High-throughput multi-row parallel high-speed scanning high-resolution microscopic imaging device and method based on adaptive optics

Abstract

The invention discloses a high-throughput multi-row parallel high-speed scanning high-resolution microscopic imaging device and method based on adaptive optics. A spatial light modulator is used for carrying out pixel-level regulation and control on a light beam phase to generate a strip-shaped light spot, and a special arrangement mode of a detector array is used for realizing a one-to-many relationship between a scanning line and an imaging line and realizing improvement of an imaging speed, a signal-to-noise ratio of an imaging result and a resolution ratio; a full-digital area array wavefront regulation and control method is utilized, a spatial light modulator is used for rapidly and dynamically compensating a laser wave surface according to imaging result feedback, the laser wave surface is free of distortion, and the influence of aberration on an imaging result is reduced; designing a double telecentric Fourier transform structure capable of simultaneously keeping rotational symmetry of object plane and image plane light beams, and realizing distortionless mapping of scanning light beam quality and a linear signal system; a switchable design of two scanning modes is developed, high-flux, high-resolution and high-speed light field scanning is realized, and the advantages of large visual field, high resolution and high frame rate imaging are achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of high-speed scanning microscopic imaging, and in particular to a high-throughput multi-row parallel high-speed scanning high-resolution microscopic imaging device and method based on adaptive optics. Background Art

[0002] In recent years, the fields of biomedicine, materials science and semiconductors have developed rapidly, and the demand for observation and research of microstructures has increased. These fields have put forward higher requirements on the resolution, field of view and imaging speed of imaging technology. Optical imaging methods have become an ideal choice for clinical applications and scientific research due to their advantages of non-contact, non-destructive and real-time observation.

[0003] Confocal laser scanning microscopy (CLSM) is an advanced optical imaging technique that can provide high-resolution and high-contrast images. It reconstructs three-dimensional images by excluding light outside the focal plane, thereby significantly improving the clarity and details of the image, and is widely used in the fields of medicine and scientific research. However, the scanning field of conventional confocal microscopes is small, and due to the one-to-one correspondence between the scanning time sequence position and the pixel position, as well as the limitation of the scanning time of the scanning galvanometer, it is difficult to achieve high-throughput and high-speed scanning. There is a bottleneck in the principle that the imaging field of view, resolution, and imaging speed are mutually restricted. In addition, the interaction between the light beam and the medium during propagation often leads to non-ideal phase changes, especially in scanning imaging with a large field of view, which in turn affects the quality of the imaging results. Summary of the invention

[0004] The purpose of the present invention is to provide a high-throughput multi-row parallel high-speed scanning high-resolution microscopic imaging device and method based on adaptive optics to address the deficiencies of the prior art. In view of the problem that high-speed and high-throughput scanning reduces the signal-to-noise ratio and resolution of microscopic imaging, a high-resolution adaptive parallel detection confocal microscopic imaging method is studied to break through the principle bottleneck of the mutual restriction of imaging field of view, resolution, and imaging speed, and achieve the balance of high resolution, high signal-to-noise ratio, and high-speed and high-throughput scanning microscopic imaging; in view of the problem that large-field scanning aberration reduces imaging resolution and signal-to-noise ratio, key technologies such as dual telecentric Fourier distortion-free scanning and fast and slow mirror overlapping scanning are carried out to achieve large-field, high-resolution, and high-speed light field scanning. This will provide demand guarantees in the fields of medical care, materials, semiconductors, etc., and promote the rapid development of related fields.

[0005] The objective of the present invention is achieved through the following technical solutions:

[0006] According to a first aspect of the present specification, there is provided a high-throughput multi-row parallel high-speed scanning high-resolution microscopic imaging device based on adaptive optics, the device comprising a laser control module, a focal spot modulation module, a high-speed scanning module and a parallel detection module;

[0007] The laser control module is used to emit illumination light to illuminate the sample, and detect it through the parallel detection module;

[0008] The focal spot modulation module uses a spatial light modulator to perform phase control on the scanning beam, modulates the beam into a long strip focal spot to cover the detector array, and uses the spatial light modulator to perform surface array wavefront control to control the scanning beam aberration;

[0009] The high-speed scanning module is used to realize focal spot beam scanning, and is composed of a switchable orthogonal galvanometer galvanometer system and a resonance and galvanometer galvanometer system. Each galvanometer system includes an X-direction galvanometer, which is denoted as a fast mirror, and a Y-direction galvanometer, which is denoted as a slow mirror. A double telecentric 4f mirror group is placed between the fast and slow mirrors to ensure the conjugation of the fast and slow mirrors.

