Device for realizing high-flux fluorescence detection based on galvanometer scanning

Abstract

The invention relates to the technical field of optical microscopy, in particular to a device for realizing high-flux fluorescence detection based on galvanometer scanning, and the device comprises an illumination module which is used for generating a multi-focus plane illumination light beam; the multi-focus plane illumination light beam can enter a first surface of the galvanometer through the dichroscope, and the galvanometer can reflect the multi-focus plane illumination light beam to the objective lens module, so that the multi-focus plane illumination light beam is focused on a sample through the objective lens module to form multi-point illumination; emitted light emitted by a sample through excitation is collected by the objective lens module, then enters a first surface of the galvanometer and is reflected to the dichroscope, the dichroscope can reflect the emitted light to the detection module, signal light enters a second surface of the galvanometer and then can be reflected to the image acquisition module, and the image acquisition module is used for acquiring and imaging the signal light. The method has the effect of solving the technical problems that the existing fluorescence detection speed and resolution are difficult to consider and the resolution improvement of a high-throughput scheme is limited.

Description

Technical Field

[0001] This application relates to the field of optical microscopy, and in particular to a high-throughput fluorescence detection device based on galvanometer scanning. Background Technology

[0002] Currently, mainstream fluorescence microscopy imaging techniques are mainly divided into two categories: wide-field microscopy and scanning confocal microscopy. Wide-field microscopy illuminates the entire field of view simultaneously and uses a camera (such as CCD or sCMOS) for global imaging. Its advantages include fast imaging speed and suitability for observing dynamic processes such as living cells. However, because the entire thickness of the sample is excited, the detector collects a large amount of defocused fluorescence signals from outside the focal plane, resulting in high background, low contrast, and limited resolution in the image.

[0003] Scanning confocal microscopy (such as laser scanning confocal microscopy, LSCM) uses a single-point laser to scan the sample point by point, and places a pinhole at the position conjugate to the illumination focal point in the probe optical path. This pinhole can effectively filter out defocused fluorescence signals, thereby obtaining optical slice images with high contrast and high resolution. However, its disadvantage is that the point-by-point scanning method results in a very slow imaging speed, making it difficult to meet the high-throughput observation requirements of large field-of-view samples or rapid biological processes.

[0004] To balance imaging speed and resolution, the industry has proposed several multifocal scanning techniques. These techniques use devices such as microlens arrays to split a single illumination beam into a multifocal array, enabling parallel scanning of the sample and thus improving imaging speed to some extent.

[0005] Regarding the aforementioned technologies: While multifocal scanning technology attempts to improve imaging speed through parallel scanning, challenges remain in signal light collection and processing. Firstly, efficiently filtering out defocus signals from multi-point emitted fluorescence signals to ensure confocal effect at all scanning points is a challenge in optical path design. Secondly, obtaining a complete final image requires stitching together the various sub-images acquired during the scanning process, which places high demands on scanning uniformity and image processing algorithms. More importantly, while existing multifocal systems improve scanning speed, they are still constrained by the diffraction limit, making further breakthroughs in resolution difficult.

[0006] Therefore, how to design a novel optical path structure that can achieve multi-focal parallel detection to ensure high throughput, effectively perform confocal filtering, and further improve imaging resolution by utilizing the scanning mechanism itself is a technical problem that urgently needs to be solved in the field of microscopic imaging. Summary of the Invention

[0007] To address the technical challenges of balancing fluorescence detection speed and resolution, and the limitations on resolution improvement in high-throughput solutions, this application provides a high-throughput fluorescence detection device based on galvanometer scanning.

[0008] This application provides a high-throughput fluorescence detection device based on galvanometer scanning, which adopts the following technical solution: A high-throughput fluorescence detection device based on galvanometer scanning includes: an illumination module, a scanning module, an objective lens module, a detection module, and an image acquisition module, wherein the illumination module is used to generate a multifocal plane illumination beam; The scanning module includes a galvanometer and a dichroic mirror arranged in sequence, with the dichroic mirror located between the galvanometer and the illumination module; The multifocal plane illumination beam can be incident on the first surface of the galvanometer through the dichroic mirror, and the galvanometer can reflect the multifocal plane illumination beam to the objective lens module so that the multifocal plane illumination beam is focused on the sample through the objective lens module to form multi-point illumination; The emitted light from the sample is collected by the objective lens module, incident on the first surface of the galvanometer, and reflected to the dichroic mirror. The dichroic mirror can reflect the emitted light to the detection module. The signal light is incident on the second surface of the galvanometer and can be reflected to the image acquisition module. The image acquisition module is used to acquire and image the signal light.

