoacoustic signal and convert it into an electrical signal. The surface of the planar focused ultrasonic transducer is provided with specific electrodes, a matching layer and a waterproof shielding layer. The amplifier is used to amplify the electrical signal output by the planar focusing ultrasonic transducer, and the data acquisition card digitizes the amplified signal. The signal generator outputs a periodic pulse signal, which synchronizes the external trigger input of the laser with the TriggerIn of the acquisition card. The signal generator and the LabVIEW program work together to control the laser pulse emission, the movement of the 3D scanner, and the data acquisition process to achieve the scanning and imaging of the sample. The lens assembly is located downstream of the laser, the filter pinhole is located downstream of the lens assembly, the mirror assembly is located downstream of the filter pinhole, the optical diffraction element is located downstream of the mirror assembly, and the focusing lens is located downstream of the optical diffraction element. The homemade 3D scanner is positioned downstream of the focusing lens, and the acoustic coding mask is positioned between the planar focusing ultrasonic transducer and the sample on the homemade 3D scanner. The planar focusing ultrasonic transducer and the sample are immersed in the medium on the homemade 3D scanner. Multiple focused light spots scan samples on a self-made 3D scanner, and the volume expansion of the samples caused by absorbing the light beam excites photoacoustic waves that are transmitted through the medium. The planar focusing ultrasonic transducer acquires photoacoustic waves transmitted through a medium, and is electrically connected to the data acquisition card via the amplifier; The data acquisition card is electrically connected to the imaging processing terminal to output digital photoacoustic signals to the imaging processing terminal. The imaging processing terminal processes the digital photoacoustic signals acquired by the data acquisition card to obtain an imaging image of the sample. The signal generator is connected to the laser and the data acquisition card.

[0005] Preferably, the lens group consists of a pair of plano-convex lenses, and the diameter of the spatial filter pinhole is 15 μm.

[0006] Preferably, the system uses custom optical diffraction elements and focusing lenses to convert a single incident beam into multiple focused spots along a two-dimensional direction at the original focal plane.

[0007] Preferably, the laser is used to image different tissue samples using lasers of different wavelengths and different pulse durations, or different wavelengths of lasers can be output by changing the laser. The optical diffraction element and focusing lens can be replaced or adjusted according to the required imaging resolution and number of focal points. After replacement or adjustment, the system needs to be recalibrated.

[0008] Preferably, the optical diffraction element is configured as a thin glass plate to facilitate integration into existing optical systems without requiring complex setup.

[0009] Preferably, a stepped acoustic coding mask is placed in the ultrasonic path between the planar focused ultrasonic transducer and the sample to encode photoacoustic signals of different focal spots.

[0010] Preferably, the custom LabVIEW program can adjust the acquisition delay, scanning range, scanning step size, sampling length, and data acquisition frequency according to imaging requirements, and can monitor the working status of each component of the system in real time, and provide alarms and corresponding handling when abnormalities occur.

[0011] Preferably, the homemade 3D scanner includes: A three-dimensional Cartesian coordinate system motion platform consisting of three linear motion modules, wherein the three linear motion modules are the X-axis module, the Y-axis module, and the Z-axis module; A motor control box electrically connected to the motion platform; And a host computer that is communicatively connected to the motor control box; The linear motion module is a PA150 precision linear slide, and its driving component is a two-phase 57 stepper motor; the linear motion module is equipped with a lead screw transmission mechanism that converts the rotary motion of the stepper motor into linear motion. The motor control box is used to receive control commands from the host computer and generate corresponding pulse signals and direction signals to drive the stepper motor in the linear motion module. Driven by the pulse signal, the stepper motor, through the lead screw transmission mechanism, can achieve a minimum linear displacement step size of 0.625μm. The host computer is a computer with LabVIEW installed, used to set the scanning area and generate control commands containing motion direction, number of steps and speed parameters, which are sent to the motor control box via serial communication.

[0012] The LabVIEW program can also adjust the acquisition delay, scanning range, scanning step size, sampling length, and data acquisition frequency according to imaging requirements, and can monitor the working status of each component of the system in real time, and issue alarms and take corresponding actions when abnormalities occur.

[0013] A two-dimensional multifocal optical resolution photoacoustic microscopy imaging method, the method comprising: The laser beam is output by controlling the laser through a signal generator; The laser beam is expanded by a pair of plano-convex lenses and spatially filtered through a pinhole. The filtered laser beam is converted into multiple focused spots along a two-dimensional direction at the original focal plane by a customized optical diffraction element and focusing lens; First, the position of the focused light spot is calibrated. A single focused light spot scans a sample immersed in water and mounted on a self-made 3D scanner. The sample excited by the focused light spot generates a photoacoustic signal. The photoacoustic signal is received by a planar focused ultrasonic transducer after propagating through an acoustic coding mask. It is amplified by two low-noise amplifiers and acquired by a high-speed acquisition card. The focused light spot is calibrated, and the position of each optical focus is identified by observing the maximum signal value. The Aline signal formed by each focused light spot scanning the sample is extracted to form the basic system matrix H. An enhanced regularization algorithm is designed to effectively separate mixed signals. Then, multiple focused light spots simultaneously scan the sample immersed in water, which is mounted on a self-made 3D scanner consisting of three precision linear slides. The sample excited by the multiple focused light spots generates multiple photoacoustic signals. The photoacoustic signals are received by a planar focused ultrasonic transducer after propagating through an acoustic coding mask, amplified by two low-noise amplifiers, and acquired by a high-speed acquisition card. Finally, the acquired mixed signals are decoded and the image is reconstructed by a custom fast iterative shrinkage threshold algorithm.