[0010] The parallel detection module uses a detector array arranged in a long strip shape to detect the long strip illumination area, so as to achieve a multi-line imaging effect in one scan.

[0011] Furthermore, the device also includes a signal acquisition and control module, which is used to output signals for controlling other modules, so as to achieve orderly execution of tasks of each module and synchronization between scanning and detection acquisition.

[0012] Furthermore, in the focal spot modulation module, a closed-loop feedback active optical wavefront control method is used to control the scanning beam aberration, specifically:

[0013] Selecting an aberration test function, generating a digital phase pattern that can estimate and correct beam aberration, loading the pattern on a spatial light modulator, capturing an image and calculating an image evaluation index to evaluate the effect of the generated phase pattern in compensating for aberrations;

[0014] The phase size that needs to be corrected again is estimated based on the image evaluation index, and a new round of correction is implemented. According to the selected aberration test function, a new phase pattern generated by the estimated phase size is loaded on the spatial light modulator, and a new round of image acquisition is performed, and the image evaluation index is calculated; the correction is repeated until the image evaluation index reaches a satisfactory aberration optimization effect.

[0015] Furthermore, when the focal spot modulation module performs aberration optimization, the beam wavefront mode is digitally expressed by the Zernike coefficients, a Zernike aberration test function is used, and a PSF sharpness index is used accordingly to evaluate the effect of the generated phase pattern compensating for aberrations.

[0016] Furthermore, in the high-speed scanning module, during the process of the X-direction galvanometer scanning a line of outbound and return strokes, the point voltage signal output by the Y-direction galvanometer does not change, so that the light spot stays in the Y direction; after completing a line of scanning, the Y-direction galvanometer changes the voltage value of the distance M times the pixel size to achieve scanning in the X direction and the Y direction in an M-fold ratio.

[0017] Furthermore, in the parallel detection module, the detector array is an avalanche photodiode APD fiber array, the position corresponding to a single pixel should be tightly surrounded by the surrounding fiber array, and adjacent fiber probes are arranged in a tangent manner.

[0018] Furthermore, in the parallel detection module, assuming that the number of rows scanned at one time is i, there are i pixels at the detection end, and the information of each pixel is obtained from the surrounding APDs.

[0019] Furthermore, the device also includes two polarization beam splitters, two reflectors and two double telecentric 4f mirror groups;

[0020] After the laser control module emits the detection light, it is modulated into light of a single polarization degree by a 1 / 2 wave plate, and after passing through the first polarization beam splitter, the polarization degree of the light can be modulated by the spatial light modulator;

[0021] The phase of the light beam is modulated by loading a phase pattern on the spatial light modulator, and the outgoing light beam is modulated into a long strip focal spot without aberration. The light beam is then expanded by the first double telecentric 4f lens group. The expanded light beam is modulated into linear polarized light by a 1 / 2 wave plate, and then passes through the second polarization beam splitter. The reflected light beam is used as the illumination light incident on the sample surface, and then passes through the high-speed scanning module for light beam scanning. The outgoing light beam is deflected by the first reflector and expanded by the second double telecentric 4f lens group, and then deflected by the second reflector. After passing through the 1 / 4 wave plate, it enters the objective lens and is incident on the sample surface.

[0022] The signal light reflected by the sample enters the detection optical path and is incident on the detector array of the parallel detection module. The computer generates an image of corresponding pixel size according to the time series based on the detected signal.

[0023] Furthermore, the first double telecentric 4f lens group, the second double telecentric 4f lens group and the two double telecentric 4f lens groups in the high-speed scanning module jointly achieve complete conjugation of the fast lens, the slow lens and the objective lens entrance pupil.

