Multi-scale photoacoustic microscope based on stepless adjustment relay optical path and imaging method thereof

Abstract

The application discloses a kind of multiscale photoacoustic microscopes and imaging method thereof.The device includes laser source component, light spot optimization component, relay optical path modulation scanning component, reflective imaging port component, detection component, signal processing component and computer.The application uses spatial transmission pulsed laser as excitation light, uses two-dimensional scanning galvanometer for optical scanning, uses microscopic objective focusing, uses adjustable relay optical path to adjust the scanning field scale and focal point scale of excitation light steplessly, while keeping resolution at the same level, combined with diameter rotation scanning method, uses line focusing ultrasonic transducer detection, realizes multiscale high-resolution three-dimensional imaging.

Description

Technical Field

[0001] This invention relates to the fields of optical design, automatic control and imaging technology, and specifically to a multi-scale photoacoustic microscope and its imaging method based on a continuously adjustable relay optical path. Background Technology

[0002] Single-shot, large-field-of-view imaging of biological tissues is of great significance for rapidly identifying regions of interest, quickly acquiring information about the entire organ, and simultaneously imaging physiological information about the relationships between different regions in living organisms. At the same time, the ability to acquire fine structures within regions of interest can greatly expand the biological questions that photoacoustic imaging systems can investigate.

[0003] To acquire fine structural or physiological information of biological tissues within a large field of view, high-resolution and wide-field-of-view photoacoustic images are required. However, current photoacoustic microscopy systems are often limited by the scanning mechanism or the inherent spatial bandwidth product of the optical excitation system, failing to meet the needs of high-resolution imaging across multiple regions and scales within a large field of view. Traditional optical-resolution photoacoustic microscopy typically employs high-performance mechanical scanning devices to achieve stable and precise scanning of the imaging end or sample, but the large driving force and the mass of the moving object severely limit the imaging speed. Replacing mechanical scanning with optical scanning can significantly improve imaging speed. Current optical scanning structures are mainly divided into two types: back-lens scanning and front-lens scanning. The rear-lens scanning structure requires a sufficient working distance to place the scanning mirror, thus limiting the use of high numerical aperture objectives and making it difficult to achieve high-resolution imaging. Its field of view is mainly limited by the flat-field scanning range or the maximum scanning angle of the scanning mirror. In contrast, the front-lens scanning structure has a smaller working distance requirement, allowing the use of high numerical aperture for high-resolution imaging. In existing systems, the use of line-focusing ultrasonic transducers has significantly expanded the acoustic detection field of view, so that the field of view of the front-lens scanning photoacoustic imaging system is only limited by the optical excitation range. However, the existing front-lens scanning system scans directly at a certain distance in front of the lens entrance pupil, without making full use of the lens's field of view.

[0004] To address the above issues, domestic and international research groups have explored various methods for large-field-of-view, high-resolution imaging under different scanning mechanisms. Some methods, such as multiple scans using mechanical movement combined with image stitching, have proven effective in improving the overall system's large-area, high-resolution imaging capabilities, but these methods significantly increase the complexity of the system and post-processing. Furthermore, once these systems are built, their resolution and maximum imaging field of view cannot be adjusted, making it difficult to meet the needs of imaging biological tissues at different scales. Dual-mode photoacoustic microscopy systems, which combine optical and acoustic resolution, only change the field of view in the depth direction, and the lateral resolution deteriorates severely at large depths. Switching-focus photoacoustic microscopy systems using scanning lenses and microscope objectives can provide two optical excitation modes, but the significant difference in working distance between the two modes makes sample repositioning during switching difficult. Summary of the Invention

[0005] (a) Technical problems to be solved To improve the large field-of-view, high-resolution imaging performance of a single system and achieve multi-scale imaging, this invention proposes a novel imaging method with stepless adjustment of field of view and resolution. Specifically, it is a multi-scale photoacoustic microscope and its imaging method based on a stepless adjustment relay optical path. It can adapt to imaging regions of interest at different scales of biological tissues and detail resolution requirements at different scales, enabling the system to obtain more sample information and greatly increasing the application value of the system.

[0006] (II) Technical Solution To address its technical problems, this invention provides a multi-scale photoacoustic microscope and its imaging method based on a continuously adjustable relay optical path. The specific technical solution is as follows.

