Three-dimensional parallel confocal fluorescence microscopic imaging system and microscopic imaging method

Abstract

The invention discloses a three-dimensional parallel confocal fluorescent microscopic imaging system and a microscopic imaging method, which are characterized in that a light source module emits decoherence monochromatic laser with uniform light intensity to pass through a multi-focus uniform flat field illumination module, and dot array illumination light is input into an illumination scanning light beam through a dichroscope to scan the light beam; the light enters the aberration correction module for optical aberration correction, then enters the microscopic illumination module to reach a to-be-observed sample, fluorescence generated by the to-be-observed sample serves as dot array imaging light to be reversely propagated along an illumination light path, passes through the aberration correction module and the illumination scanning module to be de-scanned, and then is transmitted through the dichroscope; the high-resolution point array imaging light is input into the photon recombination module, high-resolution point array imaging light is generated, and the generated high-resolution point array imaging light reaches the imaging module through the imaging scanning module. High-speed imaging detection and aberration correction can be realized at the same time, so that the resolution and the signal-to-noise ratio are improved.

Description

Technical Field

[0001] The present application relates to the field of microscopic imaging technology, and in particular to a three-dimensional parallel confocal fluorescence microscopic imaging system and a microscopic imaging method. Background Art

[0002] As a non-contact, non-destructive detection method, confocal technology is widely used in biological research and medical fields, with excellent optical sectioning and three-dimensional reconstruction capabilities. However, in practical applications, wavefront aberrations can lead to a significant decrease in imaging quality, affecting the accuracy and stability of imaging. Especially in the imaging process of thick sample tissues, due to the inhomogeneity of the internal refractive index, the existing confocal microscopy system cannot effectively and accurately image the internal structure of the sample.

[0003] Although some technologies have been tried to solve the wavefront aberration problem, most commercial systems still face the problem of being unable to overcome the aberration caused by refractive index inhomogeneity in complex tissue structures. Due to the lack of effective aberration correction methods, these problems seriously restrict the imaging accuracy and stability of the system. Therefore, how to overcome the influence of refractive index inhomogeneity on imaging quality through effective means without damaging the sample is still a challenge in current technology. Summary of the invention

[0004] The purpose of the embodiments of the present application is to provide a three-dimensional parallel confocal fluorescence microscopy system and a microscopy method, which are used to solve the problems of low imaging resolution and signal-to-noise ratio in existing ordinary microscopy systems and confocal microscopy systems due to wavefront aberration introduced by the uneven refractive index of samples, especially thick sample tissues.

[0005] According to a first aspect of an embodiment of the present application, a three-dimensional parallel confocal fluorescence microscopy imaging system is provided, comprising:

[0006] Light source module, multi-focus uniform flat field illumination module, dichroic mirror, illumination scanning module, aberration correction module, microscopic illumination module, photon recombination module, imaging scanning module, imaging module, the light source module emits monochromatic laser through the multi-focus uniform flat field illumination module to obtain point array illumination light with uniform light intensity, the point array illumination light is input into the illumination scanning module through the dichroic mirror to scan the light beam, and then enters the aberration correction module for optical aberration correction, and after correction, enters the microscopic illumination module to reach the sample to be observed, the fluorescence generated by the sample to be observed is used as the point array imaging light to propagate in the reverse direction along the illumination light path, and after aberration correction, the light beam is sent to the microscopic illumination module to reach the sample to be observed, and ... microscopic illumination module is sent to the microscopic illumination module to propagate in the reverse direction along the illumination light path, and the light beam is sent to the microscopic illumination module to propagate The module is descanned by the illumination scanning module, and then transmitted through the dichroic mirror and input into the photon recombination module to generate high-resolution point array imaging light. The generated high-resolution point array imaging light passes through the imaging scanning module and reaches the imaging module; wherein the aberration correction module comprises a second Fourier lens, a deformable mirror and a third Fourier lens, the second Fourier lens converges the array illumination light spot into a plurality of overlapping light beams on its Fourier plane, the second Fourier lens and the third Fourier lens form a 4F system with overlapping spectrum planes, and the deformable mirror is placed at the middle spectrum plane position to correct the aberration of the light spot.