[0024] According to the second aspect of this specification, there is provided a high-throughput multi-row parallel high-speed scanning high-resolution microscopic imaging method based on the device as described in the first aspect, the method comprising:

[0025] The laser control module emits laser, and the high-speed scanning module starts scanning at the same time;

[0026] The laser emitted by the laser control module reaches the spatial light modulator of the focal spot modulation module, and through the closed-loop feedback active optical wavefront control method, the corresponding phase is loaded on the spatial light modulator, so that the light beam becomes an aberration-free light beam that can be focused into a long strip focal spot; then the light beams are combined, and after passing through the high-speed scanning module, they are focused onto the sample surface by the objective lens to illuminate the long strip focal area, and the reflected light carrying the detection signal is detected and collected by the parallel detection module.

[0027] Compared with the prior art, the present invention has the following advantages and innovations:

[0028] 1. The present invention realizes the multi-row correspondence between the galvanometer position and the image information by adjusting the shape of the light beam focal spot with a phase-type spatial light modulator and arranging the multi-row shapes of the detector. On the basis of improving the signal-to-noise ratio and resolution of the confocal microscope, the scanning imaging speed is greatly improved, thus making up for the deficiency of the limited scanning speed of the galvanometer.

[0029] 2. The present invention utilizes a fully digital array wavefront control method and uses a spatial light modulator to quickly and dynamically compensate the laser wavefront based on imaging result feedback, thereby achieving distortion-free laser wavefront and rapid dynamic compensation of aberrations, ensuring distortion-free laser scanning of the entire field of view, and making up for the loss of scanning information due to insufficient scanning beam quality.

[0030] 3. The design of the present invention can realize a double telecentric Fourier transform structure that maintains rotational symmetry between the object plane and the image plane beam, and achieves distortion-free mapping of the scanning beam quality and the linear signal system. It realizes complete conjugation of the fast mirror, slow mirror and the objective lens entrance pupil, avoiding problems such as uneven intensity distribution and distortion of the scanning field of view that exist in conventional confocal systems when scanning.

[0031] 4. The present invention develops a switchable design of two scanning modes to achieve high-throughput, high-resolution, and high-speed light field scanning, achieving the advantages of large field of view, high resolution, and high frame rate imaging. It solves the contradiction between the scanning speed, field of view, and resolution of conventional confocal scanning solutions. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0033] Figure 1 A schematic diagram of a scanning principle shown in an exemplary embodiment of the present invention;

[0034] Figure 2A phase pattern for realizing the elongation of the focal spot shown in an exemplary embodiment of the present invention;

[0035] Figure 3 A closed-loop feedback active optical wavefront control flow chart showing an exemplary embodiment of the present invention;

[0036] Figure 4 A schematic diagram of high-speed and high-throughput scanning imaging with a large field of view is shown as an exemplary embodiment of the present invention;

[0037] Figure 5 This is a diagram showing the arrangement of an APD fiber array according to an exemplary embodiment of the present invention;

[0038] Figure 6 This is a device optical path diagram showing an exemplary embodiment of the present invention. DETAILED DESCRIPTION

[0039] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. It should be noted that the features in the following embodiments and implementation methods can be combined with each other without conflict.

[0040] The present invention provides a high-throughput multi-row parallel high-speed scanning high-resolution microscopic imaging device and method based on adaptive optics. Aiming at the problem that high-speed and high-throughput scanning reduces the microscopic imaging signal-to-noise ratio and resolution, a high-resolution adaptive parallel detection confocal microscopic imaging method is studied to break through the principle bottleneck of the mutual restriction of imaging field of view, resolution and imaging speed, and achieve high resolution, high signal-to-noise ratio, high-speed and high-throughput scanning microscopic imaging. Aiming at the problem that large-field scanning aberration reduces imaging resolution and signal-to-noise ratio, key technologies such as dual-telecentric Fourier distortion-free scanning and fast and slow mirror overlapping scanning are carried out to achieve large-field, high-resolution, high-speed light field scanning.

