An interference scattering image-based drift correction system and its control system

Abstract

The embodiments of the present application relate to the field of microscopic imaging technology, and in particular to a drift correction system based on interference scattering images and its control system. The system provided by the present application has a simple structure and uses only one laser beam to achieve drift correction in the XYZ three-dimensional directions. The system uses focus locking to actively lock the Z-direction drift in real time, reducing the complexity of evaluating the Z-direction drift of the sample. The system allows XY direction drift and realizes three-dimensional drift correction based on objective lens illumination combined with interference scattering imaging and post-processing. This can effectively avoid problems such as vibration introduced when switching equipment and compensation error of the translation stage, thereby improving the drift correction effect. In addition, the system is easy to build and can be assembled into existing imaging systems as a plug-in, with good compatibility.

Description

Technical Field

[0001] Embodiments of the present application relate to the field of microscopy imaging technology, and in particular, to a drift correction system based on interference scattering images and its control system. Background Art

[0002] Microscopy imaging technology, especially super-resolution microscopy imaging technology, is very sensitive to the position information of the sample. During the data acquisition process, the sample will inevitably drift due to mechanical vibration, temperature change, etc., resulting in blurred artifacts in the reconstructed image and significantly reducing the final imaging quality. Summary of the Invention

[0003] The following is an overview of the subject matter described in detail herein. This overview is not intended to limit the scope of protection of the claims.

[0004] The main purpose of the embodiments of the present disclosure is to propose a drift correction system based on interference scattering images and its control system, which uses focus locking to actively and real-time lock the Z-direction drift, reduces the complexity of evaluating the Z-direction drift amount of the sample, allows the XY-direction drift, and realizes three-dimensional drift correction based on objective lens illumination combined with interference scattering imaging and then post-processing. It can effectively avoid problems such as vibration introduced when switching devices and displacement stage compensation errors, and improve the drift correction effect.

[0005] To achieve the above object, a first aspect of the embodiments of the present disclosure proposes a drift correction system based on interference scattering images, the system includes:

[0006] A first laser module for generating a first laser;

[0007] A first acquisition module for collecting fluorescence reflected from the objective lens to obtain a first image; the fluorescence is generated by the first laser irradiating the sample on the sample slide through the objective lens and collected by the objective lens;

[0008] A second laser module for generating a second laser;

[0009] An objective lens illumination module for focusing the second laser to the rear focal plane of the objective lens at a set focal length, so that the second laser irradiates the sample slide in an approximately parallel light manner to obtain the reflected light and illumination light generated by the sample slide, as well as the first scattered light generated by the illumination light irradiating the surface of the slide, the second scattered light generated by the illumination light irradiating the sample, and the interference signal generated after the interference of the first scattered light and the second scattered light;

[0010] A photoelectric position detector for detecting the reflected light and outputting a corresponding voltage according to the reflected light;

[0011] An axial nano-displacement stage, sleeved on the objective lens, is used to lock the axial distance between the objective lens and the sample slide according to the voltage.

[0012] A second acquisition module is used to acquire the first scattered light, the second scattered light and the interference signal to obtain a second image.

[0013] A drift correction module is used to calculate the drift amount of the sample according to the first image, and reconstruct a super-resolution image after drift correction according to the drift amount and the first image.

[0014] In some embodiments, the second laser module includes a laser, an optical fiber coupler and a polarization-maintaining fiber jumper. The laser is used to generate linearly polarized laser, the optical fiber coupler is used to couple the linearly polarized laser into the polarization-maintaining fiber jumper, and the polarization-maintaining fiber jumper generates the second laser.

[0015] In some embodiments, the objective lens illumination module includes a first doublet lens, a polarization beam splitter prism and a rotatable quarter-wave plate. The second laser sequentially passes through the first doublet lens, the polarization beam splitter prism and the rotatable quarter-wave plate and is focused on the rear focal plane of the objective lens.

[0016] In some embodiments, the high-precision drift correction system based on the interference scattering image further includes a diaphragm stop and a second doublet lens. The first scattered light, the second scattered light and the interference signal are sequentially transmitted through the diaphragm stop and the second doublet lens to the second acquisition module.