[0009] By employing the above technical solution, the illumination module generates a multifocal plane illumination beam. This beam is incident on the first surface of the galvanometer via a dichroic mirror, and the galvanometer reflects it to the objective lens module, thus forming multi-point illumination on the sample. This multi-point illumination method allows the illumination source to more uniformly excite the fluorescence of the sample, improving illumination efficiency and uniformity within the field of view compared to traditional single-point illumination. The emitted light from the sample is collected by the objective lens module and reflected again by the first surface of the galvanometer to the dichroic mirror. The dichroic mirror reflects the emitted light to the detection module, and finally, the signal light is reflected by the second surface of the galvanometer to the image acquisition module for acquisition and imaging. The signal light is scanned twice by the galvanometer. Combined with the algorithms and image processing used by the image acquisition module to acquire and image the signal light, an image with twice the wide-field resolution can be obtained. This helps to solve the technical problem of the difficulty in balancing fluorescence detection speed and resolution, and the limitation of resolution improvement in high-throughput schemes.

[0010] Optionally, the illumination module includes a light-emitting element, a first objective lens, a first tube lens, and a first compound eye lens arranged in sequence. The first compound eye lens is positioned close to the dichroic mirror. The light emitted by the light-emitting element passes through the first objective lens and the first tube lens and then enters the first compound eye lens to form the multifocal plane illumination beam.

[0011] By adopting the above technical solution, the light emitted by the light-emitting element passes through the first objective lens and the first tube lens. The first objective lens and the first tube lens can adjust the light emitted by the light-emitting element, such as magnifying the light spot. Then the light is incident on the first compound eye lens, which can focus the illumination beam to form a multifocal plane illumination beam. The multifocal plane illumination beam can provide more uniform and efficient illumination for subsequent scanning and detection, which helps to improve the illumination effect on the sample, thereby improving the imaging quality and detection accuracy of the entire high-throughput fluorescence detection device.

[0012] Optionally, the illumination module includes a square optical fiber connected to the light-emitting element and located between the light-emitting element and the first objective lens.

[0013] By adopting the above technical solution, the square optical fiber can perform preliminary shaping and transmission of the light emitted by the light-emitting element, so that the light enters the subsequent first objective lens and first tube lens more uniformly. This ensures that a more stable and uniform multi-focal plane illumination beam can be formed when passing through the first compound eye lens, providing better illumination conditions for subsequent multi-point illumination of the sample, and helping to improve the detection accuracy and imaging quality of the entire high-throughput fluorescence detection device.

[0014] Optionally, a second tube mirror is disposed between the first compound eye lens and the dichroic mirror, a fifth tube mirror is disposed between the objective lens module and the galvanometer, a third tube mirror is disposed between the detection module and the dichroic mirror, and a fourth tube mirror is disposed between the detection module and the galvanometer. The third tube mirror is used to receive the emitted light and cause the emitted light to be incident on the detection module. The fourth tube mirror is used to receive the signal light and cause the signal light to be incident on the second surface of the galvanometer. A seventh tube mirror is disposed between the image acquisition module and the galvanometer.

[0015] By adopting the above technical solution, a second tube mirror is placed between the first compound eye lens and the dichroic mirror, which can effectively transmit and adjust the multifocal plane illumination beam, ensuring that it can enter the subsequent optical path in a suitable state; a fifth tube mirror is placed between the objective lens module and the galvanometer, which helps to achieve optical matching between the objective lens module and the galvanometer, so that the multifocal plane illumination beam is accurately focused on the sample to form multi-point illumination; a third tube mirror is placed between the detector module and the dichroic mirror, which can receive the emitted light and accurately incident it onto the detector module, ensuring the effective transmission of the emitted light; a fifth tube mirror is placed between the detector module and the galvanometer. The fourth tube mirror receives the signal light and accurately directs it onto the second surface of the galvanometer, facilitating further transmission and processing of the signal light. A seventh tube mirror is placed between the image acquisition module and the galvanometer, ensuring the signal light illuminates the target surface of the image acquisition module in a suitable manner, improving the quality and accuracy of image acquisition. Through the coordinated arrangement of multiple tube mirrors, a rational optical path structure is formed, ensuring efficient and accurate transmission of the multifocal plane illumination beam, emitted light, and signal light throughout the device. This, in turn, enhances the performance of the entire high-throughput fluorescence detection device, achieving clearer and more accurate fluorescence detection imaging.

[0016] Optionally, the objective module includes a sixth tube and a microscope objective arranged in sequence. The sixth tube is positioned close to the fifth tube. The fifth tube and the second tube form a first 4f system. The sixth tube and the microscope objective form a second 4f system. The third tube and the fifth tube form a third 4f system. The seventh tube and the fourth tube form a fourth 4f system.