[0007] According to a second aspect of an embodiment of the present application, a microscopic imaging method of the above-mentioned three-dimensional parallel confocal fluorescence microscopic imaging system is provided, comprising:

[0008] (1) The light intensity of the light beam emitted by the light source module is uniformized by a multi-focus uniform flat field illumination module and focused into a multi-focus array as a point array illumination light; the point array illumination light is input into the illumination scanning module through a dichroic mirror, and then passes through an aberration correction module and an illumination module to reach and focus on the plane of the sample to be detected;

[0009] (2): The fluorescence generated by the sample to be observed propagates in reverse as a point array imaging light through the illumination optical path and the aberration correction module, then passes through the dichroic mirror to enter the photon recombination module, passes through the imaging scanning module to reach the imaging module, and is focused on the imaging module;

[0010] (3): The imaging module collects the fluorescence signal;

[0011] (4): Control the illumination scanning module and the imaging scanning module to scan, and repeat steps (1) to (3) until the entire field of view is scanned;

[0012] (5): The computer of the imaging module evaluates the aberration through an aberration estimation algorithm, loads the corresponding correction item onto the deformable mirror, and completes the aberration correction.

[0013] The technical solution provided by the embodiments of the present application may have the following beneficial effects:

[0014] In optical imaging systems, the thickness of the sample has a direct impact on the image quality, especially when facing thick samples, the imaging system often faces complex optical effects. For example, during the transmission of the light beam, thick samples will produce refraction, scattering and absorption of the propagation of the incident light due to their different physical thickness. These effects will not only cause image distortion, but may also cause focus shift and reduced resolution. In severe cases, it may even make the imaging system unable to accurately capture the details of the sample. Specifically, when light penetrates a thick sample, it needs to undergo multiple refractions, and each refraction will change the path and propagation speed of the light. Due to the uneven thickness of different areas of the sample, the degree of refraction of the light will also be different, which will cause the light from various areas of the sample to focus on different positions, thereby causing aberration problems.

[0015] The appearance of aberrations will directly affect the imaging quality and resolution of the system, especially in application scenarios requiring high resolution. Aberrations caused by thick samples may cause image blur, loss of details or defocused imaging, affecting the final image clarity and accuracy. For example, in fields such as microscopic imaging or biomedical imaging, thick samples often hinder detailed microstructure observation, affecting the resolution and precise measurement of the target area. In order to solve these problems, traditional optical systems usually use mechanical adjustment or multiple exposure methods to try to compensate for aberrations. However, these methods often require sophisticated equipment and complex operations, which not only increase the cost and difficulty of operation of the system, but also reduce the stability and operability of the system.

[0016] In order to solve this problem, the embodiment of the present application introduces an aberration correction module, which uses active optical adjustment methods to overcome the aberration effects caused by thick samples. Specifically, the aberration correction module can monitor the optical properties of the sample, and dynamically adjust the deformable mirror in the aberration correction module according to the optical aberration introduced by the thick sample, thereby effectively compensating for the optical aberration caused by the thick sample. Through a high-precision deformable mirror, the module can automatically identify and correct the refraction, scattering and other effects caused by thick samples, ensuring that the light beam can be accurately focused and imaged during the imaging process, avoiding optical distortion caused by uneven sample thickness.

[0017] Specifically, the aberration correction module corrects the aberration problem caused by the change of sample thickness by optimizing the beam propagation path and adjusting the beam propagation angle and optical path. In addition, the module can also automatically perform real-time calibration during the imaging process to ensure that the system can always maintain clear imaging quality in different sample areas. This dynamic adjustment method not only effectively eliminates the optical distortion caused by thick samples, but also avoids the cumbersome operation of manual adjustment or multiple exposures required in traditional methods. Through the aberration correction module, the system can ensure that high-resolution and high-contrast imaging effects are always obtained under samples of different thicknesses and types, significantly improving the robustness of the system. In many high-precision adaptive optical imaging systems, it is often necessary to rely on wavefront sensors such as Shack-Hartmann wavefront sensors or Michelson interferometers to monitor the system status in real time and provide feedback, so as to dynamically adjust the parameters or optical path of the aberration compensation element (deformable mirror) to overcome aberration problems. However, sensor-dependent systems often bring a series of additional challenges, especially in terms of system complexity and cost. First, the sensor itself needs to be highly integrated with other optical components in the system, and complex algorithms are needed to analyze sensor data in real time to perform precise optical path adjustment. The design of this structure usually requires a lot of hardware support and software algorithms, which increases the size, weight and power consumption of the system. At the same time, the installation, debugging and maintenance of the sensor itself also require additional time and cost, which not only increases the overall complexity of the system, but also makes the system more prone to failure and maintenance problems during long-term use.