[0041] The first innovation of the present invention is to achieve multi-line imaging through single illumination. At the illumination end, the present invention uses an adaptive optical device - spatial light modulator (SLM) to precisely control the phase of the scanning light beam, modulate the light beam into a long strip focal spot, and illuminate a long strip area on the sample surface. At the detection end, a detector array arranged in a long strip is used to detect the long strip illumination area, so as to achieve the imaging effect of obtaining multiple lines in one scan, that is, when the galvanometer scans one line in the X direction, it can achieve multiple line scanning in the Y direction, such as Figure 1 As shown, the number of lines required for scanning is reduced, greatly increasing the imaging speed.

[0042] The second innovation of the present invention is to utilize a fully digital array wavefront control method, using a spatial light modulator to quickly and dynamically compensate the laser wavefront based on imaging result feedback, thereby achieving distortion-free laser wavefront and rapid dynamic compensation of aberrations, ensuring distortion-free laser scanning of the entire field of view, and reducing the impact of scanning beam aberrations on scanning results.

[0043] The third innovation of the present invention is to design a double telecentric Fourier transform structure that can realize the rotational symmetry of the object plane and the image plane light beam at the same time, so as to realize the distortion-free mapping of the scanning beam quality and the linear signal system. Since the incident pupil of the double telecentric lens is located at infinity, the lens can accurately reflect the image of the real object, and the distortion can be strictly controlled, with almost zero distortion. The use of two sets of double telecentric lenses can realize the complete conjugation of the scanning fast mirror, the scanning slow mirror and the objective lens entrance pupil, and realize zero distortion imaging among the three. In a conventional confocal imaging system, the scanning fast mirror and the scanning slow mirror are placed close to each other. After the optical system between them and the objective lens is imaged, the conjugate plane of the objective lens entrance pupil is located in the middle position of the fast and slow mirrors. The complete conjugation of the fast mirror, the slow mirror and the objective lens entrance pupil is not realized, which will lead to problems such as uneven distribution of imaging field intensity and distortion during scanning imaging. The double telecentric Fourier distortion-free scanning method designed by the present invention realizes the complete conjugation of the fast mirror, the slow mirror and the objective lens entrance pupil, avoiding the problems such as uneven distribution of scanning field intensity and distortion when the conventional confocal system is scanned.

[0044] The fourth innovation of the present invention is to use an optical fiber array to detect optical signals, replacing the traditional confocal pinhole, to achieve the acquisition of high signal-to-noise ratio information.

[0045] In addition, the present invention has also developed a switchable design of two scanning modes to achieve high-throughput, high-resolution, and high-speed light field scanning, so that a set of imaging devices can have the advantages of large field of view, high resolution, and high frame rate imaging. The adaptive correction technology of inter-row misalignment in high-speed resonant galvanometer bidirectional scanning imaging is studied. In high-speed resonant galvanometer bidirectional scanning confocal imaging, there is a complex inter-row image misalignment phenomenon. The adaptive correction technology of inter-row image misalignment based on image features is studied to adaptively compensate for the image misalignment of different areas in high-speed resonant galvanometer bidirectional scanning confocal imaging.

[0046] The present invention provides a high-throughput multi-row parallel high-speed scanning high-resolution microscopic imaging device based on adaptive optics, comprising the following modules: a signal acquisition and control module, a laser control module, a focal spot modulation module, a high-speed scanning module and a parallel detection module. The five modules work in coordination to achieve high-speed parallel multi-row high-throughput microscopic imaging.

[0047] The signal acquisition and control module includes a control host and a signal acquisition card, which are used to output signals for controlling other modules, realize the orderly execution of tasks of each module and the synchronization between scanning and detection acquisition.

[0048] The laser control module includes a laser and an optical path thereof, and is responsible for emitting illumination light to illuminate the sample, and performing detection through the parallel detection module.