[0007] A multi-scale photoacoustic microscope based on a continuously adjustable relay optical path is characterized by comprising: a laser source assembly, a spot optimization assembly, a relay optical path modulation and scanning assembly, a reflective imaging port assembly, a detection assembly, a signal processing assembly, and a computer. The laser source component emits at least two parallel pulse beams of different wavelengths along the optical axis. The beams pass sequentially through the spot optimization component, the relay optical path modulation scanning component, and the reflective imaging port component to illuminate the imaging target, thereby exciting the photoacoustic signal of the imaging target and triggering the signal synchronously. The beam optimization component is used for beam shaping and expansion; The relay optical path modulation scanning assembly includes a two-dimensional scanning galvanometer, an adjustable relay optical path, and a microscope objective. The two-dimensional scanning galvanometer is used for scanning the light beam. The adjustable relay optical path includes a converging lens and a sleeve lens along the optical axis from the object side to the image side, which are used to adjust the working mode of the system. The microscope objective is used to focus the light beam onto the imaging target. During the adjustment process from the extreme high resolution mode to the extreme large field of view mode, the converging lens moves axially along the optical axis from the object side to the image side. The structure of the relay optical path modulation and scanning component conforms to the following relationship. :, where d GT D0 is the axial distance between the optical scanning center of the two-dimensional scanning galvanometer and the center of the principal surface of the sleeve lens, and D0 is the beam diameter at the optical scanning center of the two-dimensional scanning galvanometer. Obj f is the entrance pupil diameter of the microscope objective. TL The effective focal length of the sleeve lens; The structure of the relay optical path modulation and scanning component conforms to the following relationship: Where D0 is the beam diameter at the optical scanning center of the two-dimensional scanning galvanometer, and D... Objf is the entrance pupil diameter of the microscope objective. L The effective focal length of the converging lens; The structure of the relay optical path modulation and scanning component conforms to the following relationship. :, where f L d is the effective focal length of the converging lens. LT The axial distance between the center of the main surface of the converging lens and the center of the main surface of the sleeve lens; The reflective imaging port assembly includes a photoacoustic signal coaxial coupling device and a two-dimensional displacement component; the photoacoustic signal coaxial coupling device has optical transmission and acoustic reflection characteristics, and is used to realize the coaxial coupling of the excitation beam and the photoacoustic signal, and to transmit the photoacoustic signal generated by the imaging target to the detection component; the two-dimensional displacement component is used to move the sample to be imaged; The detection assembly includes a line-focusing ultrasonic transducer, a rotary motor, a transmission gear set, and a motor controller. The rotary motor drives the line-focusing ultrasonic transducer to rotate, which is used to keep the optical focal point and the acoustic focal line always coincided when the excitation beam rotates and scans, so as to realize the three-dimensional detection of the photoacoustic signal of the sample. The signal processing component is used to perform electrical processing and data acquisition on the signals captured by the detection component, and the acquired data is transmitted to the computer for further processing. The computer is used to implement scanning control of the galvanometer in the relay optical path modulation scanning assembly, displacement control of the converging lens, synchronous control of the rotating motor in the detector assembly, and also for photoacoustic signal reconstruction and image processing.

[0008] Preferably, the relay optical path modulation scanning component operates as follows: a multi-wavelength parallel beam is scanned by a two-dimensional scanning galvanometer, modulated by an adjustable relay optical path, and then focused onto the sample by a microscope objective. The adjustable relay optical path includes a converging lens and a sleeve lens, used to conjugate the optical scanning center of the galvanometer to a limited range after the front focal point of the microscope objective, while adjusting the collimated laser beam at the galvanometer to a diverging beam at the entrance pupil of the microscope objective. The converging lens is fixed on an axial displacement stage, which is used to move the converging lens along the optical axis from one to two focal lengths in front of the sleeve lens. This is used to adjust various characteristics of the beam in front of the microscope objective, including the relative position of the conjugate point of the optical scanning center and the focal point in front of the microscope objective, the beam divergence angle and radius at the entrance pupil of the microscope objective, and so on. This allows for stepless adjustment of the optically excited field of view, effective numerical aperture, actual working distance, and stepless adjustment of the field of view and lateral resolution. While significantly expanding the field of view, the resolution is kept at the same level, thus achieving multi-scale high-resolution imaging.

[0009] Preferably, the relay optical path modulation scanning component can switch between operating modes—extreme large field of view mode, extreme high resolution mode, and any mode in between—by steplessly adjusting the position of the converging lens. In the extreme large field of view mode, the distance between the converging lens and the sleeve lens is minimized, the distance between the conjugate point of the optical scanning center and the front focal point of the microscope objective is minimized but still located behind the front focal point, the beam divergence angle and beam radius at the entrance pupil of the microscope objective are maximized, thereby resulting in the maximum actual working distance of optical excitation, the maximum field of view, the minimum effective numerical aperture, and the maximum optical focal scale, ultimately achieving extreme large field of view imaging. In extreme high resolution mode, the distance between the converging lens and the sleeve lens is maximized, the distance between the conjugate point of the optical scanning center and the front focal point of the microscope objective is maximized and located after the entrance pupil of the microscope objective, the beam divergence angle and the beam radius are minimized at the entrance pupil of the microscope objective, thereby resulting in the minimum actual working distance of optical excitation, the minimum field of view, the maximum effective numerical aperture, and the minimum optical focal size, ultimately achieving extreme high resolution imaging.

[0010] Preferably, the laser source assembly includes a function generator and multiple wavelength fast nanosecond pulse lasers electrically connected to each other. The function generator provides the external trigger timing of the laser pulses and synchronizes the acquisition timing of the system. The output beams of the multiple wavelength fast nanosecond pulse lasers are incident on the spot optimization assembly along the optical axis, and focused onto the imaging target by the relay optical path modulation scanning assembly and the reflective imaging port assembly to excite its photoacoustic signal. The beam optimization component, along the optical axis from the object side to the image side, includes an aperture and a spatial light filter, used for shaping and expanding the pulsed laser beam, conforming to the following relationship: Where D0 is the beam diameter at the optical scanning center of the two-dimensional scanning galvanometer, M is the beam radius magnification of the spatial light filter, and D Iris The diameter of the beam emitted from the aperture; The signal processing component includes an electrical amplifier, a bandpass filter, and a data acquisition card. The electrical signal output by the detection component is amplified by the electrical amplifier, filtered by the bandpass filter, and then converted from analog to digital by the data acquisition card before being transmitted to the computer for further processing.