[0017] In some embodiments, the first scattered light, the second scattered light and the interference signal also pass through the rotatable quarter-wave plate and the polarization beam splitter prism before passing through the diaphragm stop.

[0018] In some embodiments, the drift correction system based on the interference scattering image further includes a first dichroic mirror, a first reflector, a second dichroic mirror, a filter wheel, a microscope tube lens and a plano-convex cylindrical lens. The fluorescence sequentially passes through the first dichroic mirror, the reflector, the second dichroic mirror, the filter wheel, the microscope tube lens and the plano-convex cylindrical lens from the objective lens to the first acquisition module.

[0019] In some embodiments, both the first acquisition module and the second acquisition module are cameras.

[0020] In some embodiments, the first acquisition module and the second acquisition module acquire images synchronously or with frequency division.

[0021] In some embodiments, the drift correction module calculates the drift amount of the sample using a cross-correlation algorithm.

[0022] To achieve the above-mentioned objectives, the second aspect of the embodiment of the present disclosure proposes a Micro-FPGA-based control system, in which the Micro-FPGA-based control system integrates the drift correction system based on the interferometric scattering image described in the first aspect. The Micro-FPGA-based control system controls the drift correction system based on the interferometric scattering image according to the EMU plug-in of the microscope control software Micro-Manager.

[0023] In its first aspect, the present application provides a drift correction system based on interferometric scattering images. This system has a simple structure, utilizing only a single laser beam to achieve drift correction in the X, Y, and Z directions. It is easy to build and can be installed as a plug-in into existing imaging systems, offering excellent compatibility. Furthermore, the system uses focus lock to actively lock Z-direction drift in real time, reducing the complexity of assessing sample Z-direction drift. While allowing X, Y directions to drift, the system achieves three-dimensional drift correction based on objective-lens illumination combined with interferometric scattering imaging and post-processing. This effectively avoids issues such as vibration introduced during device switching and stage compensation errors, while also improving the drift correction effect.

[0024] The second aspect of the present application provides a control system based on Micro-FPGA, which can realize automatic data acquisition and automatic reconstruction of super-resolution images. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following briefly introduces the drawings required for use in the embodiments or related technical descriptions. Obviously, the drawings described below are only some embodiments of the embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0026] Figure 1 This is a schematic structural diagram of a drift correction system based on interferometric scattering images provided in one embodiment of the present application;

[0027] Figure 2 is a schematic diagram of interference between first scattered light and second scattered light provided by one embodiment of the present application;

[0028] Figure 3 is an image captured by a second acquisition module provided in one embodiment of the present application;

[0029] Figure 4 is a schematic diagram of an electronic unit Micro-FPGA provided by one embodiment of the present application;

[0030] Figure 1 Label introduction in

[0031] 101. First acquisition module; 102. Second acquisition module; 103. Quadrant photodiode array; 104. Objective lens; 1041. Rear focal plane; 105. Sample slide; 106. Axial nano-displacement stage; 107. First laser module; 108. 785nm laser; 109. Fiber optic coupler; 110. Polarization-maintaining fiber jumper; 201. First dichroic mirror; 202. Second dichroic mirror; 203. First reflector; 204. Second reflector; 205. Third reflector; 206. First doublet lens; 207. Second doublet lens; 301. Polarizing beam splitter prism; 302. Quarter-wave plate; 303. Diaphragm stop; 304. Filter wheel; 305. Microscope tube lens; 306. Plano-convex cylindrical lens. Specific implementation mode

[0032] In order to make the objectives, technical solutions and advantages of the present application clearer, the present application will be further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0033] It should be noted that although the functional modules are divided in the device schematic diagram and the logical order is shown in the flowchart, in some cases, the steps shown or described can be executed in a different order from the module division in the device or the flowchart. Terms such as "first" and "second" in the specification, claims and the above-mentioned drawings are used to distinguish similar objects and do not necessarily need to describe a specific order or sequence.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which this application belongs. The terms used herein are only for the purpose of describing the embodiments of this application and are not intended to limit this application.

