Microscopy system and image reconstruction method

Abstract

This application discloses a microscopic system and image reconstruction method, relating to the field of microscopic imaging. In this system, diffracted light obtained by diffraction of a laser beam through sectors in a rotating sector grating is incident on a splitter mirror; the diffracted light exiting the splitter mirror is incident on a polarization modulation device; the polarization modulation device is used to adjust the polarization state of the diffracted light; a converging lens is used to focus the polarization-adjusted diffracted light onto the back focal plane of the objective lens; the sample is placed on the front focal plane of the objective lens; the polarization-adjusted diffracted light coherently superimposes on the sample surface to form fringe structured light; the fringe structured light excites the sample to generate fluorescence; the fluorescence is incident on the objective lens; the fluorescence collected by the objective lens is focused onto a camera through a tube lens; the camera acquires the original image; and a computer is used to perform super-resolution reconstruction based on the original image. This application reduces the system's control precision requirements and eliminates the problems of low diffraction efficiency, severe wavefront distortion, high system cost, and high synchronization complexity associated with digital devices.

Description

Technical Field

[0001] This application relates to the field of microscopic imaging, and in particular to a microscopic system and image reconstruction method. Background Technology

[0002] In Super-Resolution Structured Illumination Microscopy (SR-SIM), excitation light with a specific spatial distribution (such as sinusoidal fringes) excites a fluorescent sample. The high-frequency structural fringes superimpose with the fine sample structure to produce low-frequency moiré fringes. These moiré fringes can be observed, resolved, and recorded using an optical microscope. By combining the frequency, phase, and other parameters of the structural fringes, spatial information exceeding the optical diffraction limit can be obtained. SR-SIM does not rely on specific fluorescent dyes, is compatible with traditional fluorescence microscopy modes, and has advantages such as low phototoxicity and high resolution, making it particularly suitable for subcellular scale super-resolution imaging and long-term live-cell imaging.

[0003] The core challenge in building an SR-SIM system is the generation and modulation of structural fringes. Traditionally, diffraction gratings are used to generate coherent light, which is then superimposed on the sample surface to form sinusoidal fringes. The rotation and phase shift of these sinusoidal fringes depend on the mechanical rotation and translation of the grating itself, placing extremely high demands on the speed, accuracy, and synchronous control of the rotary stage and translation stage. In recent years, due to the advantages of rapidly developing digital devices, such as flexible programmability and fast response speed, light modulation schemes that simulate real diffraction gratings using pixel structures from digital devices like spatial light modulators (SLMs) and digital micromirror devices (DMDs) have become the mainstream approach for SR-SIM. However, the pixel structure scale of digital devices is comparable to the wavelength of light, which leads to severe "pixel structure artifacts" and wavefront distortion during diffraction. This manifests as stray diffraction orders and reduced beam quality, significantly decreasing light energy utilization. Furthermore, SR-SIM systems based on digital devices are often expensive, and the synchronization and cooperation of multiple active devices within the system are complex. These technical and cost hurdles limit the further development and widespread application of SR-SIM. Therefore, a solution is needed that can reduce the requirements for control precision and eliminate the problems caused by digital devices. Summary of the Invention

[0004] The purpose of this application is to provide a microscopic system and image reconstruction method that can reduce the system's requirements for control precision and eliminate problems such as low diffraction efficiency of digital devices, severe wavefront distortion, high system cost, and high synchronization complexity.

[0005] To achieve the above objectives, this application provides the following solution: In a first aspect, this application provides a microscope system, comprising: Laser array, beam expander system, rotating sector grating, rotating mount, splitter mirror, polarization modulation device, converging lens, objective lens, tube lens, camera, and computer; The laser array, the beam expander, the rotating sector grating, the splitter, and the polarization modulation unit are arranged sequentially. The rotating sector grating is mounted on a rotating base. The rotating base drives the rotating sector grating to rotate around its geometric center. The rotation axis of the rotating sector grating is parallel to the principal optical axis. The diffracted light obtained by the laser beam being diffracted by the sectors in the rotating sector grating is incident on the splitter. The diffracted light exiting the splitter is incident on the polarization modulation device. The polarization modulation device is used to adjust the polarization state of the diffracted light. The converging lens is used to focus the polarization-adjusted diffracted light onto the back focal plane of the objective lens. The sample is placed on the front focal plane of the objective lens. The polarization-adjusted diffracted light coherently superimposes on the sample surface to form a fringe structured light. The fringe structured light excites the sample to generate fluorescence. The fluorescence is incident on the objective lens. The fluorescence collected by the objective lens is focused onto the camera through a tube lens. The camera obtains the original image. The computer is used to reconstruct the image based on the original image.

[0006] In one embodiment, the laser array includes: a plurality of single-wavelength lasers; one of the single-wavelength lasers operates at the same time.