[0011] Preferably, the laser source component is used to emit pulsed laser beams of different wavelengths; the beam spot optimization component processes each wavelength beam separately, specifically, the aperture is used to adjust the energy of each wavelength beam, the spatial light filter is used to remove high-frequency noise from the light source and expand the beam, ensuring that the beam fill rate of the microscope objective entrance pupil is close to 1 when the relay optical path modulation scanning component is in extreme high resolution mode; the beam spot optimization component also includes a dichroic mirror and a reflecting mirror for merging beams of different wavelengths.

[0012] Preferably, in the relay optical path modulation scanning assembly, the two-dimensional scanning galvanometer uses one two-dimensional scanning galvanometer or two one-dimensional scanning galvanometers; when using one two-dimensional scanning galvanometer, the optical scanning center is the mechanical rotation center of the galvanometer; when using two one-dimensional scanning galvanometers, the galvanometers are orthogonally combined, and the optical scanning center is the geometric center of the line connecting the mechanical rotation centers of the two one-dimensional scanning galvanometers. If a telescope system is further added between the two one-dimensional scanning galvanometers to achieve pupil matching, the optical scanning center is the mechanical rotation center of the second scanning galvanometer.

[0013] Preferably, in the reflective imaging port assembly, the photo-acoustic signal coaxial coupling device is used to excite the transmission of the light beam and the reflection of the photo-acoustic signal. Its structure includes a bottom quartz glass sealing sheet, internal deionized water and tilted embedded quartz glass acoustic reflector, upper polyethylene sealing film and surface coated with ultrasonic coupling gel; the two-dimensional displacement component is used to move the sample to be imaged and keep the region of interest at the center of the imaging field of view.

[0014] Preferably, in the detection assembly, the focal line center point of the line-focusing ultrasonic transducer coincides with the center point of the optical excitation focusing scanning field of view. The detection assembly operates as follows: the optical focal line scans the acoustic focal line of the line-focusing transducer, and the ultrasonic transducer detects the depth signal of a single diameter of the optical excitation field of view. After the single diameter line detection is completed, the optical focal line scans around the center point of the optical excitation field of view, and the acoustic focal line moves in sync with it until the diameter line detection of the entire field of view is completed. At this point, the line-focusing ultrasonic transducer rotates 180° around its central axis to acquire the depth signal of the entire optical excitation field of view, thus achieving three-dimensional detection.

[0015] Preferably, in the signal processing component, the signal is first preprocessed by the electrical amplifier and bandpass filter to improve the signal-to-noise ratio, and then synchronously acquired by the signal acquisition card; the computer is electrically connected to the two-dimensional scanning galvanometer controller, the motor controller, and the signal acquisition card for synchronous control of the entire system.

[0016] This invention also discloses a multi-scale photoacoustic microscopy imaging method based on a continuously adjustable relay optical path, which uses the aforementioned multi-scale photoacoustic microscope based on a continuously adjustable relay optical path for imaging, and the steps are as follows: Step S1: Preprocess the imaging area of ​​the biological sample and apply ultrasonic coupling gel, fix the sample on the sample holder of the two-dimensional displacement component, and attach the imaging area tightly to the polyethylene film. Step S2 involves single-shot imaging in the extreme large field-of-view mode. Specifically, the adjustable relay optical path is configured in the extreme large field-of-view mode, i.e., the converging lens is moved to the corresponding position in the extreme large field-of-view mode. For any wavelength, the laser is triggered by the function generator to emit pulsed laser light. The beam passes sequentially through the aperture, the spatial light filter, and then through the dichroic mirror to be combined with other wavelength lasers. It is then scanned by the two-dimensional scanning galvanometer, relayed by the relay optical path, and finally focused by the microscope objective to excite the photoacoustic signal of the sample. The acoustic signal is reflected by the quartz glass plate and converted into an electrical signal by the line-focusing ultrasonic transducer. The electrical signal is pre-processed by the electrical amplifier and filter, acquired by the signal acquisition card, and stored and processed by the computer. When the optical focal point completes the photoacoustic signal excitation of one optical focal plane, the acoustic focal line rotates synchronously by 180° to complete the acquisition of three-dimensional information of the sample within the three-dimensional focal area. Step S3: Quantitatively move the sample using a two-dimensional displacement component, repeat step S2 until signal acquisition of the entire large-scale imaging area is completed; reconstruct and register the image using a computer to obtain a large-scale three-dimensional image of the sample. Step S4: Determine the center and range of the region of interest (ROI) in the image, and determine the required field of view for imaging the ROI based on the sampling interval. Configure the adjustable relay optical path to either the extreme high-resolution mode or any mode between the two extreme modes to perform a single imaging of the ROI. Specifically: Based on the scale requirements of the ROI for imaging field of view and resolution, control the converging lens to move to the corresponding position; quantitatively move the sample using a two-dimensional displacement component to move the center of the ROI to the center of the current mode's imaging field of view; complete the excitation, detection, processing, and acquisition of the sample's photoacoustic signal. Step S5: For all regions of interest with fine structure scattered across a large area of ​​3D images, repeat step S4 until the 3D images of the fine structure of each region are acquired. Step S6: Use a computer to reconstruct and filter the multi-scale three-dimensional image of the sample, optimize the imaging effect, register and replace the corresponding regions of the large-scale three-dimensional image with the fine structure three-dimensional image of each region, and obtain a large-scale multi-scale three-dimensional image of the sample that contains the fine structure of the region of interest.