[0035] First, introduce the technical names and components used in this application:

[0036] (1) Based on the objective lens-based illumination method, the laser beam is first expanded by a certain magnification beam expander system, and then focused onto the rear focal plane of a high-power objective lens by a focusing lens with an appropriate focal length, and finally hits the sample in the form of approximately parallel light. An obvious advantage of this illumination method is that different illumination methods can be achieved by adjusting the "focusing spot" to different positions on the objective lens, such as total internal reflection illumination, high-incidence angle illumination, and epi-illumination, and these three illumination methods can be conveniently switched according to the needs of the experiment.

[0037] (2) Interference scattering imaging technology: The illumination light illuminates the sample. The scattered light is generated at the glass slide surface and the sample by the illumination light. The scattered light generated at both places will also interfere with each other. Imaging applications are carried out using the collected interference and scattering signals.

[0038] (3) Active focusing system, for reference: TIRF Lock TM |Keeps the sample in the focal plane and maintains TIRF signal (madcitylabs.com), Piezo Focus Motor Drives / Nano-focus Systems|Voice Coil, Stage&Controller|PIFOC.

[0039] (4) Cross-correlation image registration algorithm (cross-correlation algorithm), for reference: Efficient subpixel image registration by cross-correlation - File Exchange - MATLAB Central (mathworks.cn).

[0040] (5) Micro-Manager, an open-source microscope hardware control and automation software, see https: / / micro-manager.org / .

[0041] Microscopic imaging technology, especially super-resolution microscopy, is a powerful imaging technology. This application takes single-molecule localization microscopy as an example for illustration. Single-molecule localization microscopy is one of the widely used super-resolution imaging technologies at present. Its principle is to collect fluorescent signals that sparsely flicker in space and time for high-precision spatial localization, and then reconstruct multiple frames of images collected over a long time into a single super-resolution image. This technology can break through the optical diffraction limit to observe subcellular structures at the nanoscale.

[0042] Single-molecule localization microscopy is very sensitive to the position information of the sample. During the data acquisition process, the sample will inevitably drift due to reasons such as mechanical vibration and temperature change, resulting in blurred artifacts in the reconstructed image and significantly reducing the final imaging quality.

[0043] Drift evaluation methods can generally be divided into two categories. The first category is marked evaluation. This type of method is completed by introducing reference marks (such as gold nanoparticles, polystyrene microspheres, bright fluorophores, etc.) into the sample. Thanks to the stability and brightness of the reference marks, they are easily tracked and recorded during the imaging process. However, introducing markers into the sample not only limits the imaging area but may also reduce the positioning accuracy of nearby probe molecules due to the high brightness of the markers. The second category is markerless evaluation, which has the unique advantage of not requiring additional marking compared to the first type of method. It mainly utilizes the biological characteristics of the sample itself, such as bright-field patterns, speckle patterns, diffraction patterns, differential phase contrast patterns, etc., and then calculates the drift amount of the sample from images taken at different times through corresponding algorithms.

[0044] Drift compensation methods can also generally be divided into two types. The first is real-time compensation, that is, while collecting data, the drift amount is calculated using an algorithm, and then the nano-displacement stage is moved in real time to compensate for the drift amount. Obviously, adopting this scheme will increase the complexity of the imaging system. The second is post-processing. This scheme does not require complex control of the optical system. During the imaging process, the sample is allowed to drift freely, and only when reconstructing the super-resolution fluorescence image, the calculated drift amount is compensated into the original single-molecule point data to obtain more accurate fluorescence point coordinates, thereby improving the resolution of the reconstructed image.

[0045] This application utilizes the principle of focus locking technology to achieve axial drift locking of the sample through a laser beam. For lateral movement, a fluorescence microscope based on objective illumination is combined with interference scattering imaging technology to complete lateral drift evaluation through the same laser beam, and then the cross-correlation algorithm is used to perform XY-direction drift compensation through post-processing. It has wide applicability, and the three-dimensional drift correction function can be achieved by simply modifying the commonly used single-molecule localization microscope in the laboratory.