[0018] In addition, the accuracy and response speed of the sensor often determine whether the system can respond to environmental changes or changes in sample characteristics in real time. If the sensor has poor performance or delayed response, the system may not be able to make effective adjustments in time, which may affect the imaging quality or cause unstable imaging. Therefore, sensor-based solutions often require a high degree of technical investment and precise adjustment, which is not the best choice for some application scenarios that require rapid deployment, low cost or high reliability.

[0019] In order to solve these problems, this application adopts a sensorless adaptive method, which successfully overcomes the disadvantage of high system complexity caused by the introduction of sensors. The core idea of ​​the sensorless adaptive method is to automatically adjust the optical parameters through the system's own feedback mechanism without the participation of external sensors. Specifically, this method uses the adaptive control algorithm inside the system to automatically adjust the position of optical elements, the propagation path of the light beam or other key parameters by analyzing the output results of the system (such as imaging quality, resolution, etc.), thereby achieving aberration correction and system optimization.

[0020] Unlike traditional sensor-dependent methods, sensorless adaptive methods can simplify the system structure, remove sensors and their related electronic devices, and reduce the hardware requirements and complexity of the system. Through autonomous algorithm adjustment, the system can respond to changes in optical conditions in real time without relying on additional hardware devices for data acquisition and processing. In this way, not only the size and weight of the hardware are greatly reduced, but also the manufacturing and maintenance costs of the system are reduced. In addition, due to the reduced reliance on external sensors, the reliability of the entire system is improved, avoiding the problems that may be caused by sensor failure, damage or malfunction, and improving the stability of the system and the reliability of long-term operation.

[0021] The sensorless adaptive method can also maintain the system's adaptability to different samples or environmental conditions without relying on external sensors. The system can fine-tune the optical components in real time based on the current imaging effect or measurement results to ensure optimal performance under different working conditions. This adaptive adjustment process is completed entirely within the system, simplifying user operation and system debugging, making the system easier to use and maintain.

[0022] The system not only significantly improves the imaging frame rate while ensuring imaging accuracy, but also has the advantage of being compact. By introducing an aberration correction module, the system is suitable for imaging samples of various complex structures and is highly practical.

[0023] It should be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present application and, together with the description, serve to explain the principles of the present application.

[0025] Figure 1 It is a schematic structural diagram of a three-dimensional parallel confocal fluorescence microscopy imaging system according to an exemplary embodiment.

[0026] Figure 2 is a flow chart of an aberration estimation algorithm according to an exemplary embodiment.

[0027] In the figure: 1. laser; 2. multimode square optical fiber; 3. metallographic microscope objective; 4. first Fourier lens; 5. first plane reflector; 6. first microlens array; 7. dichroic mirror; 8. first scanning lens; 9. galvanometer; 10. second scanning lens; 11. second Fourier lens; 12. deformable mirror; 13. third Fourier lens; 14. tube lens; 15. microscope objective; 16. microhole array; 17. second plane reflector; 18. first relay lens; 19. third plane reflector; 20. fourth plane reflector; 21. second relay lens; 22. fifth plane reflector; 23. second microlens array; 24. third scanning lens; 25. fourth scanning lens; 26. camera; 27. acquisition card; 28. computer. DETAILED DESCRIPTION

[0028] Exemplary embodiments will be described in detail herein, examples of which are shown in the accompanying drawings. When the following description refers to the drawings, the same numbers in different drawings represent the same or similar elements unless otherwise indicated. The implementations described in the following exemplary embodiments do not represent all implementations consistent with the present application. Instead, they are merely examples of devices and methods consistent with some aspects of the present application as detailed in the appended claims.

