Variable curvature scanning illumination device for inverted microscope and imaging method thereof

Abstract

The invention relates to a variable curvature scanning lighting device for an inverted microscope and an imaging method thereof, the device comprises the inverted microscope and a flexible arc-shaped bracket located above the inverted microscope, and a plurality of LED light sources capable of independently addressing are uniformly arranged on the flexible arc-shaped bracket along the arc direction; the curvature adjusting mechanism is used for adjusting the curvature of the flexible arc-shaped bracket and changing the NA range of illumination of each LED light source; the rotating mechanism is configured to control the flexible arc-shaped bracket to rotate around the optical axis of the inverted microscope under the current curvature; the imaging method comprises the following steps: respectively executing complete rotary scanning under different curvatures by using the device, collecting a data set covering a plurality of NA ranges and all azimuth angles, and finally obtaining a high-quality image through calculation and reconstruction. Dual programmable control over the illumination angle is achieved, and the device is particularly suitable for deep optimization imaging of three-dimensional samples such as organoids and the like and has the remarkable advantages of being compact in structure and low in cost efficiency.

Description

Technical Field

[0001] This invention relates to the fields of microscopic imaging and biomedical optics and technology, specifically to a variable curvature scanning illumination device and its imaging method for inverted microscopes. It can be used in Fourier transform microscopy (FPM), differential phase contrast (DPC), dark field and other computational imaging modes for biological samples such as three-dimensional multicellular structures, organoids, and hydrogel spheres, to achieve dual programmable scanning of zenith angle and azimuth angle. Background Technology

[0002] Computational imaging techniques such as Fourier Ptychographic Microscopy (FPM) rely on illuminating samples from multiple spatial angles to achieve high-resolution imaging beyond the diffraction limit of objectives. An ideal illumination device should provide a high numerical aperture (NA), uniform and dense Fourier spatial sampling, while being compact and easy to integrate.

[0003] Existing illumination solutions each have limitations. Planar LED light source arrays are simple in structure, but their illumination range (NA) is limited, and Fourier space sampling is non-uniform. Fixed rigid arc light sources (including rotating and non-rotating types) can provide a large zenith angle, but their fixed curvature results in a fixed illumination NA range, which cannot be dynamically adjusted according to sample size, depth, objective working distance, or specific imaging requirements. For example, a fixed rotating arc device can achieve uniform azimuth coverage through rotation, but its zenith angle sampling range is locked. Hemispherical LED light source domes can provide the most comprehensive angular coverage, but their large size, high cost, and extremely complex wiring make them difficult to integrate into space-constrained inverted microscopes, hindering their widespread application.

[0004] For three-dimensional biological samples such as organoids, the optimal illumination strategy often needs to be dynamically adjusted according to the imaging depth; for example, imaging the surface structure may require a smaller illumination angle, while penetrating the deep layers of the sample requires a large angle of oblique illumination. No existing fixed structure (whether planar, fixed arc, or rotating fixed arc) can meet the needs of such dynamically optimized illumination NA. In summary, current illumination structures generally have the following shortcomings: (1) Single degree of freedom: Existing devices may be able to adjust the zenith angle (such as variable curvature arc) or scan the azimuth angle (such as rotating arc), but cannot simultaneously achieve dynamic programmable control of both degrees of freedom. (2) Poor adaptability: The illumination NA of a fixed curvature structure is not variable and cannot adapt to different samples and imaging tasks. (3) Incomplete sampling: The lack of dual scanning degrees of freedom makes it difficult to achieve complete, flexible, and high-density coverage of Fourier space. (4) Cost and performance contradiction: High-performance sampling schemes (such as domes) are too expensive, while low-cost schemes (such as planar arrays) have insufficient performance. Summary of the Invention

[0005] The purpose of this invention is to provide a variable curvature scanning illumination device and its imaging method for inverted microscopes that integrates rotation and curvature adjustment functions, is compact, and has a high degree of freedom. It aims to solve the technical problems of existing illumination devices having only one degree of freedom, fixed illumination NA, and being unable to adapt to the dynamic imaging requirements of three-dimensional samples.

[0006] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: a variable curvature scanning illumination device for an inverted microscope, comprising an inverted microscope and a flexible arc-shaped support located above the inverted microscope, wherein multiple independently addressable LED light sources are uniformly arranged along the arc direction on the flexible arc-shaped support; and further comprising: Curvature adjustment mechanism, used to adjust the curvature of the flexible arc support to change the NA range of each LED light source; The rotation mechanism is configured to control the flexible arcuate support to rotate around the optical axis of the inverted microscope at the current curvature.

[0007] Preferably, the flexible arc-shaped support includes multiple linkage units, and adjacent linkage units are connected by hinge joints. Each linkage unit is equipped with an independently addressable LED light source. The curvature adjustment mechanism is configured to control the rotation of each linkage unit around the hinge joint to adjust the curvature of the flexible arc-shaped support as a whole.

[0008] Preferably, the curvature adjustment mechanism includes a winding structure and a drive cable. The winding structure is installed at one end of the flexible arc-shaped bracket, and one end of the drive cable is connected to the other end of the flexible arc-shaped bracket. The other end of the drive cable is connected to the winding structure. The winding structure is configured to cause the flexible arc-shaped bracket to bend and change its curvature by tightening or loosening the drive cable.