[0014] Preferably, the parameters of the acoustic coding mask are determined by simulation using the K-wave toolbox of MATLAB. By changing the mask's width, thickness, and other parameters, a signal correlation matrix is ​​established under different parameter combinations to determine the parameters that minimize the correlation of photoacoustic signals at different focal points as the final design parameters of the mask. Meanwhile, by establishing a photoacoustic coupling effect model for a two-dimensional multifocal optical resolution photoacoustic microscopy imaging system suitable for complex optical paths, the propagation, reflection, and attenuation of ultrasound waves in tissues are predicted, and the reception of ultrasound signals is determined and optimized. The variation patterns of characteristic parameters such as amplitude, spectrum, temporal characteristics, and phase of photoacoustic signals under the influence of laser energy, numerical aperture, light intensity distribution, tissue scattering, and ultrasound frequency are observed, thereby preparing optical diffraction elements to facilitate the provision of a separated multifocal optical field for subsequent steps (acoustic coding and algorithms).

[0015] Preferably, the photoacoustic coupling effect model is as follows: ;

[0016] , ; and discretization form ; ; By solving the regularization minimization problem ; In the formula, The original photoacoustic image signal (i.e., the target to be reconstructed); H: System matrix, obtained during the calibration phase, describes the contribution of each focus to the received signal; λ: Regularization parameter, used to balance the data fitting term and the image smoothing term; : The actual mixed signal acquired.

[0017] Image reconstruction is performed using a custom fast iterative shrinkage threshold algorithm.

[0018] The photoacoustic coupling effect model (including continuous and discrete forms) is mainly used to reconstruct the original photoacoustic image from the mixed acquired signals. The specific objective is: 1. Establish a signal mixing mechanism Describe how photoacoustic signals generated by multiple focal excitations are received by the same ultrasonic transducer after passing through an acoustically coded mask; In the model Let represent the acoustic propagation response function corresponding to the (i,j)th focus; This represents the distribution of the original, uncoded photoacoustic signal.

[0019] 2. Achieve signal separation and image reconstruction Through discretization This transforms the problem into a linear inverse problem; Solving problems using regularization minimization (such as TV regularization) .

[0020] Compared with existing technologies, the beneficial effects of this invention are as follows: This two-dimensional multifocal optical resolution photoacoustic microscopy imaging system outputs a laser beam, which is then expanded and spatially filtered through a pinhole by a beam-expanding unit composed of a pair of plano-convex lenses before being projected onto subsequent optical diffraction elements. These elements are placed in the optical path in front of the focusing lens and work in conjunction with it to convert a single incident beam into multiple focal points in a two-dimensional direction. The imaging sample is placed in a water immersion environment within the focal region of the focusing lens and precisely scanned and positioned using a self-made 3D scanner composed of three precision linear slides to obtain different focal points. The photoacoustic signal of the position uses a stepped acoustic coding mask located on the ultrasonic propagation path between the planar focusing ultrasonic transducer and the sample. The volume expansion of the sample due to the absorption of the beam excites photoacoustic waves, which are transmitted through the medium. The ultrasonic transducer receives the photoacoustic waves transmitted through the medium and outputs them to the amplifier for amplification to obtain an amplified signal. Then, the signal is digitized by the data acquisition card to obtain a digital photoacoustic signal. The imaging processing terminal acquires the digital photoacoustic signal and processes it to obtain an image of the sample. This achieves the goal of providing a solution for rapid OR-PAM in an economical and efficient manner, breaking the speed limitation caused by the laser repetition rate. Attached Figure Description

[0021] Figure 1 This is a structural diagram of the two-dimensional multifocal optical resolution photoacoustic microscopy imaging system of the present invention; Figure 2 This is a schematic diagram of the acoustic coding mask of the present invention; Figure 3 This is a structural diagram of the self-made 3D scanner of the present invention; Figure 4This is a flowchart of the two-dimensional multifocal optical resolution photoacoustic microscopy imaging method of the present invention; Figure 5 This is a flowchart of the algorithm of the present invention; Figure 6 This is a reconstructed image from the two-dimensional multifocal optical resolution photoacoustic microscopy imaging system of the present invention.