[0046] Referring to Figure 1 , an embodiment of this application provides a drift correction system based on interference scattering images, and this system includes the following devices:

[0047] The first laser module 107 is used to generate the first laser. The role of the first laser is to obtain a fluorescence image. The type of this laser and the specific model of the first laser module 107 are not limited in the embodiments of this application.

[0048] The first acquisition module 101 is used to collect the fluorescence reflected from the objective lens 104 to obtain the first image; the fluorescence is generated by the first laser passing through the objective lens 104 and irradiating the sample on the sample slide 105 and collected by the objective lens 104.

[0049] In some embodiments of this application, the first acquisition module 101 is an sCMOS camera, and the sCMOS camera is used to capture fluorescence images.

[0050] In some embodiments of the present application, the specific optical paths of the first laser and the fluorescence in the system include:

[0051] The first laser module 107 generates the first laser. The first laser is reflected by the first dichroic mirror 201 and focused on the rear focal plane 1041 of the objective lens 104 to illuminate the imaging sample from the rear. The sample generates fluorescence, which is collected by the objective lens 104. The fluorescence passes through the first dichroic mirror 201, the first mirror 203, the second dichroic mirror 202, the filter wheel 304 (for filtering stray light such as residual laser), the microscope tube lens 305, and the plano-convex cylindrical lens 306 until it is captured by the sCMOS camera. It should be noted that devices such as the first dichroic mirror 201 and the first mirror 203 can be arranged in the system. Since these devices are common in the field, they will not be described in detail here.

[0052] A second laser module, for generating a second laser.

[0053] In some embodiments of the present application, the second laser module includes a 785nm laser 108, an optical fiber coupler 109, and a polarization-maintaining fiber jumper 110. The 785nm laser 108 is used to generate linearly polarized laser. The optical fiber coupler 109 is used to couple the linearly polarized laser into the polarization-maintaining fiber jumper 110. The polarization-maintaining fiber jumper 110 generates the second laser. The polarization-maintaining fiber jumper 110 can be rotated as a whole to adjust the linear polarization direction of the laser. For example, the polarization-maintaining fiber jumper 110 is integrally installed on a mirror mount that can be adjusted in pitch and deflection to control the position of the emitted laser in the optical path. It should be noted that here only the common model of the 785nm laser is taken as an example, and it cannot be used as a limitation of all optional types of lasers in the present application.

[0054] The objective lens 104 type illumination module is used to focus the second laser at a set focal length on the rear focal plane 1041 of the objective lens 104, so that the second laser irradiates the sample slide 105 in an approximately parallel light manner, in order to obtain the reflected light and illumination light generated by the sample slide 105 of the sample slide, as well as the first scattered light generated by the illumination light irradiating the surface of the slide, the second scattered light generated by the illumination light irradiating the sample, and the interference signal generated after the first scattered light and the second scattered light interfere.

[0055] A photoelectric position detector, for detecting the reflected light and outputting a corresponding voltage according to the reflected light.

[0056] In some embodiments of the present application, taking the quadrant photodiode array 103 as an example of the photoelectric position detector, the quadrant photodiode array 103 is equipped with a current-voltage amplifier, which can provide differential signals of subtracting the top from the bottom and the left from the right of the array. The quadrant photodiode array 103 also provides a sum signal of the outputs of the four quadrants of the array. The differential signal is a voltage analog signal of the difference in light intensity sensed by paired photodiode elements in the array.

[0057] The axial nano-displacement stage 106 is sleeved on the objective lens 104, and the axial nano-displacement stage 106 is used to lock the axial distance between the objective lens 104 and the sample slide 105 according to the voltage.

[0058] In some embodiments of the present application, since the voltage output by the photoelectric position detector is correlated with the distance between the objective lens 104 and the slide, the voltage output by the photoelectric position detector can be used to accurately locate the distance between the objective lens 104 and the slide. Then, the axial nano-displacement stage 106 can lock the distance between the objective lens 104 and the slide in real time, avoiding errors caused by mechanical vibrations of the sample.