[0029] The terms used in this application are for the purpose of describing specific embodiments only and are not intended to limit this application. The singular forms of "a", "said" and "the" used in this application and the appended claims are also intended to include plural forms unless the context clearly indicates other meanings. It should also be understood that the term "and / or" used herein refers to and includes any or all possible combinations of one or more associated listed items.

[0030] It should be understood that although the terms first, second, third, etc. may be used in the present application to describe various information, these information should not be limited to these terms. These terms are only used to distinguish the same type of information from each other. For example, without departing from the scope of the present application, the first information may also be referred to as the second information, and similarly, the second information may also be referred to as the first information. Depending on the context, the word "if" as used herein may be interpreted as "at the time of" or "when" or "in response to determining".

[0031] Figure 1 is a schematic structural diagram of a three-dimensional parallel confocal fluorescence microscopy imaging system according to an exemplary embodiment. Figure 1As shown, the three-dimensional parallel confocal fluorescence microscopy imaging system may include: a light source module, a multi-focus uniform flat field illumination module, a dichroic mirror 7, an illumination scanning module, an aberration correction module, a microscopic illumination module, a photon recombination module, an imaging scanning module, and an imaging module. The light source module emits a monochromatic laser through the multi-focus uniform flat field illumination module to obtain a point array illumination light with uniform light intensity. The point array illumination light passes through the dichroic mirror 7 to input the point array illumination light into the illumination scanning module to scan the light beam, and then enters the aberration correction module for optical aberration correction. After correction, the light beam enters the microscopic illumination module to reach the sample to be observed. The fluorescence generated by the sample to be observed is used as a point array imaging light and propagates in the reverse direction along the illumination light path. It passes through the aberration correction module and the illumination scanning module to be descanned, and then is transmitted through the dichroic mirror 7 and input into the photon recombination module to generate a high-resolution point array imaging light. , the generated high-resolution point array imaging light passes through the imaging scanning module to reach the imaging module; wherein the dichroic mirror 7 is used to reflect the illumination light and transmit the imaging light; the aberration correction module is used to correct the optical aberration caused by the thick sample; the aberration correction module is used to correct the aberration of the light spot introduced by the thick sample to improve the robustness of the imaging of the thick sample, and the aberration correction module includes a second Fourier lens 11, a deformable mirror 12 and a third Fourier lens 13, the second Fourier lens 11 converges the array illumination light spot into a plurality of overlapping light beams on its middle spectrum plane, the second Fourier lens 11 and the third Fourier lens 13 form a 4F system with overlapping spectrum planes, the front focal plane coincides with the back focal plane of the 4F system composed of the first scanning lens 8 and the second scanning lens 10 to ensure that the properties of the light beam remain unchanged, and the deformable mirror 12 is placed in the middle spectrum plane position to correct the aberration of the light spot. The deformable mirror 12 is placed on the spectrum plane of the 4F system, and the aberration correction of all points of the dot matrix illumination can be performed simultaneously.

[0032] It can be seen from the above embodiments that the present application, through the intervention of the aberration correction module, makes the system have good robustness against the aberration caused by thick samples, avoiding the influence of the aberration caused by thick samples on the system resolution. The use of multimode square optical fiber 2 as a decoherence element reduces the high system complexity caused by the elimination of coherence. The system can meet the imaging requirements of complex sample structures while improving imaging accuracy and efficiency.

[0033] In one embodiment, the light source module is used to generate monochromatic laser light with uniform light intensity distribution.

[0034] Specifically, the light source module includes a laser 1 and a multimode square optical fiber 2, wherein the laser 1 is used to generate monochromatic laser light; the multimode square optical fiber 2 is used to receive the monochromatic laser light emitted by the laser 1 and generate a decoherent and uniform light intensity beam; the laser 1 emits a monochromatic laser light, which is coupled into the multimode square optical fiber 2, and the decoherent and uniform light intensity laser beam emitted from the other end thereof serves as the light source of the system. In this embodiment, the wavelength of the laser beam emitted by the light source module is 405 / 488 / 561 / 640nm.