[0017] (III) Beneficial Effects Compared with the prior art, the present invention has significant positive technical effects, and its beneficial effects are reflected in at least the following aspects.

[0018] (1) By designing an adjustable relay optical path for stepless adjustment of the scanning field of view and focal scale of the excitation light, the multi-scale photoacoustic microscopy imaging device based on a stepless adjustable relay optical path proposed in this invention can perform multi-scale imaging of samples while maintaining the same resolution level while significantly expanding the field of view. Compared with traditional photoacoustic microscopy imaging, the multi-scale photoacoustic microscope proposed in this invention can use the same microscope objective without changing the system components, achieving stepless adjustment of resolution and single imaging field of view while maintaining high optical resolution.

[0019] (2) Once an existing optical resolution photoacoustic microscopy system is built, its lateral resolution cannot be infinitely adjusted, and its optical excitation field of view can only be further expanded based on mechanical scanning. In order to ensure that the sampling interval matches the lateral resolution, the scanning time for a single imaging of a large field of view is proportional to the field of view area. With the multi-scale photoacoustic microscopy device based on an infinitely adjustable relay optical path proposed in this invention, when the adjustable relay optical path is configured in the extreme large field of view mode, the optical focal point scale is also increased accordingly, thereby reducing the spatial sampling frequency and reducing the scanning time for a single large field of view imaging.

[0020] (3) Existing optical resolution photoacoustic microscopy systems use fixed-scale optical focal points for scanning. Too small a focal point can lead to excessively high light energy density, increasing the risk of tissue thermal damage. The multi-scale photoacoustic microscopy device based on a continuously adjustable relay optical path proposed in this invention increases the focal point scale with the optical scanning field of view as the converging lens moves towards the image side to approach the sleeve lens. This allows for larger focal point scales in applications requiring a larger imaging field of view. On the one hand, while meeting the requirements of matching the sampling interval with the lateral resolution, the increased focal point scale can accommodate the increased sampling interval, reducing the number of focal points on the imaging plane containing the biological tissue. On the other hand, the biological sample volume excited by a single focal point is larger, resulting in lower light energy density. Therefore, the multi-scale photoacoustic microscopy device proposed in this invention can effectively reduce the laser energy density irradiated into biological tissue in extreme large field-of-view modes, thereby effectively avoiding the risk of tissue thermal damage.

[0021] (4) Applying the stepless adjustable relay optical path proposed in this invention to photoacoustic microscopy can ingeniously integrate the two major imaging features of large field of view and high resolution into the same system based on the axial movement of a single converging lens, effectively expanding the biological problems that can be observed by existing single rotating photoacoustic microscopy systems. Taking the microscope objective with a 4x lateral magnification and a numerical aperture of 0.1 used in this system as an example, when the adjustable relay optical path is configured in the extreme high resolution mode, the lateral resolution of the system can approach the optical diffraction limit of the objective lens to reach 5 micrometers, which meets the resolution requirements for capillary imaging; when the adjustable relay optical path is configured in the extreme large field of view mode, the optical excitation field of view of the system can exceed the nominal field of view of the objective lens to reach 8.5 mm, which meets the field of view requirements for mouse whole brain imaging. In addition, this system can switch between the extreme large field of view mode, the extreme high resolution mode, and any mode in between by only the axial movement of a single converging lens, which is easy to be automatically controlled by a computer and has the potential for miniaturization.

[0022] (5) The stepless adjustment relay optical path design proposed in this invention is applicable to microscope objectives with different magnifications, numerical apertures, and sizes, and is suitable for multi-scale imaging needs of samples of different scales. When applied to photoacoustic microscopy, the stepless adjustment relay optical path is suitable for different scanning modes and their corresponding imaging ports and detection components, and can meet the multi-scale imaging requirements of different scanning and detection modes. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of the system structure of the multi-scale photoacoustic microscope based on the stepless adjustment relay optical path of the present invention.

[0024] Figure 2 This is a schematic diagram of the relay optical path modulation and scanning mechanism of the present invention.

[0025] Figure 3 The images show (a) and (b) of mouse ear vascular network imaging results obtained by the multi-scale photoacoustic microscope based on the continuously adjustable relay optical path in the extreme large field-of-view mode and the local region of interest ear vascular network imaging results obtained in the extreme high resolution mode, respectively.