[0049] The focal spot modulation module is composed of a phase-type spatial light modulator and its affiliated optical path. The principle of the phase-type spatial light modulator is to phase-delay the light beam based on the liquid crystal molecules. The different voltages on each pixel of the phase-type spatial light modulator correspond to the different polarity directions of the liquid crystal of each pixel, and the light beam passing through each pixel is phase-delayed differently. Therefore, by modulating the voltage of each pixel on the phase-type spatial light modulator, an arbitrary phase pattern can be obtained, thereby realizing arbitrary phase modulation of the light beam. By loading the corresponding phase pattern onto the spatial light modulator, the phase of the laser beam can be modulated, thereby changing the spatial distribution of the focal spot. This module uses the Gerchberg-Saxton (GS) algorithm commonly used in SLM to calculate and generate the phase map required for the target image, and loads such as Figure 2 After the phase pattern shown, the focal spot is modulated from the Airy disk to a long strip focal spot to cover the detector used in the present invention. At the same time, the aberration generated on the scanning beam is actually the non-ideal change in the phase distribution of the beam during the propagation of the optical path. Therefore, the aberration of the scanning beam can be strictly controlled by using a spatial light modulator to perform surface array wavefront control. The closed-loop feedback active optical wavefront control method used is as follows Figure 3 As shown, the wavefront pattern of the light beam can be digitally expressed by the Zernike coefficients. After the illumination light passes through the optical system, it can ideally irradiate the sample in the same pattern as the illumination light. However, in reality, the wavefront of the light beam incident on the sample is different from that of the illumination light beam. By loading a suitable phase pattern on the pixelated phase modulation device, wavefront correction can be achieved. The specific closed-loop feedback process designed by the present invention is as follows: Figure 3As shown in the right figure, firstly, an aberration test function is selected, such as the Zernike aberration test function used in the present invention, to generate an estimated digital phase pattern that can correct the beam aberration, which is loaded on the phase-type spatial light modulator. After passing through the system medium, the beam wavefront with aberration will be initially compensated for the aberration with the corresponding phase loaded after passing through the spatial light modulator. Then, the image is collected, and the image evaluation index is calculated, such as the PSF sharpness index used in the present invention, to evaluate the effect of the generated phase pattern compensating for the aberration. It should be noted that the selected image evaluation index is closely related to the aberration test function. The former can reflect the size of the beam wavefront aberration, and the latter The system can evaluate the wavefront aberration optimization effect; when the correction is performed for the first time, the estimated phase pattern is loaded on the SLM, which will not have a very ideal effect on the aberration optimization, so the phase correction needs to be re-implemented; based on the evaluation of the compensation effect, the phase size that needs to be corrected again is estimated, and then a new round of correction is implemented. According to the selected aberration test function, the estimated phase size is loaded into a new phase pattern and loaded on the SLM, and then a new round of image acquisition is performed, and the image evaluation index is calculated to evaluate this round of aberration optimization effect, and then the phase size that needs to be further loaded is estimated, and the correction is repeated until the image evaluation index reaches a satisfactory aberration optimization effect.

[0050] The high-speed scanning module is used to scan the focal spot beam and is composed of a switchable orthogonal galvanometer galvanometer mirror and a resonance and galvanometer galvanometer mirror system. The structure of the scanning optical solution is as follows: Figure 4 As shown, by controlling the up, down, left and right movement of the switchable reflector 1 and the switchable reflector 2 according to the scanning requirements, the scanning light beam can be realized by passing through the orthogonal galvanometer galvanometer mirror or the resonant and galvanometer galvanometer mirror. The orthogonal galvanometer galvanometer mirror has a large scanning field and a low scanning rate, while the resonant and galvanometer galvanometer mirror has a small scanning field and a fast scanning rate. In this way, the advantages of the two scanning modes can be achieved on one system through the switchable reflector structure, that is, high-throughput, high-resolution, and high-speed light field scanning can be achieved, achieving the advantages of large field of view, high resolution, and high frame rate imaging. Among them, the resonant and galvanometer galvanometer mirror system can be used for fast preview of a small field of view, while the orthogonal galvanometer galvanometer mirror system can be used for overall imaging of a large field of view. Both scanning systems include a galvanometer mirror for scanning in the X direction and a galvanometer mirror for scanning in the Y direction, which are called a fast mirror and a slow mirror, respectively. A pair of telecentric 4f mirror groups are placed between the fast mirror and the slow mirror to ensure the conjugation of the fast and slow mirrors. The specific working process is as follows Figure 1 As shown in the figure, during the process of the X-direction galvanometer scanning a line of forward and return strokes, the point voltage signal output by the Y-direction galvanometer does not change, so that the light spot can stay still in the Y direction. After completing a line of scanning, the Y-direction galvanometer changes the voltage value equivalent to a distance five times the pixel size, which can achieve a five-fold scanning ratio between the X-direction and the Y-direction, saving nearly five times the scanning time.