[0007] In one embodiment, the beam expanding system includes: a first dichroic mirror, a first beam expanding lens, a second beam expanding lens, and a field stop arranged sequentially.

[0008] In one embodiment, the microscope system further includes: an optical spacing adjustment lens group; The optical spacing adjustment mirror group is disposed in the optical path between the rotating sector grating and the split mirror; The optical spacing adjustment lens group includes a convex lens and a concave lens; the convex lens is disposed on the optical path near the rotating sector grating; the concave lens is disposed on the optical path near the split mirror.

[0009] In one embodiment, the microscope system further includes: a spatial filter; The spatial filter is disposed in the optical path between the split mirror and the polarization modulation device.

[0010] In one embodiment, the geometry of the split-view mirror is determined by the number of structural stripe directions.

[0011] In one embodiment, the microscope system further includes a third beam expander lens; The third beam expander lens is disposed in the optical path between the spatial filter and the polarization modulation device.

[0012] In one embodiment, the polarization modulation device includes: a linear polarizer and an achromatic partitioned half-wave plate; The linear polarizer is disposed on the optical path near the splitter lens; the achromatic half-wave plate is disposed on the optical path near the converging lens.

[0013] In one embodiment, the microscope system further includes a second dichroic mirror; The second dichroic mirror is disposed in the optical path between the converging lens, the objective lens, and the tube lens.

[0014] Secondly, this application provides a method for image reconstruction of a microscopic system, wherein the method applies the microscopic system and includes: Acquire all real, raw images within a single work cycle; The frequency and orientation of structural stripes in the real original image are estimated using the cross-correlation parameter estimation method; Based on the original real image, notch filtering is performed on the stripe modulation points in the frequency domain according to the stripe frequency and direction of the structure to obtain a simulated wide-field image without stripe modulation points in the spectrum. The simulated wide-field image is multiplied with simulated structured light stripes in all directions to obtain a simulated original image. The structured light phase parameters of the real original image are then determined based on the real original image and the simulated original image. Phase separation is performed based on the structure fringe frequency, the direction, and the structured light phase parameters to obtain the zero-order and positive and negative first-order spectra after phase separation; Based on two-stage notch filtering, the zero-order and positive and negative first-order spectra after phase separation are filtered in the frequency domain to obtain the filtered zero-order and positive and negative first-order spectra. The zero-order and positive and negative first-order spectra after filtering are processed by two-step frequency domain filtering and inverse Fourier transform to obtain the final reconstructed image.

[0015] According to the specific embodiments provided in this application, this application has the following technical effects: This application provides a microscope system and image reconstruction method that replaces the two-dimensional motion of rotation and translation in traditional methods with the one-dimensional rotation of a rotating sector grating, greatly reducing the control precision requirements of the system. At the same time, by replacing digital devices with a real physical grating structure, namely a rotating sector grating, it fundamentally eliminates the problems of low diffraction efficiency, severe wavefront distortion, high system cost, and high synchronization complexity of digital devices. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 This is an optical path diagram of a microscope system according to one embodiment of this application; Figure 2 The images provided are physical diagrams, design schematics, and functional schematics of a rotating sector grating according to one embodiment of this application. Figure 3 This is a schematic diagram of a splitter in one embodiment of this application; Figure 4 This is a schematic diagram illustrating the working principle of the splitter in one embodiment of this application; Figure 5 This is a schematic diagram of a microscopic system image reconstruction method according to another embodiment of this application.

[0018] Figure label: LA1 - First single-wavelength laser, LA2 - Second single-wavelength laser, DM1 - First dichroic mirror, DM2 - Second dichroic mirror, BE1 - First beam expander lens, BE2 - Second beam expander lens, BE3 - Third beam expander lens, BE4 - Fourth beam expander lens, DP - Field stop, GR - Rotating sector grating, L1 - Convex lens, L2 - Concave lens, L3 - Converging lens, PL - Reflector, SF - Spatial filter, LP - Linear polarizer; WP - Achromatic partition half-wave plate, OBJ - Objective lens, TL - Tube lens, CA - Camera, M1 - First reflecting mirror, M2 - Second reflecting mirror, M3 - Third reflecting mirror, M4 - Fourth reflecting mirror, M5 - Fifth reflecting mirror, M6 - Sixth reflecting mirror, M7 - Seventh reflecting mirror, M8 - Eighth reflecting mirror, M9 - Ninth reflecting mirror, M10 - Tenth reflecting mirror. Detailed Implementation