[0026] In the figure, the corresponding technical component names and their reference numerals are as follows: function generator 1-1, first wavelength fast nanosecond pulse laser 1-2, second wavelength fast nanosecond pulse laser 1-3, aperture 2-1, 2-2, first wavelength spatial light filter 2-3, second wavelength spatial light filter 2-4, dichroic mirror 2-5, second wavelength reflector 2-6, two-dimensional scanning galvanometer 3-1, converging lens in extreme high resolution mode 3-2, converging lens in extreme large field of view mode 3-2', sleeve lens 3-3, first wavelength and second wavelength reflectors 3-4, microscope objective 3-5, quartz glass plate 4-1, deionized water 4-2, two-dimensional displacement component 4-3, ultrasonic transducer 5-1, rotary motor 5-2, electrical amplifier 6-1, bandpass filter 6-2, data acquisition card 6-3, computer 7, field of view in extreme high resolution mode 8-1, field of view in extreme large field of view mode 8-2. Detailed Implementation

[0027] To address its technical problems, this invention provides a multi-scale photoacoustic microscope and its imaging method based on a continuously adjustable relay optical path. The technical solution of this invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0028] like Figure 1 As shown, a multi-scale photoacoustic microscope based on a continuously adjustable relay optical path in this embodiment includes: a laser source assembly 1, a spot optimization assembly 2, a relay optical path modulation scanning assembly 3, a reflective imaging port assembly 4, a detection assembly 5, a signal processing assembly 6, and a computer 7. like Figure 1 As shown, the laser source assembly includes a function generator 1-1, a wavelength 1 pulsed laser 1-2, and a wavelength 2 pulsed laser 1-3, used to excite photoacoustic signals of multiple components of the sample. This system has multi-wavelength compatibility, allowing selection of multiple applicable optical center wavelengths based on the optical absorption spectra of each of the multiple components of interest in the sample.

[0029] The laser source component 1 can emit at least two parallel beams of different wavelengths. The parallel beams pass sequentially through the spot optimization component 2, the relay optical path modulation scanning component 3, and the reflective imaging port component 4 to reach the sample plane and excite photoacoustic signals. Based on the multi-wavelength absorption specificity, the photoacoustic signals carrying sample structure and functional information are transmitted to the detection component 5. The beam spot optimization component 2 is used to adjust the initial laser intensity, optimize the beam spot to a Gaussian shape, and expand the beam with reference to the laser emission beam spot size and the objective lens entrance pupil size. For two wavelength beams, apertures 2-1 and 2-2 adjust the light transmission energy respectively, and spatial light filters 2-3 and 2-4 filter out high-frequency noise in the beam spot respectively. The Gaussian beam spot formed after filtering is combined by dichroic mirror 2-5 and second wavelength reflector 2-6, and is incident at a 45° tilt angle on the center of the first-dimensional scanning reflector of the two-dimensional scanning galvanometer 3-1, and enters the relay optical path modulation scanning component 3. The relay optical path modulation scanning assembly 3 includes a two-dimensional scanning galvanometer 3-1, a converging lens 3-2, a sleeve lens 3-3, a full-wavelength reflecting mirror 3-4, and a microscope objective 3-5. The selection principles and positional relationships of the components are as follows: the microscope objective 3-5 with a suitable optical diffraction limit is selected based on the structural dimensions of the sample to be measured; to ensure the utilization efficiency of the numerical aperture of the microscope objective 3-5, the entrance pupil diameter D of the microscope objective 3-5 is considered. Obj The optical scanning center spot diameter D0 of the two-dimensional scanning galvanometer 3-1 and the focal length f of the sleeve lens 3-3. TL The axial distance between the optical scanning center of the two-dimensional scanning galvanometer 3-1 and the center of the principal surface of the sleeve lens 3-3 is determined to be approximately... In this example system, the optical scanning center spot diameter of the two-dimensional scanning galvanometer 3-1 is 2.2 mm, the entrance pupil diameter of the microscope objective 3-5 is 9 mm, the focal length of the sleeve lens 3-3 is 200 mm, and the axial distance between the optical scanning center of the two-dimensional scanning galvanometer 3-1 and the center of the principal surface of the sleeve lens 3-3 is set to 280 mm. The axially movable converging lens 3-2, together with the sleeve lens 3-3, constitutes an adjustable relay system used to conjugate the optical scanning center to a certain range after the entrance pupil of the objective lens. The focal length f of the converging lens 3-2 is... L Should meet In this example system, the focal length of the converging lens 3-2 is set to 75 mm. The converging lens 3-2 moves within a certain distance in front of the sleeve lens 3-3. The distance between the two should be greater than one focal length of the converging lens 3-2 and less than two focal lengths. Due to the limitations of the entrance pupil diameter of the microscope objective 3-5 and the aperture size of the converging lens 3-2, the movement range of the converging lens 3-2 is further limited. In this example system, the converging lens 3-2 moves between 110 mm and 140 mm in front of the sleeve lens 3-3. The axial movement of the converging lens 3-2 can simultaneously adjust the beam diameter, beam divergence angle at the entrance pupil of the microscope objective 3-5, and the maximum deflection angle of the focused beam at its exit pupil, thereby achieving the adjustment of the system's field of view and resolution, and ensuring that the resolution remains at the same level while increasing the field of view. In the adjustable relay optical path, the distance between the converging lens 3-2 and the sleeve lens 3-3 corresponds to the imaging field of view and resolution of the system. Therefore, when the converging lens 3-2 is moved along the optical axis, the system is configured in the extreme large field of view mode, the extreme high resolution mode, and any mode in between, thus achieving stepless adjustment of the imaging field of view and resolution. Figure 2 As shown in (a), in the extreme high-resolution mode, the spacing between the converging lens 3-2 and the sleeve lens 3-3 is adjusted to the maximum. In this example system, the spacing is set to 140 mm. The distance between the conjugate point of the optical scanning center and the front focal point of the microscope objective is the maximum and it is located after the entrance pupil of the microscope objective. The beam divergence angle and beam radius at the entrance pupil of the microscope objective are the smallest, resulting in the smallest actual working distance of optical excitation, the smallest field of view, the largest effective numerical aperture, and the smallest optical focal size, ultimately achieving extreme high-resolution imaging. In this example system, the field of view 8-1 of the extreme high-resolution mode is 2.3 mm, and the lateral resolution is 5.09 μm. The imaging effect on the vascular network of the mouse ear is as follows. Figure 3 As shown in (b); Figure 2 As shown in (b), in the extreme large field-of-view mode, the spacing between the converging lens 3-2 and the sleeve lens 3-3 is adjusted to the minimum. In this example system, the spacing is set to 110 mm. The distance between the conjugate point of the optical scanning center and the front focal point of the microscope objective is the smallest, but it is still located behind the front focal point. The beam divergence angle and beam radius at the entrance pupil of the microscope objective are the largest, resulting in the largest actual working distance of optical excitation, the largest field of view, the smallest effective numerical aperture, and the largest optical focal size, ultimately achieving extreme large field-of-view imaging. In this example system, the field of view 8-2 of the extreme large field-of-view mode is 8.5 mm, and the lateral resolution is 9.24 micrometers. The imaging effect on the vascular network of the mouse ear is as follows: Figure 3 As shown in (a); The quartz glass plate 4-1 and deionized water 4-2 contained in the reflective imaging port assembly 4 have similar optical refractive indices but significantly different acoustic impedances, exhibiting optical transparency and acoustic reflection characteristics. This is used to achieve the coincidence of the scanning centers for optical excitation and acoustic reception. The deionized water 4-2 is used to achieve acoustic coupling from the sample to the detector assembly, ensuring the sensitivity of the detector assembly and a high signal-to-noise ratio of the received photoacoustic signal. The two-dimensional displacement assembly 4-3 has a light-transmitting aperture and is fixed to the sample, allowing the sample to move laterally in two dimensions along a direction perpendicular to the optical axis, adjusting the position of the region of interest. The ultrasonic transducer 5-1 included in the detection component 5 is used to convert the detected photoacoustic signal into an electrical signal. The rotary motor 5-2 drives the ultrasonic transducer 5-1 to rotate through a gear set, ensuring that the acoustic focal line and the optical focal point always coincide during the optical scanning process, thus ensuring a good signal-to-noise ratio throughout the scanning process. This patent is independent of the selected detection component and its scanning method. The ultrasonic transducers in the adapted detection components include, but are not limited to, point-focusing ultrasonic transducers, line-focusing ultrasonic transducers, and flat-field ultrasonic transducers. The adapted scanning methods include, but are not limited to, grating scanning, rotational scanning with a field diameter or radius, and spiral scanning. The structures of the reflective imaging port component 4 and the detection component 5 are mutually compatible, and their structural design can be adjusted according to the selected ultrasonic transducer type and scanning method. The signal processing component 6 is used to connect to the computer component 7 and to process, display and store the photoacoustic image of the sample. The selection of the electrical amplifier 6-1 and bandpass filter 6-2 included therein is based on the signal-to-noise ratio of the photoacoustic signal, the spectral characteristics and sensitivity of the ultrasonic transducer, etc. The specific embodiments described in this application are merely illustrative examples of the main ideas of the present invention. Those skilled in the art to which this invention pertains may make various modifications or additions to the described specific embodiments or use similar methods to substitute them, without departing from the spirit of the invention or exceeding the scope defined by the appended claims.