[0017] By adopting the above technical solutions, the fifth and second tubes constitute the first 4f system, which enables beam collimation and image transmission, ensuring the stability and accuracy of the multifocal plane illumination beam during transmission, and allowing the illumination light to reach the objective module more precisely. The sixth tube and the microscope objective constitute the second 4f system, which can focus the multifocal plane illumination beam reflected by the scanning module onto the sample, forming clear and uniform multi-point illumination, effectively exciting the sample's fluorescence. The third and fifth tubes constitute the third 4f system, which can accurately transmit the emitted light from the sample to the detection module, facilitating subsequent processing of the emitted light. The seventh and fourth tubes constitute the fourth 4f system, which can accurately transmit the signal light after confocal filtering by the detection module to the image acquisition module, enabling the image acquisition module to acquire clear and accurate signal light images, thereby improving the imaging quality and detection accuracy of the entire high-throughput fluorescence detection device.

[0018] Optionally, the fifth and seventh endoscopes are respectively configured as replaceable structures.

[0019] Optionally, the detection module is located on the side of the scanning module away from the objective lens module. The detection module includes a first reflecting mirror, a second compound eye lens, a pinhole array, and a second reflecting mirror arranged in sequence. The first reflecting mirror is arranged close to the dichroic mirror, and the second reflecting mirror is arranged close to the galvanometer. The microlenses of the second compound eye lens are arranged in a one-to-one correspondence with the pinholes of the pinhole array, and the center of the second compound eye lens is aligned with the center of the corresponding pinhole.

[0020] By adopting the above technical solution, the microlenses of the second compound eye lens are set one-to-one with the pinholes of the pinhole array, and the center of the second compound eye lens is aligned with the center of the corresponding pinhole. After being reflected by the scanning module, the emitted light first passes through the first reflecting mirror and then is focused by the second compound eye lens. Due to the corresponding setting and center alignment of the second compound eye lens with the pinhole array, the emitted light can be accurately filtered through the pinhole array to remove stray light and background noise, forming a purer signal light. Finally, the signal light is reflected by the second reflecting mirror to the subsequent modules, thereby effectively improving the confocal filtering effect of the emitted light, improving the quality of the signal light, and laying the foundation for the image acquisition module to acquire clearer and more accurate images.

[0021] Optionally, the detection module includes an adjustable aperture layer disposed between the second compound eye lens and the pinhole array, the adjustable aperture layer being used to adjust the aperture of the pinholes.

[0022] By employing the above technical solution, an adjustable aperture layer is set between the second compound eye lens and the pinhole array, allowing for adjustment of the pinhole aperture and thus changing the amount of signal light passing through it. In different detection scenarios, such as detecting samples with different fluorescence intensities, adjusting the pinhole aperture can control the intensity of the signal light entering the subsequent image acquisition module, avoiding poor image acquisition results due to excessively strong or weak signal light. This improves the adaptability of the entire high-throughput fluorescence detection device to different samples and enhances its detection accuracy.

[0023] Optionally, the adjustable aperture layer includes a transparent substrate and a plurality of liquid crystal cells disposed on the transparent substrate. Each liquid crystal cell corresponds one-to-one with a microlens of the second compound eye lens. An electrode is disposed within the liquid crystal cell for changing the light transmittance of its central region. By controlling the voltage applied to the electrode, the size of the central transparent region of the liquid crystal cell is adjusted to achieve aperture adjustment of the small hole.

[0024] By adopting the above technical solution, multiple liquid crystal units corresponding one-to-one with the microlenses of the second compound eye lens are set on the transparent substrate. Each liquid crystal unit contains electrodes that can alter the light transmittance of its central region. When different voltages are applied to the electrodes, the orientation of the liquid crystal molecules within the liquid crystal unit changes, thereby adjusting the size of the central transparent region of the liquid crystal unit. The change in the size of the central transparent region of the liquid crystal unit directly corresponds to a change in the aperture diameter, thus achieving aperture diameter adjustment. This allows for flexible adjustment according to actual needs to adapt to different detection scenarios and requirements, improving the applicability and flexibility of the device.

[0025] Optionally, the electrode is a ring electrode, and each liquid crystal cell is connected to an independent control circuit. The control circuit uses a matrix addressing method to control each liquid crystal cell.

[0026] By adopting the above technical solution and setting the electrodes as circular electrodes, the adjustment of the central transparent area of ​​the liquid crystal cell under voltage is made more uniform and stable. Each liquid crystal cell is connected to an independent control circuit, enabling precise control of each liquid crystal cell and allowing for personalized adjustment of the aperture at different positions according to actual needs. The control circuit uses a matrix addressing method to control each liquid crystal cell, reducing wiring complexity, which is particularly suitable for high-density arrays. This avoids interference and wiring difficulties caused by a large number of connections, improving the reliability and stability of the system. This allows for more flexible and efficient adjustment of the aperture to meet the needs of high-throughput fluorescence detection devices in different detection scenarios.