[0019] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0020] To make the above-mentioned objectives, features and advantages of this application more apparent and understandable, the application will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0021] In one exemplary embodiment, such as Figure 1 As shown, a microscopy system is provided, comprising: a laser array, a beam expander, a rotating sector grating GR, a rotating mount, a refraction mirror PL, a polarization modulation device, a converging lens L3, an objective lens OBJ, a tube lens TL, a camera CA, and a computer; the laser array, the beam expander, the rotating sector grating GR, the refraction mirror PL, and the polarization modulation unit are arranged sequentially; the rotating sector grating GR is mounted on the rotating mount; the rotating mount is used to drive the rotating sector grating GR to rotate around its geometric center; the rotation axis of the rotating sector grating GR is parallel to the principal optical axis; the laser beam is diffracted by sectors in the rotating sector grating GR. Light is incident on the splitter lens PL; diffracted light exiting the splitter lens PL is incident on the polarization modulation device; the polarization modulation device is used to adjust the polarization state of the diffracted light; the converging lens L3 is used to focus the polarization-adjusted diffracted light onto the back focal plane of the objective lens OBJ; the sample is placed on the front focal plane of the objective lens OBJ; the polarization-adjusted diffracted light coherently superimposes on the sample surface to form fringe structured light; the fringe structured light excites the sample to generate fluorescence; the fluorescence is incident on the objective lens OBJ; the fluorescence collected by the objective lens OBJ is focused by the tube lens TL onto the camera CA; the camera CA acquires the original image; the computer is used to reconstruct the image based on the original image. Figure 1 In the diagram, the 561 nm excitation light is represented in yellow, and the 473 nm excitation light is represented in blue. After being combined by the dichroic mirror DM1, the paths of the two lasers almost overlap. The sample emission fluorescence is represented in red. The colored box shows a schematic diagram of the rotating sector grating GR in operation. The center of the grating is fixed directly above the incident laser. The red area is the working sector, and each sector of the rotating sector grating GR enters this area in turn under the drive of the rotating mount. The blue arrows in the diagram represent the alignment direction of each diffracted beam.

[0022] In another exemplary embodiment, the laser group includes: a plurality of single-wavelength lasers; one of the single-wavelength lasers operates at the same time.

[0023] In another exemplary embodiment, the beam expanding system includes: a first dichroic mirror DM1, a first beam expanding lens BE1, a second beam expanding lens BE2, and a field stop DP arranged sequentially.

[0024] In another exemplary embodiment, the microscope system further includes: an optical spacing adjustment lens group; the optical spacing adjustment lens group is disposed in the optical path between the rotating sector grating GR and the split mirror PL; the optical spacing adjustment lens group includes a convex lens L1 and a concave lens L2; ​​the convex lens L1 is disposed in the optical path near the rotating sector grating GR; the concave lens L2 is disposed in the optical path near the split mirror PL.

[0025] In another exemplary embodiment, the microscope system further includes a spatial filter SF; the spatial filter SF is disposed in the optical path between the splitter mirror PL and the polarization modulation device.

[0026] In another exemplary embodiment, the geometry of the split-view mirror PL is determined by the number of structural stripe directions.

[0027] In practical applications, the aforementioned microscopy system further includes a third beam expander lens BE3; the third beam expander lens BE3 is disposed in the optical path between the spatial filter SF and the polarization modulation device. The polarization modulation device includes a linear polarizer LP and an achromatic partitioned half-wave plate WP; the linear polarizer LP is disposed in the optical path near the splitter lens PL; the achromatic partitioned half-wave plate WP is disposed in the optical path near the converging lens L3. The microscopy system also includes a fourth beam expander lens BE4, which is disposed in the optical path between the achromatic partitioned half-wave plate WP and the converging lens L3.

[0028] The microscope system also includes a second dichroic mirror DM2; the second dichroic mirror DM2 is disposed in the optical path between the converging lens L3, the objective lens OBJ, and the tube lens TL.

[0029] In practical applications, ten reflectors are also used. The first reflector M1 and the second reflector M2 are used to reflect the laser emitted by the second single-wavelength laser LA2, so that it is incident on the first dichroic mirror DM1 and the laser emitted by the first single-wavelength laser LA1 is combined. The third reflector M3 and the fourth reflector M4 are sequentially arranged between the second beam expander lens BE2 and the field stop DP. The fifth reflector M5 is arranged between the linear polarizer LP and the spatial filter SF. The sixth reflector M6 is arranged between the spatial filter SF and the third beam expander lens group BE3. The seventh reflector M7, the eighth reflector M8 and the ninth reflector M9 are arranged between the fourth beam expander lens group BE4 and the converging lens L3. The tenth reflector M10 is arranged between the second dichroic mirror DM2 and the objective lens OBJ.

[0030] This application replaces the two-dimensional motion of rotation and translation in the traditional method with the one-dimensional rotation of the rotating sector grating GR, greatly reducing the control accuracy requirements of the system. At the same time, this application replaces digital devices with a real physical grating structure, fundamentally eliminating the problems of low diffraction efficiency, severe wavefront distortion, high system cost, and high synchronization complexity of digital devices.