[0035] In one embodiment, the multi-focus uniform flat-field illumination module is used to modulate monochromatic laser light with uniform light intensity distribution into point array illumination light with uniform light intensity.

[0036] Specifically, the multi-focal uniform flat field illumination module comprises: a metallographic microscope objective lens 3, a first Fourier lens 4, a first plane reflector 5 and a first microlens array 6. The light beam emitted from the light source module converges through the metallographic microscope objective lens 3 to reach the first Fourier lens 4, passes through the first Fourier lens 4, and is reflected by the first plane reflector 5 to form a light field with uniform light intensity on the plane where the first microlens array 6 is located, and a multi-focal array with uniform light intensity is generated on the back focal plane of the first microlens array 6. The distance between the exit pupil of the metallographic microscope objective lens 3 and the first Fourier lens 4 is the focal length of the first Fourier lens 4, so that the spacing of the point array illumination light generated by the first microlens array 6 is conserved. The exit end of the multi-mode square optical fiber 2 should be placed at the working distance position of the metallographic microscope objective lens 3 to achieve the best imaging effect; the exit pupil position of the metallographic microscope objective lens 3 should be at one times the focal length of the first Fourier lens 4, so that the light spot generated by the first microlens array 6 has the best telecentricity.

[0037] In one embodiment, the illumination scanning module is used to project point array illumination light onto a sample to be observed and realize rapid scanning; the illumination scanning module comprises a first scanning lens 8, a galvanometer 9, and a second scanning lens 10; the first scanning lens 8 converges the point array illumination light into overlapping light beams, which are received by the regular reflection surface of the galvanometer 9 and reflected, and are input into the illumination module through the second scanning lens 10; the first scanning lens 8 and the second scanning lens 10 constitute a 4F system, and the use of the 4F system can reduce scanning errors and improve imaging quality. The intermediate spectrum surface of the 4F system constituted by the first scanning lens 8 and the second scanning lens 10 is an overlapping beam of several light beams, which is the position with the smallest area of ​​the entire light beam. Projecting it onto the scanning galvanometer 9 for scanning can make the scanning process more stable, and the required area of ​​the scanning galvanometer 9 is minimized.

[0038] In one embodiment, the microscopic illumination module is used to shrink the laser illumination spot and project it onto the sample plane to form a shrunken uniform illumination spot.

[0039] Specifically, the microscopic illumination module includes a tube lens 14 and a microscope objective lens 15 . The tube lens 14 and the microscope objective lens 15 form an asymmetric 4F system to reduce the size of the light spot.

[0040] In one embodiment, the photon recombination module is used to reduce the spot diameter of the point array imaging light to generate high-resolution point array imaging light.

[0041] Specifically, the photon recombination module includes: a microhole array 16 and a second microlens array 23, wherein the microhole array 16 is used to eliminate out-of-focus light and stray light of the point array imaging light; and the second microlens array 23 is used to reduce the diameter of all light spots in the point array imaging light to generate high-resolution point array imaging light; wherein a light beam transfer module for coordinating imaging and extending the light path is arranged between the microhole array 16 and the second microlens array 23. In this embodiment, the micropore diameter selected for the micropore array is 55um, which is used to pass the point array imaging light and block the out-of-focus light. The point array imaging light passes through the micropore array to generate the point array imaging light that excludes the out-of-focus light and the stray light. The second microlens array 23 is used to reduce the diameter of all the light spots in the point array imaging light to half of the diameter of the light spots in the absence of the second microlens array 23, so as to improve the resolution of the system; the light beam transfer module composed of the first relay lens 18, the second relay lens 21, the second plane reflector 17, the third plane reflector 19, the fourth plane reflector 20 and the fifth plane reflector 22 is arranged between the microlens array and the micropore array, so as to cooperate with the imaging and extend the light path.

[0042] Furthermore, the beam transfer module includes a first relay lens 18 and a second relay lens 21 , which conjugate the imaging light of the point array located at the microhole array 16 to the back focal plane of the second microlens array 23 .

[0043] In one embodiment, the imaging scanning module is used to project the point array imaging light onto the imaging module and realize rapid imaging.