[0027] In summary, this application includes at least one of the following beneficial technical effects: 1. By cooperating with the illumination module, scanning module, objective lens module, detection module and image acquisition module, clearer and more accurate images can be obtained by removing stray light and improving signal quality, realizing high-throughput fluorescence detection and meeting the needs of scanning detection imaging of biological cell samples, etc., thus helping to overcome the technical bottleneck of existing fluorescence detection speed and resolution being difficult to balance and high-throughput scheme resolution improvement being limited. 2. By setting an adjustable aperture layer between the second compound eye lens and the pinhole array, the aperture of the pinhole can be adjusted, thereby changing the amount of signal light passing through the pinhole, so as to improve the adaptability of the entire high-throughput fluorescence detection device to different samples and the accuracy of detection. Attached Figure Description

[0028] Figure 1 This is a schematic diagram of the overall structure of a high-throughput fluorescence detection device based on galvanometer scanning in Embodiment 1 of this application.

[0029] Figure 2 This is a schematic diagram of the structure of the first compound eye lens in Embodiment 1 of this application.

[0030] Figure 3 This is a side view of the second compound eye lens and pinhole array in Embodiment 1 of this application.

[0031] Figure 4 This is a schematic diagram of the structure of the second compound eye lens and the pinhole array in Embodiment 1 of this application.

[0032] Figure 5 This is a schematic diagram of the overall structure of a high-throughput fluorescence detection device based on galvanometer scanning in Embodiment 2 of this application.

[0033] Figure 6 This is a side view of the second compound eye lens, the pinhole array, and the adjustable aperture layer in Embodiment 2 of this application.

[0034] Figure 7 This is a schematic diagram of the adjustable pore size layer in Embodiment 2 of this application.

[0035] Explanation of reference numerals in the attached figures: 1. Illumination module; 11. Light-emitting element; 12. First objective lens; 13. First tube lens; 14. First compound eye lens; 15. Square optical fiber; 16. Second tube lens; 2. Scanning module; 21. Galvanometer; 22. Dichroic mirror; 3. Objective lens module; 31. Sixth tube lens; 32. Microscope objective lens; 33. Fifth tube lens; 4. Detection module; 41. First reflecting mirror; 42. Second compound eye lens; 43. Pinhole array; 44. Second reflecting mirror; 45. Third tube lens; 46. Fourth tube lens; 47. Adjustable aperture layer; 471. Transparent substrate; 472. Liquid crystal unit; 473. Electrode; 5. Image acquisition module; 51. Seventh tube lens. Detailed Implementation

[0036] The following is in conjunction with the appendix Figure 1-7 This application will be described in further detail.

[0037] This application discloses a high-throughput fluorescence detection device based on galvanometer scanning.

[0038] It should be noted that, in the description of this invention, the terms "center," "longitudinal," "lateral," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," and "outer," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing this invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this invention.

[0039] Example 1: Refer to Figure 1A high-throughput fluorescence detection device based on galvanometer scanning includes an illumination module 1, a scanning module 2, an objective lens module 3, a detection module 4, and an image acquisition module 5. The illumination module 1 generates a multifocal plane illumination beam, which is reflected by the scanning module 2 and incident on the objective lens module 3, where it is focused onto the sample to form multi-point illumination. The emitted light from the sample is collected by the objective lens module 3 and returned to the scanning module 2. The scanning module 2 reflects the emitted light to the detection module 4, where it performs confocal filtering to form a signal light. This signal light is incident on the scanning module 2 and reflected by it to the image acquisition module 5, which acquires and images the signal light. This structural combination enables the device to efficiently achieve high-throughput fluorescence detection, improving both the speed and quality of imaging.

[0040] The illumination module 1 includes a light-emitting element 11, a first objective lens 12, a first tube lens 13, a first compound eye lens 14, and a square optical fiber 15 arranged sequentially. The square optical fiber 15 is connected to the light-emitting element 11 and is located between the light-emitting element 11 and the first objective lens 12. The light-emitting element 11 uses a four-wavelength laser (405nm / 488nm / 561nm / 640nm). In other embodiments, the light-emitting element 11 can also be equipped with lasers of different wavelengths according to user requirements to meet different detection needs.

[0041] The square optical fiber 15 can transmit and shape the light emitted by the light-emitting element 11, so that the laser emitted by the light-emitting element 11, after being output through the square optical fiber 15, can form a square light spot with uniform intensity distribution. In this embodiment, the square optical fiber 15 can be made of materials such as quartz optical fiber.

[0042] The first objective lens 12 is used to perform preliminary focusing on the light output from the square optical fiber 15. In this embodiment, the first objective lens 12 is a 50X objective lens. In other embodiments, the first objective lens 12 may also be an objective lens with special optical properties, such as an achromatic objective lens, to improve the focusing effect of the light.

[0043] The first tube lens 13 has a focal length of 240mm. The function of the first tube lens 13 is to further magnify the light spot focused by the first objective lens 12. Its material can be common optical materials such as optical glass.