[0022] In the diagram: 1. Laser; 2. Lens assembly; 3. Filter pinhole; 4. Mirror assembly; 5. Optical diffraction element; 6. Focusing lens; 7. Self-made 3D scanner; 8. Acoustic coding mask; 9. Planar focusing ultrasonic transducer; 10. Amplifier; 11. Data acquisition card; 12. Signal generator; 13. Imaging processing terminal. Detailed Implementation

[0023] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] Example 1 Please see Figures 1-6 The present invention provides a technical solution: a two-dimensional multifocal optical resolution photoacoustic microscopy imaging system.

[0025] Figure 1 The figure shows a schematic diagram of the structure of a two-dimensional multifocal optical resolution photoacoustic microscopy imaging system provided in an embodiment of the present invention. The system includes: a laser 1, a lens assembly 2, a filter pinhole 3, a mirror assembly 4, an optical diffraction element 5, a focusing lens 6, a self-made 3D scanner 7, an acoustic coding mask 8, a planar focusing ultrasonic transducer 9, an amplifier 10, a data acquisition card 11, a signal generator 12, and an imaging processing terminal 13. Laser 1 is used to generate a laser beam that illuminates the imaging object, and the laser beam is sequentially passed through a beam expanding lens group and a spatial filter pinhole 3 before illuminating subsequent optical elements. The optical diffraction element 5 can convert a single incident beam of light into multiple focal points in a two-dimensional direction, and the focusing lens 6 is used to focus the beam so that the focal point achieves a predetermined resolution on the original focal plane. Acoustic coding mask 8, a stepped acoustic coding mask 8, is located on the ultrasonic path between the cylindrical planar focusing ultrasonic transducer 9 and the sample. It spatially codes the photoacoustic signals at different focal points by introducing position-related delay. The planar focused ultrasonic transducer 9 is used to receive the encoded photoacoustic signal and convert it into an electrical signal. The surface of the planar focused ultrasonic transducer 9 is provided with specific electrodes, a matching layer and a waterproof shielding layer. Amplifier 10 is used to amplify the electrical signal output by the planar focusing ultrasonic transducer 9, and data acquisition card 11 digitizes the amplified signal; The signal generator 12 outputs a periodic pulse signal, which synchronizes the external trigger input of the laser 1 with the TriggerIn of the acquisition card. The signal generator 12 and the custom LabVIEW program work together to control the laser pulse emission, the movement of the 3D scanner, and the data acquisition process to achieve the scanning and imaging of the sample. Lens assembly 2 is located downstream of laser 1, filter pinhole 3 is located downstream of lens assembly 2, mirror assembly 4 is located downstream of filter pinhole 3, optical diffraction element 5 is located downstream of mirror assembly 4, and focusing lens 6 is located downstream of optical diffraction element 5. The self-made 3D scanner 7 is positioned downstream of the focusing lens 6, and the acoustic coding mask 8 is positioned between the planar focusing ultrasonic transducer 9 and the sample on the self-made 3D scanner 7. The planar focusing ultrasonic transducer 9 and the sample are immersed in the medium on the self-made 3D scanner 7. Multiple focused light spots scan the sample on the self-made 3D scanner 7. The volume expansion of the sample caused by the absorption of the light beam excites photoacoustic waves, which are then transmitted through the medium. The planar focusing ultrasonic transducer 9 acquires photoacoustic waves transmitted through the medium, and is electrically connected to the data acquisition card 11 via the amplifier 10; The data acquisition card 11 is electrically connected to the imaging processing terminal 13 to output digital photoacoustic signals to the imaging processing terminal 13; The imaging processing terminal 13 processes the digital photoacoustic signals acquired by the data acquisition card 11 to obtain an imaging image of the sample; The signal generator 12 is connected to the laser 1 and the data acquisition card 11.

[0026] In the aforementioned two-dimensional multifocal optical resolution photoacoustic microscopy imaging system, the laser beam is expanded and spatially filtered through a beam-expanding unit composed of a pair of plano-convex lenses before being projected onto the subsequent optical diffraction element 5. The optical diffraction element 5 is placed in the optical path in front of the focusing lens 6 and works in conjunction with the focusing lens 6 to convert the single incident beam into multiple focal points in a two-dimensional direction. The imaging sample is placed in a water immersion environment within the focal area of ​​the focusing lens 6 and is precisely scanned and positioned by a self-made 3D scanner 7 composed of three precision linear slides to obtain photoacoustic signals at different positions. A stepped acoustic coding mask 8 is used, which is located on the ultrasonic propagation path between the planar focusing ultrasonic transducer 9 and the sample. The volume expansion of the sample due to the absorption of the beam excites photoacoustic waves, which are transmitted through the medium. The ultrasonic transducer receives the photoacoustic waves transmitted through the medium and outputs them to the amplifier 10 for amplification to obtain an amplified signal. The signal is then digitized by the data acquisition card 11 to obtain a digital photoacoustic signal. The imaging processing terminal 13 acquires the digital photoacoustic signal and processes it to obtain the imaging image of the sample.