[0044] Specifically, the imaging scanning module includes a third scanning lens 24, a back reflection surface of the galvanometer 9, and a fourth scanning lens 25. The third scanning lens 24 converges the point array imaging light into overlapping light beams, which are received by the back reflection surface of the galvanometer 9 and reflect the converged light spots, and then are re-diverged into point array imaging light through the fourth scanning lens 25 and input into the imaging module. The third scanning lens 24 and the fourth scanning lens 25 constitute a 4F system;

[0045] In one embodiment, the imaging module is used to image and process the received sample fluorescence information.

[0046] Specifically, the imaging module includes a camera 26, an acquisition card 27 and a computer 28. The camera 26 receives the point array imaging light and transmits the signal to the computer 28 through the acquisition card 27; the computer 28 is used to control the galvanometer 9 to scan and process the acquired signal to generate a sample image through the acquisition card 27, and run the aberration correction algorithm to control the shape of the deformable mirror 12, thereby realizing the correction of optical aberrations.

[0047] From the above description and Figure 1 It can be seen that the illumination scanning module uses the positive reflection surface of the galvanometer 9, while the imaging scanning module uses the back reflection surface of the galvanometer 9, that is, the illumination scanning module and the imaging scanning module share a scanning galvanometer 9. This structure can reduce system complexity, reduce mechanical errors, and improve the consistency of illumination and imaging;

[0048] The basic principle of optical transmission in this embodiment is as follows:

[0049] The laser 1 emits a laser beam of a specific wavelength, which is emitted through the multimode square optical fiber 2 to form a uniform light beam with decoherence. The uniform light beam is imaged through the metallographic microscope objective 3 and the first Fourier lens 4 to form a uniform light beam with reduced NA and enlarged diameter, which is projected on the front surface of the first microlens array 6, and generates a dot matrix illumination with uniform light intensity and good telecentricity on its focal plane; the dot matrix illumination light is reflected by the dichroic mirror 7 and enters the illumination scanning module, which quickly scans the illumination light, and then corrects the spot aberration through the aberration correction module, and then projects it onto the sample through the microscopic illumination module to excite fluorescence, and the fluorescent point array returns along the illumination light path, is transmitted through the dichroic mirror 7 into the photon recombination module, and enters the imaging module through the imaging scanning module for imaging.

[0050] The embodiment of the present invention also provides a microscopic imaging method of the above-mentioned three-dimensional parallel confocal fluorescence microscopic imaging system, comprising:

[0051] (1) The light intensity of the light beam emitted by the light source module is uniformized by the multi-focus uniform flat field illumination module and focused into a multi-focus array as the point array illumination light; the point array illumination light is input into the illumination scanning module through the dichroic mirror 7, and then passes through the aberration correction module and the microscopic illumination module to reach and focus on the plane of the sample to be detected;

[0052] (2): The fluorescence generated by the sample to be observed propagates in reverse as a point array imaging light through the illumination light path and the aberration correction module, then passes through the dichroic mirror 7 to enter the photon recombination module, passes through the imaging scanning module to reach the imaging module, and is focused on the imaging module;

[0053] (3): The imaging module collects the fluorescence signal;

[0054] (4): Control the illumination scanning module and the imaging scanning module to repeat steps (1) to (3) until the entire field of view is scanned;

[0055] (5): The computer 28 of the imaging module evaluates the aberration through an aberration estimation algorithm, loads the corresponding correction item onto the deformable mirror 12, and completes the aberration correction.

[0056] In one embodiment, the flowchart of the aberration estimation algorithm is as follows: Figure 2 As shown, the processing steps are as follows:

[0057] S1: Set the Zernike polynomial order m of the aberration to be corrected;

[0058] Specifically, the Zernike term corresponding to the aberration that needs to be corrected may be set according to the imaging effect of the system.

[0059] S2: Take the Zernike term of order i , the coefficient is , loaded onto the deformable mirror 12;

[0060] Specifically, the i-th order Zernike polynomial can be written as

[0061]

[0062] S3: Using the camera 26 to form an image, and using the computer 28 to calculate the image quality evaluation function at this time ;

[0063] Specifically, the image quality evaluation index can be calculated according to the following formula:

[0064]

[0065] In the formula, is the horizontal and vertical coordinates of the image pixel, is the system laser wavelength, is the numerical aperture of the microscope, is a circular domain function, The variance is The unit two-dimensional Gaussian function, It is the spectrum of the wide-field image, that is, the result of the two-dimensional Fourier transform of the wide-field image. Represents the modulo operation.