[0044] Reference Figure 1 and Figure 2The first compound eye lens 14 is composed of multiple microlenses. The light emitted by the light-emitting element 11 passes through the square optical fiber 15, the first objective lens 12, and the first tube lens 13 before entering the first compound eye lens 14 to form a multifocal plane illumination beam. In this embodiment, the arrangement of the microlenses in the first compound eye lens 14 can be a regular matrix arrangement or other special arrangements to better form the desired multifocal plane illumination beam.

[0045] A second tube mirror 16 is disposed between the first compound eye lens 14 and the scanning module 2. The scanning module 2 includes a galvanometer 21 and a dichroic mirror 22 disposed sequentially, with the dichroic mirror 22 located between the galvanometer 21 and the second tube mirror 16.

[0046] In this embodiment, the galvanometer 21 is a high-speed galvanometer with double-sided coating. The side used to reflect the multifocal plane illumination beam is called the first side, and the opposite side is called the second side, thereby enabling rapid changes in the propagation direction of the light to scan the sample. The coating material can be a metal film or a dielectric film, etc., to improve the reflectivity and stability of the galvanometer 21.

[0047] Dichroic mirror 22 has special optical properties, which can selectively reflect or transmit light according to the wavelength of light. For example, it can transmit light for illumination beams and reflect light for emitted light. Its material and coating process will affect its optical properties.

[0048] Reference Figure 1 The objective module 3 includes a sixth tube mirror 31 and a microscope objective mirror 32 arranged sequentially. The sixth tube mirror 31 is positioned close to the galvanometer mirror 21, and a fifth tube mirror 33 is positioned between the sixth tube mirror 31 and the galvanometer mirror 21. In this embodiment, the sixth tube mirror 31 and the fifth tube mirror 33 are identical tube mirrors with the same focal length. The sixth tube mirror 31 can be a tube mirror from a microscope system adapted to the microscope objective mirror 32, and the fifth tube mirror 33 is provided in this device.

[0049] For users upgrading their systems, this device can quickly replace the corresponding fifth microscope 33 with the one that matches the user's microscope, thus meeting the needs of different users and different scenarios. The microscope objective 32 is used to focus the multifocal plane illumination beam onto the sample. Its magnification and numerical aperture parameters affect the resolution and quality of the image. For example, microscope objectives 32 with different magnifications can be selected, such as 10x, 20x, 40x, etc.

[0050] When the multifocal plane illumination beam passes through the second tube 16 and illuminates the dichroic mirror 22, the beam can then pass through the dichroic mirror 22 and enter the first surface of the galvanometer 21. The galvanometer 21 reflects the beam to the fifth tube 33, allowing it to pass sequentially through the fifth and sixth tubes 33 and enter the microscope objective 32. The beam is then focused onto the sample by the microscope objective 32, achieving multi-point illumination. The path of the multifocal plane illumination beam is defined as the illumination optical path.

[0051] When the sample is illuminated by a multifocal plane illumination beam, it produces fluorescence with a longer wavelength. The fluorescence, as emitted light, is collected by the microscope objective 32 and then passes sequentially through the sixth tube mirror 31 and the fifth tube mirror 33 back to the first surface of the galvanometer 21, where it is reflected by the galvanometer 21 to the dichroic mirror 22. The dichroic mirror 22 reflects the emitted light to the detection module 4, thus separating the emitted light from the illumination light path.

[0052] Reference Figure 1 The detection module 4 is located on the side of the scanning module 2 away from the objective lens module 3. The detection module 4 includes a first reflecting mirror 41, a second compound eye lens 42, a pinhole array 43, and a second reflecting mirror 44 arranged sequentially. The first reflecting mirror 41 is positioned close to the dichroic mirror 22, and the second reflecting mirror 44 is positioned close to the galvanometer mirror 21. The first reflecting mirror 41 and the second reflecting mirror 44 are used to change the direction of light propagation. The first reflecting mirror 41 and the second reflecting mirror 44 can be ordinary plane mirrors or mirrors with special reflective properties, such as mirrors with high reflectivity.

[0053] Reference Figure 1 A third tube mirror 45 is disposed between the first reflecting mirror 41 and the dichroic mirror 22, and a fourth tube mirror 46 is disposed between the second reflecting mirror 44 and the galvanometer 21.

[0054] Reference Figure 3 and Figure 4 The second compound eye lens 42 is spaced apart from and parallel to the pinhole array 43. The microlenses of the second compound eye lens 42 are arranged one-to-one with the pinholes of the pinhole array 43. The center of the second compound eye lens 42 is aligned with the center of the corresponding pinhole, thereby enabling confocal filtering of the emitted light to remove defocused fluorescence signals and improve the contrast and resolution of the image.