[0027] The technical method in this embodiment addresses the challenge of limited imaging speed in high-resolution OR-PAM by proposing a two-dimensional multifocal optical resolution photoacoustic microscopy imaging system. Through an optical diffraction element 5 and a focusing lens 6, a single incident beam is converted into multiple focused spots in a two-dimensional direction. An acoustic coding mask 8 is designed to propagate multiple photoacoustic signals generated by the sample excited by the multiple focused spots, introducing position-related phase and making each photoacoustic signal uniquely identifiable in the mixed photoacoustic signal. The mixed photoacoustic signal is a linear combination of photoacoustic signals from a single point. Photoacoustic signals from different beams are separated through spatial and temporal coding. Compared with traditional OR-PAM with the same laser repetition rate, the imaging speed is significantly improved.

[0028] In a more specific embodiment, laser 1 is selected as a 532nm nanosecond pulsed laser with a wavelength of 532nm, a repetition frequency of 10kHz, and a pulse width of 2ns. The above-mentioned two-dimensional multifocal optical resolution photoacoustic microscopy imaging system can use lasers of different wavelengths and different pulse durations to image different tissue samples, and can also output lasers of different wavelengths by changing laser 1.

[0029] In a more specific embodiment, the planar focusing ultrasonic transducer 9 has a center frequency of 7.85 MHz and a -6 dB bandwidth of 72%. In a more specific embodiment, amplifier 10 is selected from two ZFL-500LN+ RF low noise amplifiers 10, with a frequency range of 0.1-500MHz, a gain of approximately 24dB, and a noise figure of 2.9dB; In a more specific embodiment, the data acquisition card 11 uses a 5124 acquisition card with a sampling frequency of 200MS / s (200 million samples per second) and an acquisition resolution of 12 bits.

[0030] In a more specific embodiment, the lens group consists of a pair of plano-convex lenses, and the diameter of the spatial filter pinhole 3 is 15 μm.

[0031] In a more specific embodiment, the optical diffraction element 5 is a two-dimensional cascaded grating beam splitter with a beam splitting mode of 2×2, a beam splitting point spacing of 1000μm, a light-transmitting aperture of Ф20mm, a working wavelength of 532nm, and a beam splitting angle of 1.15°. The optical diffraction element 5 and the focusing lens 6 can be replaced or adjusted according to the required imaging resolution and number of focal points. After replacement or adjustment, the system calibration operation needs to be re-performed.

[0032] In a more specific embodiment, the parameters of the acoustic coding mask 8 are determined through simulation using the K-wave toolbox in MATLAB. By changing parameters such as the mask's width and thickness, signal correlation matrices are established under different parameter combinations to determine the parameters that minimize the correlation of photoacoustic signals at different focal points as the final design parameters of the mask. In this embodiment, the acoustic coding mask 8 is made of PLA material. In the simulation, the sound velocity of water is 1480 m / s and its density is 1000 kg / m³, while the sound velocity of PLA material is 2200 m / s and its density is 1240 kg / m³. The transducer center frequency is 8 MHz. Please refer to [link / reference]. Figure 2 In this embodiment, the acoustic coding mask 8 includes three steps with a height difference of 0.5 mm between adjacent steps. The acoustic coding mask 8 introduces spatial modulation on the propagation path of the photoacoustic signal to change the propagation time and phase of the photoacoustic signal at different incident positions, so that the signals from different focal points exhibit distinguishable coding features at the receiving end. This can reduce the correlation between different signals, thereby achieving the separation of multifocal photoacoustic signals during decoding and reconstruction.

[0033] For a more specific embodiment, please refer to Figure 3The self-made 3D scanner 7 consists of three PA150 precision linear slides designed by Zoli Hanguang and a motor control box. The PA150 precision linear slides use two-phase 57 stepper motors with a step angle of 1.8° and a minimum scanning step size of 0.625μm. The three PA150 precision linear slides correspond to the X, Y, and Z directions respectively. By combining them, they achieve three-dimensional positioning and scanning. The two-phase 57 stepper motors receive pulse signals output from the control box. Each pulse corresponds to a fixed step angle (1.8°), which is converted into linear motion by a lead screw transmission mechanism. Under microstepping drive, the minimum linear displacement step size is 0.625μm, ensuring micron-level positioning accuracy. The LabVIEW program generates control commands (including motion direction, number of steps, speed, etc.) according to the set scanning area. The commands are sent to the motor control box through the serial port. The control box parses the commands and converts them into a pulse signal sequence. The stepper motor completes the displacement according to the number and frequency of pulses. The direction of motion is determined by the direction signal, thereby realizing precise motion control of the 3D scanner.

[0034] In a more specific embodiment, the medium is deaerated water, and the sample is immersed in the medium.