[0031] The principle of the microscopic system provided in this application is as follows.

[0032] Each single-wavelength laser can emit a single-mode laser of a set wavelength. Multiple lasers emitting different wavelengths are combined by the first dichroic mirror DM1, ensuring that the propagation paths of the different colors coincide. Generally, only one laser operates at a time. The laser beam is expanded and collimated after passing through the first beam expander BE1 and the second beam expander BE2. The field stop DP limits the beam size within the working sector of the rotating sector grating GR. The beam undergoes diffraction after passing through the rotating sector grating GR, forming multiple orders of diffracted light (…). Figure 1 (within the box).

[0033] The design and manufacturing method of a rotating sector grating (GR) is as follows: A circular grating substrate is divided into M×N equally divided sectors. Different binary fringes are etched in each sector to form a diffraction grating. The period d is determined by the wavelength of the light and the required resolution improvement of the system. M is the number of directions of the structured light, typically 2 or 3; N is the number of phases of the structured light, typically 3 or 5. A schematic diagram of a rotating sector grating (GR) is shown below. Figure 2 As shown (taking a 3×3 sector as an example). The rotating sector grating GR is mounted in a precision piezoelectric rotating base, allowing it to rotate around its geometric center. In the optical path, the geometric center of the rotating sector grating GR is directly above the principal optical axis, at a distance R from it, and the rotation axis is parallel to the principal optical axis. At any given time, the laser illuminates only one sector of the rotating sector grating GR, called the working sector. The laser is diffracted by this sector, and the arrangement directions of the diffracted beams are marked with blue arrows. Figure 1 The sector edges shown in the box (taking a 3×3 sector as an example).

[0034] In the system described in this application, the ±1st order diffracted light ultimately interferes with the sample plane through the objective lens OBJ, while diffracted light of other orders is filtered out by the spatial filter SF. The angle between the ±1st order diffracted light and the principal optical axis when it exits the diffraction grating is called the diffraction angle of the diffraction grating for that wavelength. The optical spacing adjustment lens group consists of a convex lens L1 and a concave lens L2, ensuring beam collimation while adjusting the beam diameter and the angle between the beam and the principal optical axis. The ±1st order diffracted light is then incident on the splitter mirror PL after passing through the optical spacing adjustment lens group.

[0035] The splitter lens (PL) is a lens (prism) specifically designed for SIM (simulated surface treatment). Its geometry is determined by the number M of structural fringe directions required by the SIM. Since the structural fringes on the sample surface are formed by the interference of ±1st order diffracted light, and the ±1st order diffracted light is symmetrical about the principal axis, the splitter lens (PL) should have 2M side faces. For the case where M=3 (e.g....), Figure 3 The geometry of the splitter mirror PL is a flat-topped hexagonal truncated pyramid (PL). Figure 3The ±1st order diffracted light incident from the bottom surface of the prism exits from a pair of opposite sides of the splitter lens PL. The thickness and wedge angle of the prism change the propagation direction of the beam without altering its collimation. The angle between the ±1st order diffracted light exiting from the splitter lens PL and the principal axis increases. After passing through the third beam expander lens BE3 and the fourth beam expander lens BE4, the diameter of each beam increases to ensure a larger illumination field of view on the sample plane. The polarization adjustment device consists of a linear polarizer LP and an achromatic partitioned half-wave plate WP, adjusting the polarization state of the ±1st order diffracted light to s-polarization. The converging lens L3 focuses the ±1st order diffracted light onto the back focal plane of the objective lens OBJ. The sample is placed on the front focal plane of the objective lens OBJ, and the ±1st order diffracted light coherently superimposes on the sample surface, forming a beam with a specific spatial frequency k and phase. The striped structured light excites the sample to produce fluorescence. The objective lens OBJ collects the emitted fluorescence, which is then focused by the tube lens TL onto the camera CA, which captures a raw image. The rotating sector grating GR rotates to place the next sector on the principal optical axis at the "working sector" position. Figure 1 (Marked in red sector). The phase of the structured light changes accordingly, and the camera CA acquires a total of M×N original images with different structured light directions and phases; the computer performs phase calibration on the original images to perform high-precision structured illumination super-resolution reconstruction to obtain the final super-resolution image.

[0036] like Figure 1 As shown, the 561 nm excitation light is represented in yellow, and the 473 nm excitation light is represented in blue. After being combined by the dichroic mirror DM1, the paths of the two lasers almost overlap. The sample emission fluorescence is represented in red. The black box shows a schematic diagram of the rotating sector grating GR. The center of the grating is fixed directly above the incident laser. The red area is the working sector, and each sector of the rotating sector grating GR enters this area in turn under the drive of the rotating mount. The blue arrows in the diagram represent the alignment direction of each diffraction order.