Claims

1. A multi-scale photoacoustic microscope based on a continuously adjustable relay optical path, characterized in that, include: Laser source assembly, beam optimization assembly, relay optical path modulation and scanning assembly, reflective imaging port assembly, detection assembly, signal processing assembly, and computer; The laser source component emits at least two parallel pulse beams of different wavelengths along the optical axis. The beams pass sequentially through the spot optimization component, the relay optical path modulation scanning component, and the reflective imaging port component to illuminate the imaging target, thereby exciting the photoacoustic signal of the imaging target and triggering the signal synchronously. The beam optimization component is used for beam shaping and expansion; The relay optical path modulation scanning assembly includes a two-dimensional scanning galvanometer, an adjustable relay optical path, and a microscope objective. The two-dimensional scanning galvanometer is used for scanning the light beam. The adjustable relay optical path includes a converging lens and a sleeve lens along the optical axis from the object side to the image side, which are used to adjust the working mode of the system. The microscope objective is used to focus the light beam onto the imaging target. During the adjustment process from the extreme high resolution mode to the extreme large field of view mode, the converging lens moves axially along the optical axis from the object side to the image side. The structure of the relay optical path modulation and scanning component conforms to the following relationship: ,in The axial distance between the optical scanning center of the two-dimensional scanning galvanometer and the center of the main surface of the sleeve lens is denoted as . The diameter of the beam at the optical scanning center of the two-dimensional scanning galvanometer is given. The entrance pupil diameter of the microscope objective is [missing information]. The effective focal length of the sleeve lens; The structure of the relay optical path modulation and scanning component conforms to the following relationship: ,in The diameter of the beam at the optical scanning center of the two-dimensional scanning galvanometer is given. The entrance pupil diameter of the microscope objective is [missing information]. The effective focal length of the converging lens; The structure of the relay optical path modulation and scanning component conforms to the following relationship: ,in The effective focal length of the converging lens. The axial distance between the center of the main surface of the converging lens and the center of the main surface of the sleeve lens; The reflective imaging port assembly includes a photoacoustic signal coaxial coupling device and a two-dimensional displacement component; the photoacoustic signal coaxial coupling device has optical transmission and acoustic reflection characteristics, and is used to realize the coaxial coupling of the excitation beam and the photoacoustic signal, and to transmit the photoacoustic signal generated by the imaging target to the detection component; the two-dimensional displacement component is used to move the sample to be imaged; The detection assembly includes a line-focusing ultrasonic transducer, a rotary motor, a transmission gear set, and a motor controller. The rotary motor drives the line-focusing ultrasonic transducer to rotate, which is used to keep the optical focal point and the acoustic focal line always coincided when the excitation beam rotates and scans, so as to realize the three-dimensional detection of the photoacoustic signal of the sample. The signal processing component is used to perform electrical processing and data acquisition on the signals captured by the detection component, and the acquired data is transmitted to the computer for further processing. The computer is used to implement scanning control of the galvanometer and displacement control of the converging lens in the relay optical path modulation scanning assembly, synchronous control of the rotating motor in the detector assembly, and also for photoacoustic signal reconstruction and image processing.