[0035] Compared with existing high-resolution OR-PAM imaging technology, the above-mentioned two-dimensional multifocal optical resolution photoacoustic microscopy imaging system has the following characteristics: 1. The sample is placed at the focal point of the optical path, enabling multiple target points to be excited simultaneously with a single laser. Furthermore, the optical diffraction element 5, as a thin glass plate, can be easily integrated into existing optical systems without complex and expensive setups. 2. An acoustic coding mask 8 is designed to separate photoacoustic signals from different beams through spatial and temporal coding. 3. Compared with the traditional OR-PAM method, the two-dimensional multifocal optical resolution photoacoustic microscopy imaging system greatly improves the imaging speed while only incurring marginal additional costs to the system (i.e., 3D printing PLA acoustic coding mask 8). The degree of improvement depends on the number of effective light spots formed by the optical diffraction element 5. As long as the laser power is sufficient, the number of effective light spots can be further increased.

[0036] Example 2 Please see Figure 4 , Figure 4 This is a flowchart illustrating the two-dimensional multifocal optical resolution photoacoustic microscopy imaging method provided in an embodiment of the present invention. The embodiment also provides a two-dimensional multifocal optical resolution photoacoustic microscopy imaging method, wherein the two-dimensional multifocal optical resolution photoacoustic microscopy imaging method is applied to the aforementioned two-dimensional multifocal optical resolution photoacoustic microscopy imaging system, such as... Figure 4 As shown, the method is divided into two parts: the first part, the calibration part, includes steps S1-S7, and the second part, the imaging part, includes steps T1-T6.

[0037] Part One: Calibration Section S1: The laser beam is output by the laser 1 through the signal generator 12; Specifically, in this embodiment, the signal generator 12 can control the laser 1 to output a laser with a wavelength of 532nm. Different wavelengths and different pulse durations of lasers can be used for imaging different tissue samples. For example, a laser with a wavelength of 266nm, a pulse repetition rate of 10kHz, and a pulse duration of 2ns can be used for a specific tissue sample. For different samples, the laser 1 can also be replaced and the wavelength changed, with the sample having the largest absorption coefficient and the strongest photoacoustic signal as the standard.

[0038] S2: The laser beam is converted into multiple focused spots along the two-dimensional direction at the original focal plane through a series of dimming operations; Specifically, the laser beam is extended by a pair of plano-convex lenses and spatially filtered through a pinhole. After filtering, the laser beam is converted into multiple focused spots along the two-dimensional direction at the original focal plane by a customized optical diffraction element 5 and a focusing lens 6.

[0039] S3: Position calibration of the focused spot; a single focused spot scans the phantom sample immersed in water and mounted on the self-made 3D scanner 7; the sample excited by the focused spot generates photoacoustic signals. Specifically, during the system calibration phase, the remaining focal points, except for the target focal point, are sequentially blocked using a single-point scanning mode, leaving only a single light spot to excite the sample. A high-precision 3D scanner is used to gradually scan and record the position of the maximum photoacoustic signal received by the planar focusing ultrasonic transducer 9 to determine the actual coordinates of each focal point on the sample plane. Subsequently, a two-dimensional Gaussian fitting is used to center the light intensity distribution, with a position error of less than ±2µm. The calibration data (a dataset containing spatial coordinates and Aline signals, etc.) is used to construct the system matrix H and is used for signal registration and position correction in subsequent imaging. S4: The photoacoustic signal is transmitted through the acoustic coding mask 8 and received by the planar focusing ultrasonic transducer 9, and then output to the amplifier 10 for amplification to obtain the amplified signal; S5: The amplified signal is digitized by the data acquisition card 11 to obtain a digital photoacoustic signal; S6: Calibrate the focused spot and observe the maximum signal value to identify the position of each optical focus; The A-line signals formed by the scanning samples of each focused spot are extracted to form the basic system matrix H. Specifically, in this embodiment, the three focused spots other than the target focused spot are blocked, while the target focused spot to be extracted is retained. The target focused spot is then scanned, and the A-line signals generated by the scanned samples of the target focused spot are extracted and stored in the system matrix H. The A-line signals formed by the scanned samples of the four focused spots are extracted one by one to form the complete basic system matrix H. The basic system matrix H is an M×N matrix, where M is the number of sampling points and N is the number of focal points. The j-th column of the system matrix H represents the A-line signal formed by the scanned sample when the j-th focal point is excited alone.

[0040] After obtaining the system matrix H, a propagation model based on photoacoustic signals (photoacoustic coupling effect model) is constructed: ; , ; and discretization form ; ; By solving the regularization minimization problem ;; S7: Design an enhanced regularization algorithm for effectively separating mixed signals.