[0037] like Figure 2 As shown, where Figure 2 i) in the image is a microscopic image of the grating (central part). Figure 2 ii) in the diagram shows the grating design: the circular substrate is evenly divided into nine sectors, denoted as sectors I-IX, and binary fringes with different directions and phases are etched in each sector. The fringe period (grating constant) is... The area highlighted in red is the "working sector." Different sectors take turns entering the working sector as the grating rotates. The diameter of the collimated laser beam is... The center of the grating is fixed directly above the laser beam, and the distance from the center of the grating to the center of the laser beam is... Between grating sectors, the direction of the stripes is distinguished by the color of the area marker, and the phase of the stripes is distinguished by the saturation of the area marker. Figure 2iii)-v) in the diagram are functional illustrations of the grating sector. When the corresponding sector enters the working sector, the fringe direction and the laser beam diffraction direction are as follows: Figure 2 As shown in iii)-v).

[0038] like Figure 3 As shown, the splitter mirror PL changes the beam propagation direction while maintaining beam collimation, where P represents a precision-polished light-transmitting plane. Figure 3 Image (a) is a front view of the splitter lens PL. Figure 3 (b) is a side view of the splitter lens PL. Figure 4 This is a schematic diagram of the working principle of the splitter lens PL. In the optical path shown in this figure, the thicker the beam of the incident objective lens OBJ, the larger the corresponding imaging field of view. Figure 4 In the diagram, 'a' represents the imaging field of view when the grating diffraction angle is large. Figure 4 In the diagram, 'b' represents the imaging field of view when the grating diffraction angle is small. Figure 4 In the diagram, 'c' represents a schematic representation of how the split-view mirror (PL) increases the effective diffraction angle. Figure 4 In the diagram, 'd' represents chromatic aberration and its correction. The splitter lens PL expands the microscope's field of view. To allow two coherent diffracted beams to enter the pupil behind the objective lens OBJ, the distance between the two beams should remain constant for a given objective lens OBJ. Figure 4 Figure a shows that the larger the grating diffraction angle, the coarser the diffracted beam after transformation by the lens group, and the larger the system's field of view. Figure 4 Figure b shows that when the grating diffraction angle is very small, in order to maintain the beam spacing, the diffracted beam is very thin after being transformed by the lens group, and the system field of view is very small. Figure 4 In Figure c, the application of the splitter lens PL changes the propagation direction of the diffracted beam without changing its collimation. While ensuring the same spacing at BFP, the change of the lens group can increase the diameter of the diffracted beam and expand the system's field of view. Figure 4 The d-plot in the figure shows that fine-tuning the position of the splitter lens (PL) can effectively correct chromatic aberration.

[0039] In another exemplary embodiment, this application provides more detailed and specific data on the microscopy system.

[0040] I. Laser source and beam expansion system.

[0041] In this example, lasers with wavelengths of 473 nm and 561 nm are used for fluorescence excitation. The two laser beams are combined into a single optical path via a dichroic mirror DM1. A set of achromatic lenses (BE1, ...) f =30 mm; BE2, fThe beam diameter is expanded to 3.3 times its original size (100 mm) to ensure uniform illumination of the working sector of the grating disk. To prevent diffraction from other grating sectors, an aperture DP is placed in front of the grating disk.

[0042] II. Rotating sector grating GR and rotating mount.

[0043] The grating constant for each sector is 62.5 μm, and according to the grating equation, the corresponding diffraction angle at a wavelength of 561 nm is 0.514°. Line pairs with different directions and phases are first generated in Mathematica and exported as DWG format, which can then be edited in AutoCAD, and a binary transmission rotating sector grating GR is customized based on the drawings. The grating is mounted on a rotating bracket with a resonant piezoelectric motor. To ensure the positional accuracy of the grating, a precise adjustment is performed using an XY translation stage. The diffracted beam is first filtered through an achromatic lens (L1, f =200 mm) converge, and then are focused by a concave lens L2 (L2, =200 mm) f The collimated beam is then collimated (=-50 mm) and then enters the splitter lens PL.

[0044] III. Split mirror (PL) and polarization modulation element.

[0045] A splitter mirror (PL) is a transparent, flat-topped, hexagonal frustum-shaped refractive element with six angled, polished sides and a hexagonal polished platform. The angled sides refract the illumination beam into three directions, while the platform transmits the 0th-order diffracted light. The apex angle of the splitter mirror (PL) is defined as the angle between each angled side and the platform plane; the larger the apex angle, the stronger the refraction of the incident light. In this example, a splitter mirror (PL) made of NBK-7 glass with an apex angle of 16.65° is used. To achieve multicolor imaging, the splitter mirror (PL) is mounted on a dovetail rail. After the splitter mirror (PL), a custom-made spatial filter (mask) blocks the 0th-order light, while the ±1st-order diffracted light continues to propagate. The collimated ±1st-order beams are collimated at L3 (…). f =19 mm) intersects before. L3 and L4 ( f =300 mm) to form a 4 f The system magnifies the beam diameter by 15.8 times. To improve the modulation depth of the structured illumination fringes, s-polarized light is required. The laser output itself has good linear polarization characteristics; after passing through the linear polarizer LP, the polarization purity is further improved with minimal energy loss. Subsequently, the polarized light passes through an achromatic six-segment waveplate, where different sectors can rotate the polarization direction of the incident light to an s-polarized state. The subsequent reflectors (M7–M9) must ensure that the system's principal optical axis is strictly parallel to the optical platform to avoid severe polarization degradation caused by reflection.