[0059] The second acquisition module 102 is used to acquire the first scattered light, the second scattered light, and the interference signal to obtain a second image.

[0060] In some embodiments of the present application, the optical path of the second laser in the system includes:

[0061] The second laser passes through the first doublet lens 206, the third mirror 205, the polarization beam splitter prism 301, and the rotatable quarter-wave plate 302 and is focused on the rear focal plane 1041 of the objective lens 104. Among them, the first doublet lens 206 can be integrally installed on a mirror mount with adjustable pitch and deflection, and the linear polarization direction of the laser can be adjusted by overall rotation in combination with the polarization-maintaining fiber jumper 110. The polarization beam splitter prism 301 and the rotatable quarter-wave plate 302 can improve the utilization rate of the 785 illumination light. It should be noted that devices such as the first doublet lens 206, the third mirror 205, the polarization beam splitter prism 301, and the rotatable quarter-wave plate 302 can all be set in the system. Since these devices are common devices in the field, they will not be described in detail here.

[0062] In some embodiments of the present application, the optical path of the reflected light in the system includes:

[0063] The reflected light passes through the first mirror 203, the second dichroic mirror 202, the rotatable quarter-wave plate 302, the polarization beam splitter prism 301, and the second mirror 204 to the photoelectric position detector. The polarization beam splitter prism 301 and the rotatable quarter-wave plate 302 can be used to adjust the reflected light.

[0064] In some embodiments of the present application, the optical paths of the first scattered light, the second scattered light, and the interference signal in the system include:

[0065] The scattered light and the interference light are captured by the second acquisition module 102 after passing through the first mirror 203, the second dichroic mirror 202, the rotatable quarter-wave plate 302, the polarization beam splitter prism 301, the aperture stop 303 (for filtering reflected light and stray light), and the second doublet lens 207.

[0066] A drift correction module is used to calculate the drift amount of the sample according to the first image, and to reconstruct the super-resolution image after drift correction according to the drift amount and the first image. The drift correction module calculates the drift amount of the sample according to multiple frames of the second images, and also uses the first image itself for drift evaluation (there are corresponding evaluation algorithms in the art). Finally, the two are compared to eliminate some abnormal data to prevent the two acquisition optical paths from collecting the same sample asynchronously during the acquisition process.

[0067] The present system mainly realizes the following functions:

[0068] (1) Adopting a focus locking technique to achieve axial drift locking of the sample through a beam of laser. The implementation process is as follows:

[0069] A second laser is generated by the second laser module, and the second laser is focused onto the rear focal plane 1041 of the objective lens 104 with a set focal length through the objective lens illumination module, so that the second laser irradiates the sample slide 105 in an approximately parallel light manner, and the reflected light generated by the slide can be obtained. The reflected light mainly serves to achieve locking in the axial direction (Z-axis direction).

[0070] According to the focus locking principle, the reflected light beam of the slide is detected by the photoelectric position detector. The output voltage of the photoelectric position detector changes with the distance between the objective lens 104 and the slide. Then, the distance between the objective lens 104 and the slide can be accurately positioned according to the output voltage of the photoelectric position detector. Then, the axial nano-displacement stage 106 sleeved on the objective lens 104 can accurately control and lock the axial distance between the objective lens 104 and the slide according to the output voltage of the photoelectric position detector. It should be noted that the axial nano-displacement stage 106 (usually the Z-axis nano-displacement stage) is well known in the art and will not be described in detail here.

[0071] (2) Through the same beam of laser, drift correction of the sample is achieved by using objective lens-based illumination combined with interference scattering imaging technology. The implementation process is as follows:

[0072] The second laser is focused to the rear focal plane 1041 of the objective lens 104 at a set focal length, so that the second laser irradiates the sample slide 105 in an approximately parallel light manner, to obtain the first scattered light (the scattered light generated by the illumination light irradiating the surface of the slide), the second scattered light (the scattered light generated by the illumination light irradiating the sample), and the interference signal (the signal generated after the first scattered light and the second scattered light interfere) generated by the sample slide 105.