2. The multi-scale photoacoustic microscope based on a continuously adjustable relay optical path according to claim 1, characterized in that: The relay optical path modulation scanning component works by scanning a multi-wavelength parallel beam with a two-dimensional scanning galvanometer, modulating it with an adjustable relay optical path, and then focusing it onto the sample with a microscope objective. The adjustable relay optical path includes a converging lens and a sleeve lens, used to conjugate the optical scanning center of the galvanometer to a limited range after the front focal point of the microscope objective, and to adjust the collimated laser beam at the galvanometer to a diverging beam at the entrance pupil of the microscope objective. The converging lens is fixed on an axial displacement stage, which is used to move the converging lens along the optical axis from one to two focal lengths in front of the sleeve lens. This is used to adjust various characteristics of the beam in front of the microscope objective, including the relative position of the conjugate point of the optical scanning center and the focal point in front of the microscope objective, the beam divergence angle and radius at the entrance pupil of the microscope objective, and so on. This allows for stepless adjustment of the optically excited field of view, effective numerical aperture, actual working distance, and stepless adjustment of the field of view and lateral resolution. While significantly expanding the field of view, the resolution is kept at the same level, thus achieving multi-scale high-resolution imaging.

3. The multi-scale photoacoustic microscope based on a continuously adjustable relay optical path according to claim 2, characterized in that: The relay optical path modulation and scanning component can switch working modes between the extreme large field of view mode, the extreme high resolution mode, and any mode in between by steplessly adjusting the position of the converging lens. In the extreme large field of view mode, the distance between the converging lens and the sleeve lens is minimized, the distance between the conjugate point of the optical scanning center and the front focal point of the microscope objective is minimized but still located behind the front focal point, the beam divergence angle and beam radius at the entrance pupil of the microscope objective are maximized, thereby resulting in the maximum actual working distance of optical excitation, the maximum field of view, the minimum effective numerical aperture, and the maximum optical focal scale, ultimately achieving extreme large field of view imaging. In extreme high resolution mode, the distance between the converging lens and the sleeve lens is maximized, the distance between the conjugate point of the optical scanning center and the front focal point of the microscope objective is maximized and located after the entrance pupil of the microscope objective, the beam divergence angle and the beam radius are minimized at the entrance pupil of the microscope objective, thereby resulting in the minimum actual working distance of optical excitation, the minimum field of view, the maximum effective numerical aperture, and the minimum optical focal size, ultimately achieving extreme high resolution imaging.

4. The multi-scale photoacoustic microscope based on a continuously adjustable relay optical path according to claim 1, characterized in that: The laser source assembly includes a function generator and multiple wavelength fast nanosecond pulse lasers electrically connected to each other. The function generator provides the external trigger timing of the laser pulses and synchronizes the acquisition timing of the system. The output beams of the multiple wavelength fast nanosecond pulse lasers are incident on the spot optimization assembly along the optical axis, and are focused onto the imaging target by the relay optical path modulation scanning assembly and the reflective imaging port assembly to excite its photoacoustic signal. The beam optimization component, along the optical axis from the object side to the image side, includes an aperture and a spatial light filter, used for shaping and expanding the pulsed laser beam, conforming to the following relationship: ,in The diameter of the beam at the optical scanning center of the two-dimensional scanning galvanometer is given. The beam radius magnification of the spatial optical filter is given. The diameter of the beam emitted from the aperture; The signal processing component includes an electrical amplifier, a bandpass filter, and a data acquisition card. The electrical signal output by the detection component is amplified by the electrical amplifier, filtered by the bandpass filter, and then converted from analog to digital by the data acquisition card before being transmitted to the computer for further processing.