[0046] IV. Fluorescence excitation and polarization detection.

[0047] Lens L5 ( f =200 mm) The ±1st order diffracted light was focused onto the back pupil (BFP) of the objective lens OBJ. The objective lenses OBJ used included: CFI Plan Apochromat Lambda D 100×Oil, NA 1.45; CFI Plan Apochromat Lambda 60×Oil, NA 1.40; CFI Plan Apochromat Lambda D 40×, NA 0.95; CFI Plan Apochromat Lambda D 20×, NA 0.8; and CFI Plan Apochromat Lambda D 10×, NA 0.45 (all from Nikon). A polarization-maintaining dichroic mirror DM2 was used to prevent degradation of the incident light polarization state. The emitted fluorescence was imaged onto the scientific-grade CMOS camera CA via a tube lens TL and passed through an emission filter. With the aid of a splitter lens PL, this example achieves a field of view (FOV) of approximately 2048×2048 pixels, covering the entire sCMOS sensor area. However, to obtain uniform illumination under a Gaussian beam, the effective field of view of Sero-SIM is approximately 1650×1650 pixels.

[0048] The calibration method for the microscopic system described in this example is similar to that of mainstream SIM systems. Since digital diffraction devices are not used, some alignment steps are omitted; however, the introduction of the rotating sector grating (GR) and the splitter mirror (PL) introduces a new calibration procedure. The rotating sector grating (GR) is mounted on a translation stage for positional adjustment along the principal optical axis or vertically, thereby ensuring phase shift accuracy.

[0049] A key step in the system alignment process is precisely adjusting the distance between L3 and the splitter lens PL. Without a spatial filter, the 0th and ±1st order diffracted beams aid in system alignment. First, during multicolor imaging, the splitter lens PL should be mounted on a dovetail rail or translation stage strictly parallel to the principal optical axis, ensuring that its translation does not alter the position of the 0th order diffracted beam. Second, temporarily replace the splitter lens PL with another convex lens L′, allowing the collimated beam to converge at its focal point. Place an interferometer behind L3 to check the beam collimation. Translate L′ along the dovetail rail, locate the collimated beam position in the interferometer, and mark the front focal point of L3, denoted as F1, using an observation screen. Then, replace L′ with the splitter lens PL and move it to a suitable position so that the ±1st order diffracted beams at a specific operating wavelength intersect precisely at F1. For a detailed diagram of chromatic aberration compensation, please refer to [link to diagram]. Figure 4 d in the text.

[0050] Phase shift correction must be performed after the complete system is established and precisely aligned. A sample with strong autofluorescence (such as onion root tip cells) is used to obtain a sufficiently clear fringe pattern. After acquiring nine raw images, the fringes can be observed in the raw images. Due to limited absolute positioning accuracy, phase shift uncertainty can be effectively compensated by introducing small rotational angular perturbations (e.g., adjusting from [120°, 160°, 200°] to [120°, 160.1°, 200.05°]). The rotating support has sufficient bidirectional positioning accuracy. The specific angular perturbation value can be determined through a small number of trials. When the grating fringe direction is tangent to its rotation direction (e.g., ...), the fringe pattern is corrected. Figure 2 When working with sectors IV, V, and VI in the grating, fine translation of the rotating sector grating GR can also be helpful.

[0051] In another exemplary embodiment, such as Figure 5 As shown, a method for image reconstruction of a microscopic system is provided. This method utilizes the microscopic system described above and includes the following components: Acquire all real, raw images within a single work cycle.

[0052] The frequency and orientation of structural fringes in the real original image are estimated using the cross-correlation parameter estimation method.

[0053] Based on the original image, notch filtering is performed on the stripe modulation points in the frequency domain according to the frequency and direction of the structure stripes to obtain a simulated wide-field image without stripe modulation points in the spectrum.

[0054] The simulated wide-field image is multiplied with simulated structured light stripes in all directions to obtain a simulated original image. The structured light phase parameters of the real original image are then determined based on the real original image and the simulated original image.

[0055] Phase separation is performed based on the structure fringe frequency, the direction, and the structured light phase parameters to obtain the zero-order and positive and negative first-order spectra after phase separation.