[0073] After the second acquisition module 102 acquires the second image containing the first scattered light, the second scattered light, and the interference signal, it calculates the drift amount of the sample according to the second image. Furthermore, the drift amount of the sample is compensated into the original fluorescence single-molecule dot data to reconstruct the drift-corrected super-resolution image.

[0074] Compared with the traditional technology, this system realizes the drift lock in the axial direction (Z direction) through focus locking. For the lateral movement, the drift evaluation is completed by using objective lens-based illumination combined with interferometric scattering imaging, and then the cross-correlation algorithm is used to perform lateral (XY direction) drift compensation through post-processing. This system has the following advantages:

[0075] (1) The structure of this system is simple. Only one laser beam (the second laser) is used to realize the drift correction in the XYZ three-dimensional directions, which is easy to build and can be assembled into the existing imaging system in the form of a plug-in, with good compatibility.

[0076] (2) This system uses focus locking to actively and real-time lock the Z-direction drift, reducing the complexity of evaluating the Z-direction drift amount of the sample; in addition, allowing the XY-direction drift, based on the objective lens-based illumination combined with interferometric scattering imaging, and then using the post-processing scheme to realize the three-dimensional drift correction, can effectively avoid problems such as vibration introduced when switching devices and displacement stage compensation errors, and improve the drift correction effect.

[0077] (3) This system integrates the software and hardware and the subsequent data processing process at the same time.

[0078] Refer to Figures 1 to 4 , an embodiment of the present application provides a drift correction method corresponding to a drift correction system based on an interferometric scattering image:

[0079] (1) Super-resolution fluorescence image acquisition;

[0080] The first laser module 107 generates the first laser, which is reflected by the first dichroic mirror 201, then focused on the rear focal plane 1041 of the objective lens 104, and finally illuminates the imaging sample. The fluorescence generated by the sample is collected by the objective lens 104, then the fluorescence passes through the first dichroic mirror 201, is reflected by the first mirror 203 and then passes through the second dichroic mirror 202, and then the residual laser and other stray light are filtered by the emission filter mounted on the filter wheel 304. After the fluorescence is collected by the microscope tube lens 305, it is finally captured by the first acquisition module 101 (sCMOS camera). In addition, a quick-disconnect plano-convex cylindrical lens 306 is placed in front of the first acquisition module 101 for three-dimensional super-resolution imaging based on astigmatism.

[0081] (2) Control and locking of the axial distance between the objective lens 104 and the glass slide;

[0082] The 785nm laser 108 emits linearly polarized laser, which is first coupled into the polarization-maintaining fiber pigtail 110 by the fiber coupler 109, and then the second laser emitted from the polarization-maintaining fiber pigtail 110 adjusts the focusing position through the movable first doublet lens 206. The polarization-maintaining fiber pigtail 110 and the first doublet lens 206 can be rotated as a whole to adjust the linear polarization direction of the second laser. At the same time, the polarization-maintaining fiber pigtail 110 and the first doublet lens 206 are integrally mounted on a frame with adjustable pitch and deflection to control the position of the emitted laser in the optical path.

[0083] In this process, the second laser passes through the polarization beam splitter prism 301, the quarter-wave plate 302, the second dichroic mirror 202, the first mirror 203 and the first dichroic mirror 201 in sequence, and then is focused on the same rear focal plane 1041 of the objective lens 104, and finally shines on the sample glass slide 105 in an approximately parallel light manner. At this time, this beam of second laser will generate reflected light and illumination light on the surface of the glass slide. The illumination light generates the first scattered light on the surface of the glass slide and the second scattered light at the sample. Refer to Figure 2 , and the scattered light generated at both places will also interfere to obtain an interference signal.