[0041] Specifically, in this embodiment, the "enhanced regularization algorithm" is preferably the Fast Iterative Shrinking Threshold Algorithm (FISTA) combined with L1 regularization. Please refer to [link to relevant documentation]. Figure 5 , Figure 5 This is a flowchart illustrating the fast iterative shrinkage threshold algorithm provided in an embodiment of the present invention, the implementation steps of which include: a) Problem modeling: For the acquired mixed signals The following optimization objectives are established: ; in, For the system matrix, Let be the coefficient matrix to be determined. This is the regularization parameter.

[0042] b) Parameter settings: The number of iterations is preferably 50-300, more preferably 100; regularization parameter In 10 -5 Up to 10 -2 Adjustable within a range, commonly used initial value is 10 -3 Step size parameter Pick ,in If it cannot be calculated directly, the retrospective shrinkage method can be used, with the shrinkage factor taken as 0.5–0.8.

[0043] Specifically, regularization parameters The selection criteria or range are determined through experience and statistical methods, and the optimal parameters are determined through the L-curve method or cross-validation.

[0044] c) Iterative process: Initialize A 0 =0、Z 0 =A 0 t0=1, execute in each iteration: 1) Calculate the residuals and gradients: , R; 2) Gradient update: ; 3) Soft thresholding and nonnegative projection: ,in ; 4) Momentum acceleration: , .

[0045] d) Termination condition: When the number of iterations is reached, or the relative change between two consecutive results is less than 10. -6 Up to 10 -3 Stop when the iteration ends, output the final result, and specify the threshold for the iteration termination condition:

[0046] The process terminates when the above formula is satisfied.

[0047] Specifically, in the experiment with blade and human hair samples, the reconstruction error curve tended to stabilize after about 6 iterations, indicating that the algorithm has good convergence and stability on real data.

[0048] e) Image backfilling: [Image backfilling] The four rows are respectively filled into 2×2 200×200 sub-blocks, and spliced ​​together to obtain a 400×400 reconstructed image.

[0049] Specifically, the stability of the calibrated system was evaluated. After running continuously for 3 hours, the same standard sample was repeatedly measured. The standard deviation of the signal strength was less than 2.5%, and the reconstructed structural similarity index (SSIM) remained above 0.98, indicating that the system maintained high stability after long-term operation.

[0050] Part Two: Imaging Section T1: The laser beam output by the laser 1 is controlled by the signal generator 12; T2: The laser beam is transformed into multiple focused spots along the two-dimensional direction at the original focal plane through a series of dimming operations; T3: The focused light spot simultaneously scans the sample immersed in water, which is mounted on the self-made 3D scanner 7. The sample excited by multiple focused light spots generates multiple photoacoustic signals. Specifically, in this embodiment, the sample is placed at the focal point of the optical path, so that a single laser can simultaneously excite multiple target points. The spacing between adjacent light spots is determined by the separation angle of the optical diffraction element 5 and the effective focal length of the focusing lens 6.

[0051] T4: The photoacoustic signal is transmitted through the acoustic coding mask 8 and received by the planar focusing ultrasonic transducer 9, and then output to the amplifier 10 for amplification to obtain the amplified signal; T5: The amplified signal is digitized by the data acquisition card 11 to obtain a digital photoacoustic signal; T6: The imaging processing terminal 13 acquires and processes digital photoacoustic signals to obtain an imaging image of the sample; Specifically, in this embodiment, the imaging processing is preferably implemented by combining the aforementioned fast iterative shrinking threshold algorithm FISTA with L1 regularization to decode the digital photoacoustic signal and reconstruct the image, thereby achieving rapid separation and reconstruction of multifocal mixed signals while maintaining high resolution.

[0052] Specifically, to verify the system's imaging performance, this embodiment images a blade with sharp edges, acquires the resulting image, and extracts the edge spread function (ESF). Then, the derivative of the ESF is calculated to obtain the line spread function (LSF). The final result shows that the system's spatial resolution can reach 3.3 μm.

[0053] Specifically, to verify the accuracy of the system's imaging, the reconstructed blade edge image was compared with the microscopic image. The edge position deviation between the two was less than 3µm, and the extracted line spread function (LSF) half-width was 3.3µm, which was consistent with the actual edge width measured by the microscope, proving the spatial accuracy and repeatability of the system's imaging results.

[0054] Please see Figure 6 , Figure 6 The image is a reconstructed image from a two-dimensional multifocal optical resolution photoacoustic microscopy system provided in an embodiment of the present invention.