[0056] The zero-order and positive and negative first-order spectra after phase separation are filtered by a two-stage notch filter to obtain the filtered zero-order and positive and negative first-order spectra.

[0057] The zero-order and positive and negative first-order spectra after filtering are processed by two-step frequency domain filtering and inverse Fourier transform to obtain the final reconstructed image. The final reconstructed image is a super-resolution image.

[0058] This application proposes a high-precision structured light reconstruction algorithm with phase calibration to accurately estimate the phase information of structured light under non-ideal conditions, achieving high-fidelity structured light reconstruction. This application has significant advantages in terms of cost, light energy utilization, imaging field of view, and system complexity.

[0059] To accurately estimate the parameters and perform super-resolution reconstruction on the original images collected by the SIM system involved in this application, this application also includes a high-precision structured light reconstruction algorithm for phase calibration, that is, a microscopy system and an image reconstruction method, including the following steps: Step 1: The M×N sectors of the rotating sector grating respectively enter the working sector under the drive of the rotating base, modulating the diffraction direction of the illumination light and the optical path difference between the ±1st order diffracted lights. Each time the rotating sector grating rotates once, the phase and (or) direction of the structural fringes generated by the interference on the sample surface change correspondingly once, and the camera exposes once to receive the fluorescence emitted by the sample. A total of M×N original images are obtained within one working cycle.

[0060] Step 2: Blocking: In the case of large-field imaging, the image is equally divided into 4 sub-blocks in a "field" shape for separate reconstruction, thereby significantly reducing parameter errors under uniform illumination conditions. If the area to be observed is small (less than 500 pixels in diameter), this step is skipped and no blocking is required.

[0061] Step 3: After processing the original image using the dark channel-based light layer cutting method, the structural fringe frequency and direction of the original image are estimated using the cross-correlation parameter estimation method. Among them Figure 5 α is the angle (direction) of the structural fringes, is the phase of the structural fringes. There are a total of three angles and three phases. Figure 5 Taking the estimation of the three phases in the first direction as an example.

[0062] Step 4: Add the original images, perform notch filtering on the fringe modulation points in the frequency domain based on the estimated structural fringe spatial frequency and direction, and obtain a simulated wide-field image without fringe modulation points in the spectrum.

[0063] Step 5: According to the estimated fringe frequency and direction, the structured light fringes in all directions are simulated by a computer and multiplied by the simulated wide-field image to obtain a simulated original image. Calculate the cross-correlation index between the simulated original image and the real original image. By maximizing the structural similarity index between the simulated original image and the real original data, the structured light phase parameters of the original image are determined. The cross-correlation index can measure the similarity between two pictures, and its expression is: .

[0064] Among them represents two images, is the pixel point position, and are the average pixel values of the images.

[0065] Step 6: Perform phase separation on all original images in each direction based on the estimated frequency, direction and phase parameters to obtain the zero-order and positive and negative first-order spectra after phase separation, and then move the positive and negative first-order spectra to the high-frequency position in the frequency domain.

[0066] Step 7: Perform frequency domain filtering using a two-stage notch filter. The first-stage filter suppresses frequency domain peaks caused by the out-of-focus background, which are distributed at the center of the positive and negative first-order spectra shifted in M ​​directions. The second-stage filter suppresses frequency domain peaks caused by phase estimation errors, which are distributed at the periphery of the positive and negative first-order spectra shifted in three directions.

[0067] Step 8: Use two-step frequency domain filtering to further reduce artifacts and preserve weak signal information; finally, obtain the final super-resolution image through inverse Fourier transform. All reconstruction processes were completed in MATLAB 2021b (MathWorks).

[0068] This application replaces digital diffraction devices with physical gratings, significantly reducing the cost of core system components while substantially improving light energy utilization. Research indicates that the core component costs for systems based on spatial light modulators or digital micromirrors are approximately $10,000 and $4,000, respectively, while the physical grating and precision piezoelectric rotator used in this application cost only $980. Measuring the ratio of positive and negative first-order diffracted light energy to the laser energy before diffraction, the diffraction efficiency of the spatial light modulator is approximately 4%, the diffraction efficiency of the digital micromirror device is approximately 9%, and the diffraction efficiency based on the binary transmission grating reaches 16%. Furthermore, replacing the binary transmission grating with a sinusoidal transmission grating or a polarization grating achieves a diffraction efficiency of 98%. Regarding the performance indicators of the microscopy system, experiments with fluorescent microspheres verified that, using 473 nm laser excitation and a 100x objective lens with a numerical aperture of 1.45, the resolution of the microscopy system constructed in this application can reach 128 nm, and the resolution in the wide-field fluorescence microscopy mode is 249 nm, representing a resolution improvement of 1.95 times, consistent with the resolution improvement of existing SIM technology. Furthermore, the system constructed in this application contains only two active devices: a precision piezoelectric rotating base and a camera, whose synchronization is controlled by a simple MATLAB program. Compared to existing SIM systems based on digital devices, this application has advantages such as simple system construction and easy control. The technical features of the above embodiments can be combined arbitrarily. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as the combination of these technical features does not contradict each other, it should be considered within the scope of this specification.