[0055] Reference Figure 1When the dichroic mirror 22 reflects the emitted light, the emitted light can pass through the third tube mirror 45 and be incident on the first reflecting mirror 41. The first reflecting mirror 41 can reflect the emitted light and cause the emitted light to be incident on the second compound eye lens 42. After being focused by the second compound eye lens 42, the emitted light can be accurately passed through the pinhole array 43 for confocal filtering, removing stray light and background noise, forming a purer signal light, improving the quality of the signal light, and laying the foundation for the image acquisition module 5 to acquire a clearer and more accurate image.

[0056] The signal light passing through the pinhole array 43 will be incident on the second reflector 44, which will reflect the signal light to the fourth tube mirror 46, and then through the fourth tube mirror 46 to the second surface of the galvanometer 21, and finally reflected by the galvanometer 21 to the image acquisition module 5.

[0057] A seventh microscope lens 51 is disposed between the image acquisition module 5 and the galvanometer 21. The fifth microscope lens 33 and the second microscope lens 16 constitute a first 4f system, and the sixth microscope lens 31 and the microscope objective lens 32 constitute a second 4f system. The third microscope lens 45 and the fifth microscope lens 33 constitute a third 4f system, and the seventh microscope lens 51 and the fourth microscope lens 46 constitute a fourth 4f system.

[0058] A 4f system is a classic relay imaging optical system, typically composed of two lenses with focal lengths of f1 and f2 (such as the tube lens in this application). Its input plane is located at the front focal plane of the first lens (focal length f1), and its output plane is located at the rear focal plane of the second lens (focal length f2). The distance between the two lenses is the sum of their focal lengths (f1 + f2). When the two lenses have equal focal lengths (both f), the total length of the system from the input plane to the output plane is 4f, hence the name 4f system. Furthermore, a 4f system can transmit the image from the input plane to the output plane without distortion at a specific magnification (M = -f2 / f1), thus avoiding image blurring or distortion.

[0059] Reference Figure 1 In this embodiment, the image acquisition module 5 uses a CCD camera to acquire and image the signal light. CCD cameras have advantages such as high sensitivity and high resolution, and their pixel count and frame rate parameters affect the quality and speed of imaging.

[0060] Furthermore, the seventh tube lens 51 is selected to match the collection target surface of the CCD camera. Different focal length tube lenses can be selected according to user needs and the size of the collection target surface of the CCD camera. In addition, the seventh tube lens 51 set in this device can be quickly replaced to match different user needs.

[0061] The implementation principle of this embodiment is as follows: When it is necessary to test the sample, the light-emitting element 11 emits a laser. After passing through the square optical fiber 15, the first objective lens 12 and the first tube lens 13, the laser is incident on the first compound eye lens 14 to form a multifocal plane illumination beam. The multifocal plane illumination beam passes through the second tube lens 16 and the dichroic mirror 22 and is incident on the first surface of the galvanometer 21. The galvanometer 21 reflects the multifocal plane illumination beam to the fifth tube lens 33. The multifocal plane illumination beam passes through the fifth tube lens 33 and the sixth tube lens 31 in sequence and is incident on the microscope objective lens 32. The microscope objective lens 32 focuses the beam onto the sample to achieve multi-point illumination.

[0062] When the sample is illuminated by a multifocal plane illumination beam, it emits light. This emitted light sequentially passes through the microscope objective 32, the sixth tube mirror 31, and the fifth tube mirror 33 before entering the first surface of the galvanometer 21. It is then reflected by the galvanometer 21 to the dichroic mirror 22, and further reflected by the dichroic mirror 22 to the third tube mirror 45. The emitted light then passes through the third tube mirror 45 and enters the first reflecting mirror 41. The first reflecting mirror 41 reflects the emitted light and directs it to the second compound eye lens 42. After being focused by the second compound eye lens 42, the emitted light can be precisely filtered through the pinhole array 43 via confocal filtering to form a purer signal light. The signal light then enters the second reflecting mirror 44, which reflects it to the fourth tube mirror 46. It then passes through the fourth tube mirror 46 and enters the second surface of the galvanometer 21. Finally, the galvanometer 21 reflects the signal light to the seventh tube mirror 51, which then enters the image acquisition module 5 to acquire an image.

[0063] This device utilizes a first compound eye lens 14 to convert a single laser beam into a multi-point light source array, achieving parallel illumination of the sample. One side of the double-sided galvanometer 21 scans this multi-point light source array, achieving rapid coverage of a large field of view. After the fluorescence signal emitted by the sample is collected, it is separated by a dichroic mirror 22 and simultaneously de-scanned by the other side of the double-sided galvanometer 21, ensuring that the fluorescence signal remains stationary in the subsequent optical path and on the camera target surface. The pinhole array 43, acting as a multi-channel confocal pinhole, effectively filters out background noise. The camera continuously acquires a series of sub-images during the scanning process. Finally, a dedicated image processing algorithm stitches and reconstructs these sub-images, and can utilize the information obtained from the secondary scan for pixel rearrangement and other super-resolution algorithms, ultimately generating a fluorescence image with a large field of view, high speed, high signal-to-noise ratio, and high resolution, achieving twice the resolution of traditional wide-field imaging.