5. The multi-scale photoacoustic microscope based on a continuously adjustable relay optical path according to claim 4, characterized in that: The laser source component is used to emit pulsed laser beams of different wavelengths; the beam spot optimization component processes each wavelength beam separately; the aperture is used to adjust the energy of each wavelength beam; the spatial light filter is used to remove high-frequency noise from the light source and expand the beam, so that the beam fill rate of the microscope objective entrance pupil is close to 1 when the relay optical path modulation scanning component is in extreme high resolution mode; the beam spot optimization component also includes a dichroic mirror and a reflecting mirror for combining beams of different wavelengths.

6. The multi-scale photoacoustic microscope based on a continuously adjustable relay optical path according to claim 1, characterized in that: In the relay optical path modulation and scanning assembly, the two-dimensional scanning mirror is one two-dimensional scanning mirror or two one-dimensional scanning mirrors.

7. The multi-scale photoacoustic microscope based on a continuously adjustable relay optical path according to claim 1, characterized in that: In the reflective imaging port assembly, the photo-acoustic signal coaxial coupling device is used to excite the transmission of the light beam and the reflection of the photo-acoustic signal. Its structure includes a bottom quartz glass sealing sheet, internal deionized water and tilted embedded quartz glass acoustic reflector, upper polyethylene sealing film and surface coated with ultrasonic coupling gel; the two-dimensional displacement component is used to move the sample to be imaged and keep the region of interest at the center of the imaging field of view.

8. The multi-scale photoacoustic microscope based on a continuously adjustable relay optical path according to claim 1, characterized in that: In the detection assembly, the focal line center point of the line-focusing ultrasonic transducer coincides with the center point of the optical excitation focusing scanning field of view. The detection assembly operates as follows: the optical focal line scans the acoustic focal line of the line-focusing transducer, and the ultrasonic transducer detects the depth signal of a single diameter of the optical excitation field of view. After the single diameter line detection is completed, the optical focal line scans around the center point of the optical excitation field of view, and the acoustic focal line moves in sync with it until the diameter line detection of the entire field of view is completed. At this point, the line-focusing ultrasonic transducer rotates 180° around its central axis to acquire the depth signal of the entire optical excitation field of view, thus achieving three-dimensional detection.

9. The multi-scale photoacoustic microscope based on a continuously adjustable relay optical path according to claim 4, characterized in that: In the signal processing component, the signal is first preprocessed by the electrical amplifier and bandpass filter to improve the signal-to-noise ratio, and then synchronously acquired by the data acquisition card; the computer is electrically connected to the two-dimensional scanning galvanometer controller, motor controller, and data acquisition card for synchronous control of the entire system.

10. A multi-scale photoacoustic microscopy imaging method based on a continuously adjustable relay optical path, characterized in that, Imaging is performed using the multi-scale photoacoustic microscope based on a continuously adjustable relay optical path as described in any one of claims 1-9, with the following steps: Step S1: Preprocess the imaging area of ​​the biological sample and apply ultrasonic coupling gel, fix the sample on the sample holder of the two-dimensional displacement component, and attach the imaging area tightly to the polyethylene film. Step S2 involves single-shot imaging in the extreme large field-of-view mode. Specifically, the adjustable relay optical path is configured in the extreme large field-of-view mode, i.e., the converging lens is moved to the corresponding position in the extreme large field-of-view mode. For any wavelength, the laser is triggered by the function generator to emit pulsed laser light. The beam passes sequentially through the aperture, the spatial light filter, and then through the dichroic mirror to be combined with other wavelength lasers. It is then scanned by the two-dimensional scanning galvanometer, relayed by the relay optical path, and finally focused by the microscope objective to excite the photoacoustic signal of the sample. The acoustic signal is reflected by the quartz glass plate and converted into an electrical signal by the line-focusing ultrasonic transducer. The electrical signal is pre-processed by the electrical amplifier and filter, acquired by the data acquisition card, and stored and processed by the computer. When the optical focal point completes the photoacoustic signal excitation of one optical focal plane, the acoustic focal line rotates synchronously by 180° to complete the acquisition of three-dimensional information of the sample within the three-dimensional focal area. Step S3: Quantitatively move the sample using a two-dimensional displacement component, repeat step S2 until signal acquisition of the entire large-scale imaging area is completed; reconstruct, register and stitch using a computer to obtain a large-scale three-dimensional image of the sample. Step S4: Determine the center and range of the region of interest (ROI) in the image, and determine the field of view required for imaging the ROI by combining the sampling interval. Based on this, configure the adjustable relay optical path to the extreme high-resolution mode or any mode between the two extreme modes, and perform a single imaging of the ROI with fine structure. Specifically, according to the scale requirements of the ROI on the imaging field of view and resolution, control the converging lens to move to the corresponding position; use a two-dimensional displacement component to quantitatively move the sample, and move the center of the ROI to the center of the imaging field of view of the current mode. Complete the excitation, detection, processing, and acquisition of photoacoustic signals from the sample; Step S5: For all regions of interest with fine structure scattered across a large area of ​​3D images, repeat step S4 until the 3D images of the fine structure of each region are acquired. Step S6: Use a computer to reconstruct and filter the multi-scale three-dimensional image of the sample, optimize the imaging effect, register and replace the corresponding regions of the large-scale three-dimensional image with the fine structure three-dimensional image of each region, and obtain a large-scale multi-scale three-dimensional image of the sample that contains the fine structure of the region of interest.

Images