[0066] S4: Change coefficient , repeat (2)-(3) several times;

[0067] Specifically, It can be taken from an arithmetic progression, such as (-a, 0, a) or (-2a, -a, 0, a, 2a).

[0068] S5: Quadratic fitting image quality evaluation index and coefficient , the independent variable is , the dependent variable is . According to the fitted expression, the coefficient of the maximum image quality evaluation index is calculated. as the coefficient of this Zernike term.

[0069] Specifically, the least squares method can be used to evaluate the image quality index and coefficient Fitting, that is, according to the discrete sequence Estimating a quadratic polynomial In .

[0070] Using loss function , so that

[0071]

[0072] The above formula can be simplified to:

[0073]

[0074] Solved

[0075]

[0076] According to the above formula, according to the coefficient used And the image quality evaluation index calculated Fit its expression. Finally, calculate the coefficient of the Zernike term when the image quality is optimal based on the fitted expression. value.

[0077] S6: Change the Zernike term to be corrected , repeat S4-S5 several times until all m Zernike coefficients are calculated, the aberration estimation is completed, all the obtained Zernike terms are superimposed and loaded on the deformable mirror 12, and the aberration correction is completed.

[0078] Those skilled in the art will readily appreciate other embodiments of the present application after considering the description and practicing the contents disclosed herein. The present application is intended to cover any modification, use or adaptation of the present application, which follows the general principles of the present application and includes common knowledge or customary techniques in the art that are not disclosed in the present application. The description and examples are intended to be exemplary only, and the true scope and spirit of the present application are indicated by the claims.

[0079] It should be understood that the present application is not limited to the precise structures that have been described above and shown in the drawings, and that various modifications and changes may be made without departing from the scope thereof. The scope of the present application is limited only by the appended claims.

Claims

1. A three-dimensional parallel confocal fluorescence microscopy imaging system, characterized in that: include: A light source module, a multi-focus uniform flat field illumination module, a dichroic mirror, an illumination scanning module, an aberration correction module, a microscopic illumination module, a photon recombination module, an imaging scanning module, and an imaging module. The light source module emits a decoherent, uniform light intensity monochromatic laser, which passes through the multi-focus uniform flat field illumination module to obtain a point array illumination light with uniform light intensity. The point array illumination light passes through the dichroic mirror to input the point array illumination light into the illumination scanning module to scan the light beam, and then enters the aberration correction module for optical aberration correction. After correction, the light beam enters the microscopic illumination module to reach the sample to be observed. The fluorescence generated by the sample to be observed is used as the point array imaging light and propagates in the reverse direction along the illumination light path. The light passes through the aberration correction module and the illumination scanning module to be descanned, and then is transmitted through the dichroic mirror and input into the photon recombination module to generate high-resolution point array imaging light. The generated high-resolution point array imaging light passes through the imaging scanning module to reach the imaging module; wherein the aberration correction module comprises a second Fourier lens, a deformable mirror and a third Fourier lens, wherein the second Fourier lens converges the array illumination light spot into a plurality of overlapping light beams on its Fourier plane, the second Fourier lens and the third Fourier lens form a 4F system with overlapping spectrum planes, and the deformable mirror is placed in the middle of the spectrum plane position to correct the aberration of the light spot.

2. The three-dimensional parallel confocal fluorescence microscopy imaging system according to claim 1, characterized in that: The light source module includes a laser and a multimode square optical fiber. The laser is used to generate monochromatic laser light. The multimode square optical fiber is used to receive the monochromatic laser light emitted by the laser and generate a decoherent and uniform light intensity beam. The laser emits monochromatic laser light and couples it into the multimode square optical fiber. The decoherent and uniform light intensity laser beam emitted from the other end of the optical fiber serves as the light source of the system.