[0064] This device utilizes the principle of image scanning microscopy. Specifically, traditional confocal microscopes use point detectors, recording only one total light intensity value at each scanning point. In contrast, this application uses a CCD camera. At each scanning position of the galvanometer 21, the CCD camera not only records the total light intensity passing through the pinhole array 43, but also records a complete image of the emission point spread function (PSF) corresponding to each focal point. Due to the descanning effect of the double-sided galvanometer 21, these PSF images are stationary on the camera target surface.

[0065] Image processing algorithms utilize this additional spatial information. For a given scan point, if the luminescent point on the sample is exactly at the center of the excitation spot, its emitted light PSF image will also be imaged on the camera at the center position; if the luminescent point deviates from the center of the excitation spot, its PSF image will also be shifted on the camera accordingly. The algorithm can analyze the centroid or peak position of the PSF image corresponding to each scan point. This positional information reveals more detailed sample structural information than the size of the excitation spot itself. Through a "pixel rearrangement" algorithm, the light intensity value recorded by each camera pixel is redistributed to a more precise position in the final image (usually a ratio of the vector sum of the scan point position and the offset position reflected by that pixel). This is equivalent to synthesizing an effective detection area much smaller than the physical size. This process significantly reduces the total point spread function (PSF) of the system, and its theoretical limit can reduce the effective PSF size of the system by half, thereby increasing the lateral resolution to twice that of traditional wide-field or confocal microscopy.

[0066] Finally, by performing the above pixel rearrangement process on a series of sub-images continuously acquired by the camera, and then stitching and reconstructing the processed sub-images, a fluorescence image with a large field of view, high speed, high signal-to-noise ratio, and ultra-high resolution can be generated.

[0067] It should be noted that image processing algorithms are conventional techniques in this field, and therefore will not be described in detail in the embodiments of this application.

[0068] Example 2: Refer to Figure 5 The difference between this embodiment and embodiment 1 is that the detection module 4 also includes an adjustable aperture layer 47.

[0069] Reference Figure 6 and Figure 7 An adjustable aperture layer 47 is disposed between the second compound eye lens 42 and the pinhole array 43. The adjustable aperture layer 47 includes a transparent substrate 471 and a plurality of liquid crystal cells 472 disposed on the transparent substrate 471. Each liquid crystal cell 472 corresponds one-to-one with a microlens of the second compound eye lens 42. In this embodiment, the liquid crystal cells 472 are made of high-speed nematic liquid crystal or polymer dispersed liquid crystal (PDLC) to achieve high contrast and fast response.

[0070] The liquid crystal cell 472 is provided with an electrode 473 for changing the light transmittance of its central area. The electrode 473 in each liquid crystal cell 472 is connected to an external independent control circuit. The control circuit adopts a matrix addressing method and can independently control each liquid crystal cell 472.

[0071] In this embodiment, electrode 473 is a ring electrode. By controlling the voltage applied to electrode 473, the size of the central transparent area of ​​liquid crystal cell 472 can be adjusted, thereby achieving aperture adjustment of the small hole.

[0072] The implementation principle of Example 2 is as follows: When no voltage is applied to electrode 473, the liquid crystal molecules in the central region surrounded by electrode 473 are in a specific orientation (e.g., a scattering state) and are opaque to light. When a voltage is applied, the electric field causes the liquid crystal molecules in the central region to deflect their orientation, becoming transparent, thereby forming an optical "pinhole". By precisely controlling the magnitude of the applied voltage, the diameter of the central transparent region can be changed, thus achieving continuous adjustment of the "pinhole" aperture size.

[0073] The adjustable aperture layer 47 allows users to quickly and in real-time adjust the size of the confocal aperture via software control, according to experimental needs. For example, when observing samples with weak fluorescence signals, the aperture diameter can be increased to collect more photons and improve the signal-to-noise ratio; when extreme resolution is required, the aperture can be decreased to suppress out-of-focus light to the greatest extent. This electronic adjustment method avoids mechanical moving parts, has a fast response speed, and high reliability, greatly improving the performance and ease of use of the device.

[0074] The above are all preferred embodiments of this application, and are not intended to limit the scope of protection of this application. Therefore, all equivalent changes made in accordance with the structure, shape and principle of this application should be covered within the scope of protection of this application.