[0051] The parallel detection module is composed of 16 avalanche photodiodes APD. The APD has a fast response speed and is suitable for high-speed optical signal detection. APD is a semiconductor device that accelerates photogenerated carriers in an electric field by applying a reverse voltage, producing an avalanche multiplication effect. When photons are incident on the APD, the generated electron-hole pairs are accelerated in the high electric field area and collide with other atoms to generate new electron-hole pairs. The new electron-hole pairs are also accelerated by the electric field, resulting in more electron-hole pairs, thereby forming an avalanche multiplication effect and achieving photocurrent amplification. The parallel detection module receives the returned light signal and converts it into an electrical signal, and transmits the signal back to the computer to detect the signal light. The arrangement of the 16 APD optical fibers and the pixel positions are as shown in the figure. Figure 5 As shown, the optical fiber transmits the optical signal through the APD to the computer, achieving a high signal-to-noise ratio effect similar to that obtained by a large pinhole composed of multiple detectors, while also having the high resolution effect obtained by the small pinhole of a single detector in the middle.

[0052] The APD fiber array has the following characteristics: In terms of arrangement, the position corresponding to a single pixel should be tightly surrounded by the surrounding fiber arrays, and adjacent fiber probes are arranged in a tangent manner. In terms of quantity, it is not limited to 16 APD fiber arrays. Theoretically, assuming that the number of rows scanned at a time is i, there are i pixels at the detection end. If the information of a pixel is Figure 5 As shown, the information is obtained from the four surrounding APDs, so the number of APD fiber arrays should be n=3i+1. Pixels can also be arranged in a hexagonal shape and the information is obtained from the middle. In this case, the number of APDs associated with a single pixel is 7, so the number of APD fiber arrays should be n=5i+2, and so on. In terms of resolution, in theory, the more APDs associated with a single pixel, the higher the signal-to-noise ratio can be, and thus the higher the resolution.

[0053] The operation process of the high-throughput multi-row parallel high-speed scanning high-resolution microscopic imaging device based on adaptive optics provided by the present invention is as follows: the signal acquisition and control module outputs a control signal to each module, first controls the laser control module to emit a laser, and controls the high-speed scanning module to start scanning. The light emitted by the laser control module reaches the spatial light modulator of the focal spot modulation module, and through the closed-loop feedback active optical wavefront control method, the corresponding phase is loaded on the spatial light modulator, so that the light beam becomes an aberration-free light beam that can be focused into a long strip focal spot. Then the light beam is combined, and after passing through the high-speed scanning module, it is focused by the objective lens onto the corresponding sample surface, illuminating the long strip focal area, and the reflected light carrying the detection signal is detected and collected by the parallel detection module, and the corresponding signal is transmitted back to the signal acquisition and control module, and imaging is achieved after system processing.

[0054] Figure 6This is an optical path diagram implemented by the device of the present invention. After the laser in the laser control module emits a detection light, it is modulated into light of a single polarization degree through a 1 / 2 wave plate. After passing through a polarization beam splitter (PBS), the polarization degree of the light can just be modulated by the SLM. The light beam is incident on the SLM, and the phase of the light beam is precisely modulated by loading a suitable phase pattern on the SLM. The outgoing light beam is modulated into an aberration-free long strip focal spot. The light beam is then expanded by a double telecentric 4f lens group consisting of two lenses with focal lengths of 100 mm and 150 mm respectively. The expanded light beam is modulated into linearly polarized light by a 1 / 2 wave plate, and then passes through a PBS. The reflected light beam is used as the illumination light incident on the sample surface. Due to the characteristics of the PBS, the reflected light is a light beam with a polarization direction parallel to the paper surface. The light beam is then scanned by the galvanometer scanning system of the high-speed scanning module. The outgoing light beam is deflected by mirror 1 and expanded by another double telecentric 4f lens group consisting of two lenses with focal lengths of 50 mm and 200 mm respectively, and then deflected by mirror 2. After passing through a 1 / 4 wave plate, it enters the objective lens and is incident on the sample surface. This 1 / 4 wave plate has two functions: 1. Make the illumination light circularly polarized; 2. Make the polarization directions of the incident illumination light and the reflected detection light perpendicular, so that the stray light reflected by the surface of the optical device will not enter the detection light path, while the detection light reflected by the sample will enter the detection light path.