3. The three-dimensional parallel confocal fluorescence microscopy imaging system according to claim 1, characterized in that: The multi-focus uniform flat field illumination module comprises: A metallographic microscope objective lens, a first Fourier lens and a first microlens array, wherein the light beam emitted from the light source module converges through the metallographic microscope objective lens to reach the first Fourier lens, passes through the first Fourier lens, is reflected by the first plane reflector, forms a light field with uniform light intensity on the plane where the first microlens array is located, and generates a multi-focal array with uniform light intensity on the back focal plane of the first microlens array, and the distance between the exit pupil of the metallographic microscope objective lens and the Fourier lens is the focal length of the first Fourier lens, so that the distance between the point array illumination light generated by the first microlens array is conserved.

4. The three-dimensional parallel confocal fluorescence microscopy imaging system according to claim 1, characterized in that: The illumination scanning module comprises a first scanning lens, a galvanometer, and a second scanning lens. The first scanning lens converges the point array illumination light into overlapping light beams, which are received by the positive reflection surface of the galvanometer and reflected, and then input into the illumination module through the second scanning lens. The intermediate spectrum plane of the 4F system formed by the first scanning lens and the second scanning lens is an overlapping light beam of several light beams.

5. The three-dimensional parallel confocal fluorescence microscopy imaging system according to claim 1, characterized in that: The microscopic illumination module comprises: a tube lens and a microscope objective lens, which form an asymmetric 4F system for reducing the laser illumination spot and projecting it onto the sample plane to form a reduced uniform illumination spot to excite the fluorescence of the sample.

6. The three-dimensional parallel confocal fluorescence microscopy imaging system according to claim 1, characterized in that: The photon recombination module comprises: A micro-aperture array for eliminating out-of-focus light and stray light of the imaging light of the dot array; and A second microlens array is used to reduce the diameters of all light spots in the point array imaging light to generate high-resolution point array imaging light; Wherein, a light beam transfer module for cooperating with imaging and extending the light path is arranged between the microhole array and the second microlens array.

7. The three-dimensional parallel confocal fluorescence microscopy imaging system according to claim 1, characterized in that: The light beam transfer module comprises a first relay lens and a second relay lens, which conjugates the imaging light of the point array located at the microhole array to the back focal plane of the second microlens array.

8. The three-dimensional parallel confocal fluorescence microscopy imaging system according to claim 1, characterized in that: The imaging scanning module includes a third scanning lens, a galvanometer back reflection surface, and a fourth scanning lens. The third scanning lens converges the point array imaging light into overlapping light beams, which are received by the galvanometer back reflection surface and reflect the converged light spots. The fourth scanning lens re-diverges the light into point array imaging light and then inputs it into the imaging module.

9. The three-dimensional parallel confocal fluorescence microscopy imaging system according to claim 1, characterized in that: The imaging module includes a camera, an acquisition card and a computer. The camera receives point array imaging light and transmits the signal to the computer through the acquisition card. The computer is used to control the galvanometer scanning through the acquisition card, process the acquired signal to generate a sample image, and run an aberration correction algorithm to control the shape of the deformable mirror, thereby realizing the correction of optical aberration.

10. The microscopic imaging method of the three-dimensional parallel confocal fluorescence microscopic imaging system according to any one of claims 1 to 9, characterized in that: include: (1) The light intensity of the light beam emitted by the light source module is uniformized by a multi-focus uniform flat field illumination module and focused into a multi-focus array as a point array illumination light; The point array illumination light is input into the illumination scanning module through the dichroic mirror, passes through the aberration correction module and the illumination module, and reaches and is focused on the plane of the sample to be detected; (2): The fluorescence generated by the sample to be observed propagates in reverse as a point array imaging light through the illumination optical path and the aberration correction module, then passes through the dichroic mirror to enter the photon recombination module, passes through the imaging scanning module to reach the imaging module, and is focused on the imaging module; (3): The imaging module collects the fluorescence signal; (4): Control the illumination scanning module and the imaging scanning module to repeat steps (1) to (3) until the entire field of view is scanned; (5): The computer of the imaging module evaluates the aberration through an aberration estimation algorithm, loads the corresponding correction item onto the deformable mirror, and completes the aberration correction.

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