[0055] This invention provides a two-dimensional multifocal optical resolution photoacoustic microscopy imaging system and method. The system includes a laser 1, a lens assembly 2, a filter pinhole 3, a mirror assembly 4, an optical diffraction element 5, a focusing lens 6, a self-made 3D scanner 7, an acoustic coding mask 8, a planar focusing ultrasonic transducer 9, an amplifier 10, a data acquisition card 11, a signal generator 12, and an imaging processing terminal 13. The aforementioned two-dimensional multifocal optical resolution photoacoustic microscopy imaging system, through the customized optical diffraction element 5 and focusing lens 6, converts a single incident beam of light into multiple focused spots in a two-dimensional direction. An acoustic coding mask 8 is designed to propagate multiple photoacoustic signals generated by the sample excited by the multiple focused spots. Photoacoustic signals from different beams are separated through spatial and temporal coding, and the average correlation coefficient of the signals is... The number decreased from 0.62 to 0.14. Compared with the traditional OR-PAM method, the spatial resolution of the two-dimensional multifocal optical resolution photoacoustic microscopy imaging system can also reach 3.3 μm, which is consistent with the traditional OR-PAM method. The imaging speed is greatly improved, about 4 times faster than the traditional single-point scanning. The optical diffraction element 5, as a thin glass plate, can be easily integrated into the existing optical system without complex and expensive settings. It provides a cost-effective solution for fast OR-PAM, breaking the speed limit caused by laser repetition rate. The proposed method can be applied to a variety of OR-PAM configurations and realize new applications of high-speed imaging, such as applications such as minimally invasive imaging catheters. It overcomes the limitation of thin shape factors in accommodating numerous ultrasound arrays in fast optical resolution photoacoustic endoscopes.

[0056] The above specific embodiments further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above are merely specific embodiments of the present invention and do not limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention are included within the protection scope of the present invention.

Claims

1. A two-dimensional multifocal optical resolution photoacoustic microscopy imaging system, characterized in that: The system includes: a laser (1), a lens assembly (2), a filter pinhole (3), a mirror assembly (4), an optical diffraction element (5), a focusing lens (6), a self-made 3D scanner (7), an acoustic coding mask (8), a planar focusing ultrasonic transducer (9), an amplifier (10), a data acquisition card (11), a signal generator (12), and an imaging processing terminal (13). The laser (1) is used to generate a laser beam that illuminates the imaging object, and the laser beam is irradiated by subsequent optical elements after passing through a beam expanding lens group and a spatial filter pinhole (3) in sequence. The optical diffraction element (5) can convert a single incident beam into multiple focal points in a two-dimensional direction, and the focusing lens (6) is used to focus the beam so that the focal point achieves a predetermined resolution on the original focal plane. The acoustic coding mask (8) is a stepped acoustic coding mask (8) located on the ultrasonic path between the cylindrical planar focused ultrasonic transducer (9) and the sample. It spatially codes the photoacoustic signals at different focal points by introducing position-related delay. The planar focused ultrasonic transducer (9) is used to receive the encoded photoacoustic signal and convert it into an electrical signal. The planar focused ultrasonic transducer (9) has specific electrodes, a matching layer and a waterproof shielding layer on its surface. The amplifier (10) is used to amplify the electrical signal output by the planar focused ultrasonic transducer (9), and the data acquisition card (11) digitizes the amplified signal. The signal generator (12) outputs a periodic pulse signal, which synchronizes the external trigger input of the laser (1) with the TriggerIn of the acquisition card. The signal generator (12) and the LabVIEW program work together to control the laser pulse emission, the movement of the 3D scanner and the data acquisition process, so as to realize the scanning and imaging of the sample. The lens assembly (2) is located downstream of the laser (1), the filter pinhole (3) is located downstream of the lens assembly (2), the mirror assembly (4) is located downstream of the filter pinhole (3), the optical diffraction element (5) is located downstream of the mirror assembly (4), and the focusing lens (6) is located downstream of the optical diffraction element (5). The homemade 3D scanner (7) is positioned downstream of the focusing lens (6), and the acoustic coding mask (8) is positioned between the planar focusing ultrasonic transducer (9) and the sample on the homemade 3D scanner (7). The planar focusing ultrasonic transducer (9) and the sample are immersed in the medium on the homemade 3D scanner (7). Multiple samples on the self-made 3D scanner (7) are scanned by the focused light spot, and the volume expansion of the sample caused by the absorption of the light beam excites photoacoustic waves to be transmitted through the medium; The planar focusing ultrasonic transducer (9) acquires photoacoustic waves transmitted through the medium and is electrically connected to the data acquisition card (11) through the amplifier (10); The data acquisition card (11) is electrically connected to the imaging processing terminal (13) to output digital photoacoustic signals to the imaging processing terminal (13). The imaging processing terminal (13) processes the digital photoacoustic signals acquired by the data acquisition card (11) to obtain an imaging image of the sample; The signal generator (12) is connected to the laser (1) and the data acquisition card (11).

2. The two-dimensional multifocal optical resolution photoacoustic microscopy imaging system according to claim 1, characterized in that: The lens group consists of a pair of plano-convex lenses, and the diameter of the spatial filter pinhole (3) is 15 μm.

3. The two-dimensional multifocal optical resolution photoacoustic microscopy imaging system according to claim 1, characterized in that: The system uses a custom optical diffraction element (5) and a focusing lens (6) to convert a single incident beam into multiple focused spots along a two-dimensional direction at the original focal plane.