Claims

1. A high-throughput fluorescence detection device based on galvanometer scanning, characterized in that, include: The system includes an illumination module (1), a scanning module (2), an objective lens module (3), a detection module (4), and an image acquisition module (5). The illumination module (1) is used to generate a multifocal plane illumination beam. The scanning module (2) includes a galvanometer (21) and a dichroic mirror (22) arranged in sequence, with the dichroic mirror (22) located between the galvanometer (21) and the illumination module (1); The multifocal plane illumination beam can be incident on the first surface of the galvanometer (21) through the dichroic mirror (22), and the galvanometer (21) can reflect the multifocal plane illumination beam to the objective lens module (3) so that the multifocal plane illumination beam is focused on the sample through the objective lens module (3) to form multi-point illumination; The emitted light from the sample is collected by the objective lens module (3), incident on the first surface of the galvanometer (21), and reflected to the dichroic mirror (22). The dichroic mirror (22) can reflect the emitted light to the detection module (4). The signal light is incident on the second surface of the galvanometer (21) and reflected to the image acquisition module (5). The image acquisition module (5) is used to acquire and image the signal light.

2. The high-throughput fluorescence detection device based on galvanometer scanning according to claim 1, characterized in that: The illumination module (1) includes a light-emitting element (11), a first objective lens (12), a first tube lens (13), and a first compound eye lens (14) arranged in sequence. The first compound eye lens (14) is arranged close to the dichroic mirror (22). The light emitted by the light-emitting element (11) passes through the first objective lens (12) and the first tube lens (13) and then enters the first compound eye lens (14) to form the multifocal plane illumination beam.

3. The high-throughput fluorescence detection device based on galvanometer scanning according to claim 2, characterized in that: The illumination module (1) includes a square optical fiber (15), which is connected to the light-emitting element (11) and located between the light-emitting element (11) and the first objective lens (12).

4. The high-throughput fluorescence detection device based on galvanometer scanning according to claim 2, characterized in that: A second tube mirror (16) is provided between the first compound eye lens (14) and the dichroic mirror (22), a fifth tube mirror (33) is provided between the objective lens module (3) and the galvanometer (21), a third tube mirror (45) is provided between the detection module (4) and the dichroic mirror (22), and a fourth tube mirror (46) is provided between the detection module (4) and the galvanometer (21). The third tube mirror (45) is used to receive the emitted light and cause the emitted light to be incident on the detection module (4). The fourth tube mirror (46) is used to receive the signal light and cause the signal light to be incident on the second surface of the galvanometer (21). A seventh tube mirror (51) is provided between the image acquisition module (5) and the galvanometer (21).

5. The high-throughput fluorescence detection device based on galvanometer scanning according to claim 4, characterized in that: The objective module (3) includes a sixth tube (31) and a microscope objective (32) arranged in sequence. The sixth tube (31) is arranged close to the fifth tube (33). The fifth tube (33) and the second tube (16) form a first 4f system. The sixth tube (31) and the microscope objective (32) form a second 4f system. The third tube (45) and the fifth tube (33) form a third 4f system. The seventh tube (51) and the fourth tube (46) form a fourth 4f system.

6. The high-throughput fluorescence detection device based on galvanometer scanning according to claim 4, characterized in that: The fifth endoscope (33) and the seventh endoscope (51) are respectively configured as replaceable structures.

7. The high-throughput fluorescence detection device based on galvanometer scanning according to claim 1, characterized in that: The detection module (4) is located on the side of the scanning module (2) away from the objective lens module (3). The detection module (4) includes a first reflecting mirror (41), a second compound eye lens (42), a pinhole array (43) and a second reflecting mirror (44) arranged in sequence. The first reflecting mirror (41) is arranged close to the dichroic mirror (22), and the second reflecting mirror (44) is arranged close to the galvanometer (21). The microlenses of the second compound eye lens (42) are arranged in a one-to-one correspondence with the pinholes of the pinhole array (43), and the center of the second compound eye lens (42) is aligned with the center of the corresponding pinhole.

8. The high-throughput fluorescence detection device based on galvanometer scanning according to claim 7, characterized in that: The detection module (4) includes an adjustable aperture layer (47), which is disposed between the second compound eye lens (42) and the pinhole array (43). The adjustable aperture layer (47) is used to adjust the aperture of the pinhole.

9. The high-throughput fluorescence detection device based on galvanometer scanning according to claim 8, characterized in that: The adjustable aperture layer (47) includes a transparent substrate (471) and a plurality of liquid crystal cells (472) disposed on the transparent substrate (471). Each liquid crystal cell (472) corresponds one-to-one with a microlens of the second compound eye lens (42). An electrode (473) is disposed in the liquid crystal cell (472) for changing the light transmittance of its central region. By controlling the voltage applied to the electrode (473), the size of the central transparent region of the liquid crystal cell (472) is adjusted to achieve aperture adjustment of the small hole.

10. The high-throughput fluorescence detection device based on galvanometer scanning according to claim 9, characterized in that: The electrode (473) is a ring electrode (473), and each liquid crystal cell (472) is connected to an independent control circuit. The control circuit uses a matrix addressing method to control each liquid crystal cell (472).

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