[0055] The above-mentioned double telecentric 4f mirror group and the double telecentric 4f mirror group between the fast and slow mirrors in the high-speed scanning module jointly realize the complete conjugation of the fast mirror, slow mirror and objective entrance pupil, realize the distortion-free mapping of the scanning beam quality and linear signal system, and avoid the problems of uneven scanning field intensity distribution and distortion that exist in conventional confocal systems when scanning.

[0056] The signal light reflected by the sample ( Figure 6 The green light (marked in the optical path diagram) enters the detection optical path and is incident on the detector array of the parallel detection module by a lens with a focal length of 200mm for signal detection. The computer will generate an image of corresponding pixel size according to the time series of the detected signal.

[0057] Among them, the spatial light modulator is directly controlled by a personal computer (PC), while the corresponding devices of other modules are controlled by an acquisition card.

[0058] The above is only a preferred embodiment of the present invention. Although the present invention has been disclosed as a preferred embodiment, it is not intended to limit the present invention. Any technician familiar with the art can make many possible changes and modifications to the technical solution of the present invention by using the above disclosed methods and technical contents without departing from the scope of the technical solution of the present invention, or modify it into an equivalent embodiment of equivalent changes. Therefore, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention, all belong to the scope of protection of the technical solution of the present invention.

Claims

1. A high-throughput multi-row parallel high-speed scanning high-resolution microscopic imaging device based on adaptive optics, characterized in that: The device comprises a laser control module, a focal spot modulation module, a high-speed scanning module and a parallel detection module; The laser control module is used to emit illumination light to illuminate the sample, and detect it through the parallel detection module; The focal spot modulation module uses a spatial light modulator to perform phase control on the scanning beam, modulates the beam into a long strip focal spot to cover the detector array, and uses the spatial light modulator to perform surface array wavefront control to control the scanning beam aberration; The high-speed scanning module is used to realize focal spot beam scanning, and is composed of a switchable orthogonal galvanometer galvanometer system and a resonance and galvanometer galvanometer system. Each galvanometer system includes an X-direction galvanometer, which is denoted as a fast mirror, and a Y-direction galvanometer, which is denoted as a slow mirror. A double telecentric 4f mirror group is placed between the fast and slow mirrors to ensure the conjugation of the fast and slow mirrors. The parallel detection module uses a detector array arranged in a long strip shape to detect the long strip illumination area, so as to achieve a multi-line imaging effect in one scan.

2. The high-throughput multi-row parallel high-speed scanning high-resolution microscopic imaging device based on adaptive optics according to claim 1, characterized in that: The device also includes a signal acquisition and control module, which is used to output signals for controlling other modules, so as to achieve orderly execution of tasks of each module and synchronization between scanning and detection acquisition.

3. The high-throughput multi-row parallel high-speed scanning high-resolution microscopic imaging device based on adaptive optics according to claim 1, characterized in that: In the focal spot modulation module, a closed-loop feedback active optical wavefront control method is used to control the scanning beam aberration, specifically: Selecting an aberration test function, generating a digital phase pattern that can estimate and correct beam aberration, loading the pattern on a spatial light modulator, capturing an image and calculating an image evaluation index to evaluate the effect of the generated phase pattern in compensating for aberrations; The phase size that needs to be corrected again is estimated based on the image evaluation index, and a new round of correction is implemented. According to the selected aberration test function, a new phase pattern generated by the estimated phase size is loaded on the spatial light modulator, and a new round of image acquisition is performed, and the image evaluation index is calculated; the correction is repeated until the image evaluation index reaches a satisfactory aberration optimization effect.