[0069] This document uses specific examples to illustrate the principles and implementation methods of this application. The descriptions of the above embodiments are only for the purpose of helping to understand the methods and core ideas of this application. Furthermore, those skilled in the art will recognize that, based on the ideas of this application, there will be changes in the specific implementation methods and application scope. Therefore, the content of this specification should not be construed as a limitation of this application.

Claims

1. A microscopy system, characterized by, The microscopic system includes: a laser array, a beam expander, a rotating sector grating, a rotating mount, a splitter mirror, a polarization modulation device, a converging lens, an objective lens, a tube lens, a camera, and a computer; The laser array, the beam expander, the rotating sector grating, the splitter, and the polarization modulation unit are arranged sequentially. The rotating sector grating is mounted on a rotating base. The rotating base drives the rotating sector grating to rotate around its geometric center. The rotation axis of the rotating sector grating is parallel to the principal optical axis. The diffracted light obtained by the laser beam being diffracted by the sectors in the rotating sector grating is incident on the splitter. The diffracted light exiting the splitter is incident on the polarization modulation device. The polarization modulation device is used to adjust the polarization state of the diffracted light. The converging lens is used to focus the polarization-adjusted diffracted light onto the back focal plane of the objective lens. The sample is placed on the front focal plane of the objective lens. The polarization-adjusted diffracted light coherently superimposes on the sample surface to form a fringe structured light. The fringe structured light excites the sample to generate fluorescence. The fluorescence is incident on the objective lens. The fluorescence collected by the objective lens is focused onto the camera through a tube lens. The camera obtains the original image. The computer is used to reconstruct the image based on the original image.

2. The microscopy system of claim 1, wherein, The laser array comprises: multiple single-wavelength lasers; one of the single-wavelength lasers operates at the same time.

3. The microscopy system of claim 1, wherein, The beam expanding system includes: a first dichroic mirror, a first beam expanding lens, a second beam expanding lens, and a field stop arranged sequentially.

4. The microscopy system of claim 1, wherein, Also includes: Optical spacing adjustment lens group; The optical spacing adjustment mirror group is disposed in the optical path between the rotating sector grating and the split mirror; The optical spacing adjustment lens group includes a convex lens and a concave lens; the convex lens is disposed on the optical path near the side of the rotating sector grating; the concave lens is disposed on the optical path near the side of the split mirror.

5. The microscopy system of claim 1, wherein, Also includes: Spatial domain filter; The spatial filter is disposed in the optical path between the split mirror and the polarization modulation device.

6. The microscopy system of claim 1, wherein, The geometry of the split mirror is determined by the number of structural fringe directions.

7. The microscopy system of claim 5, wherein, It also includes a third beam expander lens; The third beam expander lens is disposed in the optical path between the spatial filter and the polarization modulation device.

8. The microscopy system of claim 1, wherein, The polarization modulation device includes: a linear polarizer and an achromatic partitioned half-wave plate; The linear polarizer is disposed on the optical path near the splitter lens; the achromatic half-wave plate is disposed on the optical path near the converging lens.

9. The microscope system according to claim 1, characterized in that, It also includes a second dichroic mirror; The second dichroic mirror is disposed in the optical path between the converging lens, the objective lens, and the tube lens.

10. A microscopy system image reconstruction method, characterized by, The image reconstruction method for the microscopic system uses the microscopic system described in any one of claims 1-9, and the image reconstruction method for the microscopic system includes: Acquire all real, raw images within a single work cycle; The frequency and orientation of structural stripes in the real original image are estimated using the cross-correlation parameter estimation method; Based on the original real image, notch filtering is performed on the stripe modulation points in the frequency domain according to the stripe frequency and direction of the structure to obtain a simulated wide-field image without stripe modulation points in the spectrum. The simulated wide-field image is multiplied with simulated structured light stripes in all directions to obtain a simulated original image. The structured light phase parameters of the real original image are then determined based on the real original image and the simulated original image. Phase separation is performed based on the structure fringe frequency, the direction, and the structured light phase parameters to obtain the zero-order and positive and negative first-order spectra after phase separation; Based on two-stage notch filtering, the zero-order and positive and negative first-order spectra after phase separation are filtered in the frequency domain to obtain the filtered zero-order and positive and negative first-order spectra. The zero-order and positive and negative first-order spectra after filtering are processed by two-step frequency domain filtering and inverse Fourier transform to obtain the final reconstructed image.

Images