4. The two-dimensional multifocal optical resolution photoacoustic microscopy imaging system according to claim 1, characterized in that: The laser (1) uses lasers of different wavelengths and different pulse durations to image different tissue samples, or outputs lasers of different wavelengths by changing the laser (1). The optical diffraction element (5) and focusing lens (6) can be replaced or adjusted according to the required imaging resolution and number of focal points. After replacement or adjustment, the system calibration operation needs to be performed again.

5. A two-dimensional multifocal optical resolution photoacoustic microscopy imaging system according to claim 3, characterized in that: The optical diffraction element (5) is configured as a thin glass plate so that it can be easily integrated into existing optical systems without complicated setup.

6. The two-dimensional multifocal optical resolution photoacoustic microscopy imaging system according to claim 1, characterized in that: A stepped acoustic coding mask (8) is placed in the ultrasonic path between the planar focused ultrasonic transducer (9) and the sample to encode photoacoustic signals of different focal spots.

7. A two-dimensional multifocal optical resolution photoacoustic microscopy imaging system according to claim 1, characterized in that: The homemade 3D scanner includes: A three-dimensional Cartesian coordinate system motion platform consisting of three linear motion modules, wherein the three linear motion modules are the X-axis module, the Y-axis module, and the Z-axis module; A motor control box electrically connected to the motion platform; And a host computer that is communicatively connected to the motor control box; The linear motion module is a precision linear slide, and its driving component is a stepper motor; the linear motion module is equipped with a lead screw transmission mechanism that converts the rotary motion of the stepper motor into linear motion. The motor control box is used to receive control commands from the host computer and generate corresponding pulse signals and direction signals to drive the stepper motor in the linear motion module. The host computer is a computer with LabVIEW installed, used to set the scanning area and generate control commands containing motion direction, number of steps and speed parameters, which are sent to the motor control box via serial communication.

8. A two-dimensional multifocal optical resolution photoacoustic microscopy imaging method, characterized in that: The two-dimensional multifocal optical resolution photoacoustic microscopy imaging method is applied to the two-dimensional multifocal optical resolution photoacoustic microscopy imaging system as described in any one of claims 1-7, and the method includes: The laser beam is output by controlling the laser (1) through the signal generator (12); The laser beam is expanded by a pair of plano-convex lenses and spatially filtered through a pinhole. The filtered laser beam is converted into multiple focused spots along the two-dimensional direction at the original focal plane by a customized optical diffraction element (5) and a focusing lens (6); First, the position of the focused spot is calibrated. A single focused spot scans a sample immersed in water on a self-made 3D scanner (7). The sample excited by the focused spot generates a photoacoustic signal. The photoacoustic signal is transmitted through an acoustic coding mask (8) and received by a planar focused ultrasonic transducer (9). It is amplified by two low-noise amplifiers (10) and acquired by a high-speed acquisition card. The focused spot is calibrated. The position of each optical focus is identified by observing the maximum signal value. The Aline signal formed by each focused spot scanning sample is extracted to form the basic system matrix H. An enhanced regularization algorithm is designed to effectively separate mixed signals. Then, multiple focused light spots simultaneously scan the sample immersed in water, which is mounted on a self-made 3D scanner (7) consisting of three precision linear slides. The sample excited by multiple focused light spots generates multiple photoacoustic signals. The photoacoustic signals are transmitted through an acoustic coding mask (8) and received by a planar focused ultrasonic transducer (9). They are amplified by two low-noise amplifiers (10) and acquired by a high-speed acquisition card. Finally, the acquired mixed signals are decoded and the image is reconstructed by a custom fast iterative shrinkage threshold algorithm.

9. A two-dimensional multifocal optical resolution photoacoustic microscopy imaging method according to claim 8, characterized in that: The parameters of the acoustic coding mask (8) are determined by simulation using the K-wave toolbox of MATLAB. By changing the mask's width, thickness and other parameters, a signal correlation matrix under different parameter combinations is established to determine the parameters that minimize the correlation of photoacoustic signals at different focal points as the final design parameters of the mask. Meanwhile, by establishing a photoacoustic coupling effect model for a two-dimensional multifocal optical resolution photoacoustic microscopic imaging system suitable for complex optical paths, the propagation, reflection and attenuation of ultrasound in tissues are predicted, and the reception of ultrasound signals is determined and optimized. The variation law of amplitude, spectrum, time domain characteristics and phase characteristic parameters of photoacoustic signals under the influence of laser energy, numerical aperture, light intensity distribution, tissue scattering and ultrasound frequency is observed, thereby preparing optical diffraction elements (5).

10. A two-dimensional multifocal optical resolution photoacoustic microscopy imaging method according to claim 8, characterized in that: The photoacoustic coupling effect model is as follows: ； , ； and discretization form ； ； By solving the regularization minimization problem ； Image reconstruction is performed using a custom fast iterative shrinkage thresholding algorithm (FISTA).

Images