4. The high-throughput multi-row parallel high-speed scanning high-resolution microscopic imaging device based on adaptive optics according to claim 3, characterized in that: When the focal spot modulation module performs aberration optimization, the beam wavefront mode is digitally expressed by the Zernike coefficients, the Zernike aberration test function is used, and the PSF sharpness index is used accordingly to evaluate the effect of the generated phase pattern compensating the aberration.

5. The high-throughput multi-row parallel high-speed scanning high-resolution microscopic imaging device based on adaptive optics according to claim 1, characterized in that: In the high-speed scanning module, during the process of the X-direction galvanometer scanning a line of forward and return strokes, the point voltage signal output to the Y-direction galvanometer does not change, so that the light spot stays in the Y direction; After completing a line of scanning, the Y-direction galvanometer changes the voltage value of the distance M times the pixel size to achieve scanning in the X direction and the Y direction at a ratio of M times.

6. The high-throughput multi-row parallel high-speed scanning high-resolution microscopic imaging device based on adaptive optics according to claim 1, characterized in that: In the parallel detection module, the detector array is an avalanche photodiode APD fiber array, the position corresponding to a single pixel should be tightly surrounded by the surrounding fiber array, and adjacent fiber probes are arranged in a tangent manner.

7. The high-throughput multi-row parallel high-speed scanning high-resolution microscopic imaging device based on adaptive optics according to claim 6, characterized in that: In the parallel detection module, assuming that the number of rows scanned at one time is i, there are i pixels at the detection end, and the information of each pixel is obtained from the surrounding APDs.

8. The high-throughput multi-row parallel high-speed scanning high-resolution microscopic imaging device based on adaptive optics according to claim 1, characterized in that: The device also includes two polarization beam splitters, two reflectors and two double telecentric 4f mirror groups; After the laser control module emits the detection light, it is modulated into light of a single polarization degree by a 1 / 2 wave plate, and after passing through the first polarization beam splitter, the polarization degree of the light can be modulated by the spatial light modulator; The phase of the light beam is modulated by loading a phase pattern on the spatial light modulator, and the outgoing light beam is modulated into a long strip focal spot without aberration. The light beam is then expanded by the first double telecentric 4f lens group. The expanded light beam is modulated into linear polarized light by a 1 / 2 wave plate, and then passes through the second polarization beam splitter. The reflected light beam is used as the illumination light incident on the sample surface, and then passes through the high-speed scanning module for light beam scanning. The outgoing light beam is deflected by the first reflector and expanded by the second double telecentric 4f lens group, and then deflected by the second reflector. After passing through the 1 / 4 wave plate, it enters the objective lens and is incident on the sample surface. The signal light reflected by the sample enters the detection optical path and is incident on the detector array of the parallel detection module. The computer generates an image of corresponding pixel size according to the time series based on the detected signal.

9. The high-throughput multi-row parallel high-speed scanning high-resolution microscopic imaging device based on adaptive optics according to claim 8, characterized in that: The first double telecentric 4f lens group, the second double telecentric 4f lens group and the two double telecentric 4f lens groups in the high-speed scanning module jointly achieve complete conjugation of the fast lens, the slow lens and the objective lens entrance pupil.

10. A high-throughput multi-row parallel high-speed scanning high-resolution microscopic imaging method based on the device according to any one of claims 1 to 9, characterized in that: The method comprises: The laser control module emits laser, and the high-speed scanning module starts scanning at the same time; The laser emitted by the laser control module reaches the spatial light modulator of the focal spot modulation module, and through the closed-loop feedback active optical wavefront control method, the corresponding phase is loaded on the spatial light modulator, so that the light beam becomes an aberration-free light beam that can be focused into a long strip focal spot; then the light beams are combined, and after passing through the high-speed scanning module, they are focused onto the sample surface by the objective lens to illuminate the long strip focal area, and the reflected light carrying the detection signal is detected and collected by the parallel detection module.

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