[0084] The reflected light of the glass slide is detected by the quadrant photodiode array 103 after passing through the polarization beam splitter prism 301 and the second mirror 204. Its position-related output voltage changes with the change of the distance between the objective lens 104 and the glass slide, and the array output voltage is fed back to the axial nano-displacement stage 106 sleeved on the objective lens 104. The up-and-down relative movement between the objective lens 104 and the glass slide will cause the reflected light spot to move to different positions such as Z-1Z0 Z1, and finally the position of the light spot on the quadrant photodiode array 103 also moves accordingly. With the positioning ability of the axial nano-displacement stage 106, the axial distance between the objective lens 104 and the glass slide can be accurately controlled and locked.

[0085] (3) Three-dimensional drift correction;

[0086] The combination of the polarization beam splitter prism 301 and the rotatable quarter-wave plate 302 is used to adjust the signal light (the first scattered light, the second scattered light, and the interference signal) and the reflected light, and to improve the utilization rate of the illumination light (whether the linearly polarized light passes through or is reflected in the polarization beam splitter prism 301 is related to whether the linearly polarized state of the incident light is P polarization or S polarization. Light with one polarization direction, such as P polarization, will become circularly polarized light after passing through the quarter-wave plate 302 once. The circularly polarized light returned from the sample end will become linearly polarized light again after passing through the quarter-wave plate 302, but it turns 90 degrees relative to the incident direction and becomes S polarization).

[0087] After the first scattered light, the second scattered light, and the interference signal pass through the rotatable quarter-wave plate 302 and the polarization beam splitter prism 301, they also pass through the aperture stop 303 and the second doublet lens 207 and are then captured by the second acquisition module 102 (camera). Refer to Figure 1 . The aperture stop 303 is used to filter out the reflected light and stray light.

[0088] The drift correction module calculates the drift amount of the sample based on multiple frames of the second image collected by the first acquisition module 101, and then reconstructs the super-resolution image after drift correction according to the drift amount of the sample and the first image.

[0089] This system has the following advantages:

[0090] (1) The structure of this system is simple. It uses only one laser beam (the second laser) to achieve drift correction in the XYZ three-dimensional directions, is easy to build, and can be assembled into an existing imaging system in the form of a plug-in, with good compatibility.

[0091] (2) This system uses focus locking to actively and real-time lock the Z-direction drift, reducing the complexity of locking the sample in the Z direction; in addition, allowing the XY-direction drift, based on the objective lens type illumination combined with interference scattering imaging, and then using a post-processing scheme to achieve three-dimensional drift correction, can effectively avoid problems such as vibration introduced when switching equipment and displacement stage compensation errors, and improve the drift correction effect.

[0092] (3) This system integrates software and hardware and the subsequent data processing process at the same time.

[0093] In some embodiments of this application, this drift correction system based on the interference scattering image can be controlled by a customized EMU plug-in integrated into the microscope control software Micro-Manager. Refer to Figure 4, under the control of the electronic unit Micro-FPGA, automatic data acquisition can be achieved, and operations such as exposure synchronization or frequency division, excitation light synchronization, or sequential emission can be performed between the first acquisition module 101 and the second acquisition module 102 (two cameras). In addition, the analog signals (such as voltage) generated by the device (such as the quadrant photodiode array 103) are converted into digital signals for computer display for real-time monitoring. Usually, the first acquisition module 101 obtains 50,000 - 100,000 frames of images with an exposure time of 15 ms for super-resolution image reconstruction; the second acquisition module 102 obtains 12,500 - 50,000 frames of images for lateral drift evaluation. Under the control of the electronic unit Micro-FPGA, the second image acquired synchronously or with frequency division with the first image can be used to obtain the drift amount of the sample using the cross-correlation algorithm. Based on the calculated drift amount, it can be compensated into the original fluorescence single-molecule point data through the SMAP software, and then a super-resolution image corrected for drift can be reconstructed. Among them, the relevant content of the SMAP software can be found in detail in the literature Ries, J. (2020). SMAP: a modular super-resolution microscopy analysis platform for SMLM data. Nature Methods, 17(9), 870 - 872. https: / / doi.org / 10.1038 / s41592-020-0938-1 , which will not be elaborated here..

[0094] The above is a specific description of the preferred implementation of the embodiments of the present application. However, the embodiments of the present application are not limited to the above implementation manners. Those skilled in the art can make various equivalent deformations or substitutions without departing from the spirit of the embodiments of the present application, and these equivalent deformations or substitutions are all included within the scope defined by the claims of the embodiments of the present application.

Claims

1. An interference scattering image-based drift correction system, characterized in that The high-precision drift correction system based on interference scattering images includes: A first laser module for generating a first laser; A first acquisition module for acquiring the fluorescence reflected from the objective lens to obtain a first image; the fluorescence is generated by the first laser irradiating the sample on the sample slide through the objective lens and collected by the objective lens; A second laser module for generating a second laser; An objective lens illumination module for focusing the second laser to the rear focal plane of the objective lens at a set focal length, so that the second laser irradiates the sample slide in an approximately parallel light manner to obtain the reflected light and illumination light generated by the sample slide, the first scattered light generated by the illumination light irradiating the surface of the slide, the second scattered light generated by the illumination light irradiating the sample, and the interference signal generated after the first scattered light and the second scattered light interfere; A photoelectric position detector for detecting the reflected light and outputting a corresponding voltage according to the reflected light; An axial nano-displacement stage sleeved on the objective lens, and the axial nano-displacement stage is used to lock the axial distance between the objective lens and the sample slide according to the voltage; A second acquisition module for acquiring the first scattered light, the second scattered light and the interference signal to obtain a second image; A drift correction module for calculating the drift amount of the sample according to the second image, and reconstructing a super-resolution image after drift correction according to the drift amount and the first image.

2. The drift correction system based on the interference scattering image according to claim 1, wherein The second laser module includes a laser, an optical fiber coupler and a polarization-maintaining fiber jumper. The laser is used to generate linearly polarized laser light. The optical fiber coupler is used to couple the linearly polarized laser light into the polarization-maintaining fiber jumper, and the polarization-maintaining fiber jumper generates the second laser.

3. The drift correction system based on an interference scattering image according to claim 1, wherein, The objective lens illumination module includes a first doublet lens, a polarization beam splitter prism and a rotatable quarter-wave plate. The second laser is sequentially focused to the rear focal plane of the objective lens through the first doublet lens, the polarization beam splitter prism and the rotatable quarter-wave plate.

4. The drift correction system based on the interference scattering image according to claim 3, characterized in that, The high-precision drift correction system based on interference scattering images further includes a diaphragm stop and a second doublet lens. The first scattered light, the second scattered light and the interference signal are sequentially transmitted to the second acquisition module through the diaphragm stop and the second doublet lens.

5. The drift correction system based on the interference scattering image according to claim 4, wherein Before passing through the diaphragm stop, the first scattered light, the second scattered light and the interference signal also pass through the rotatable quarter-wave plate and the polarization beam splitter prism.

6. The drift correction system based on an interference scattering image according to claim 1, wherein The drift correction system based on interference scattering images further includes a first dichroic mirror, a first mirror, a second dichroic mirror, a filter wheel, a microscope tube lens and a plano-convex cylindrical lens. The fluorescence passes from the objective lens through the first dichroic mirror, the mirror, the second dichroic mirror, the filter wheel, the microscope tube lens and the plano-convex cylindrical lens to the first acquisition module.

7. The drift correction system based on an interference scattering image according to claim 1, characterized in that Both the first acquisition module and the second acquisition module are cameras.

8. The drift correction system based on the interference scattering image according to claim 7, wherein The first acquisition module and the second acquisition module acquire images synchronously or at different frequencies.

9. The drift correction system based on an interference scattering image according to any one of claims 1 to 8, characterized in that, The drift correction module calculates the drift amount of the sample by using the cross-correlation algorithm.

10. A control system based on Micro-FPGA, characterized in that, The drift correction system based on the interference scattering image according to any one of claims 1 to 9 is integrated in the Micro-FPGA-based control system, and the Micro-FPGA-based control system controls the drift correction system based on the interference scattering image according to the EMU plug-in of the microscope control software Micro-Manager.

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