[0009] Preferably, an angle sensor is installed at at least one hinge joint, and the curvature adjustment mechanism adopts a closed-loop control mechanism. Specifically, the closed-loop control mechanism is as follows: feedforward compensation control is performed based on the real-time feedback value of the angle sensor and the target curvature, so that the curvature of the flexible arc support converges to the preset target and remains stable during the acquisition.

[0010] Preferably, the rotating mechanism is used to support the flexible arc-shaped support and the curvature adjustment mechanism, and to drive the flexible arc-shaped support and the curvature adjustment mechanism to rotate as a whole around the optical axis of the inverted microscope.

[0011] Preferably, the rotating mechanism includes a stepper motor and a turntable bearing. The stepper motor is connected to the turntable bearing, and a flexible arc-shaped bracket is mounted on the turntable bearing. The stepper motor controls the rotation of the turntable bearing, which in turn drives the flexible arc-shaped bracket to rotate synchronously around the optical axis of the inverted microscope.

[0012] The present invention also discloses a variable curvature scanning imaging method for an inverted microscope, which uses a variable curvature scanning illumination device for an inverted microscope to perform scanning imaging, including the following steps: The curvature adjustment mechanism sequentially adjusts the flexible arc-shaped support to multiple different predetermined curvatures; The angle scanning cycle includes controlling the flexible arc support to rotate around the optical axis of the inverted microscope at a corresponding predetermined curvature through a rotating mechanism, and sequentially positioning it at several discrete azimuth angles. At each azimuth angle, the LED light source is lit sequentially, and the corresponding image is acquired by the camera. All images acquired at all curvatures and all angular positions are input into the imaging algorithm for reconstruction to obtain the final image.

[0013] Preferably, when the curvature adjustment mechanism controls the curvature adjustment mechanism to switch curvature, a cubic spline curve or S-shaped velocity curve is generated based on the actual value of the previous curvature and the target value of the curvature to be switched, so that continuous velocity and continuous acceleration are maintained throughout the switching process.

[0014] Preferably, a determination mechanism is executed before the angle scanning cycle is executed. The determination mechanism is as follows: if the error between the actual curvature and the target curvature is always less than a set threshold within a set stable time, then the state is determined to be stable, and the angle scanning cycle is started.

[0015] Preferably, a rapid calibration is performed before scanning imaging to correct for deviations caused by wear or temperature changes. This rapid calibration includes: Under the preset reference curvature of the flexible arc support, several LED light sources are selected from the flexible arc support for lighting, and the selected LED light sources are symmetrically distributed in the arc direction of the flexible arc support. The selected LED light sources are lit sequentially, and the corresponding imaging data is collected using a camera; Based on a pre-established LED spatial position model or illumination NA mapping relationship, the theoretical incident angle or equivalent NA value is calculated and compared with the illumination response characteristics reflected in actual imaging to obtain the geometric deviation of the current system. The curvature-NA mapping relationship or servo control parameters are corrected based on the geometric deviation.

[0016] Beneficial Effects: This invention achieves two-dimensional programmable control of the illumination solid angle by combining a flexible arc with variable curvature, a curvature adjustment mechanism, and a rotation mechanism. Curvature adjustment (controlling the zenith angle / NA range) is combined with rotational motion (scanning the azimuth angle). Specifically, rotation achieves complete azimuth coverage, while adjusting the curvature dynamically changes the sampling range and density of the zenith angle. This combination enables the invention to generate highly flexible and dense sampling patterns in Fourier space, such as depicting concentric circular sampling trajectories of arbitrary radius and density, thereby achieving seamless optimized coverage of Fourier space. This is crucial for high-fidelity image reconstruction.

[0017] In addition, the present invention is particularly suitable for imaging three-dimensional samples, and can acquire multiple datasets optimized for the sample surface (e.g., using large radius, low NA illumination) and deep structure (e.g., using small radius, high NA illumination) by switching different curvatures in the same experiment; through subsequent calculation and fusion, high-quality overall three-dimensional information of the sample from the surface to the interior can be obtained.

[0018] The device of this invention is not only cost-effective but also compact in structure, and can be easily integrated into a standard inverted microscope system, which greatly reduces the technical threshold and hardware cost of realizing advanced computational microscopy imaging. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the variable curvature scanning illumination device for an inverted microscope according to the present invention; Figure 2 This is a schematic diagram of a segmented structure of a specific embodiment of the flexible arc-shaped support of the present invention; Figure 3 This is a graph showing the variation of the numerical aperture (NA) of illumination under different curvature conditions in an embodiment of the present invention, with the geometric conditions being an arc length of 150 mm and an arc apex distance of 100 mm from the sample. Figure 4 This is a schematic diagram illustrating the application of the present invention in a three-dimensional hydrogel cell sample imaging scenario; Figure 5 This is a flowchart of the variable curvature scanning imaging method for an inverted microscope according to the present invention.

[0020] The following are the labels in the diagram: 1. Flexible arc-shaped support; 11. Linkage unit; 12. Hinge joint; 2. LED light source; 3. Curvature adjustment mechanism; 31. Drive cable; 32. Servo motor; 4. Rotation mechanism; 41. Stepper motor; 42. Turntable bearing; 5. Inverted microscope; 6. Sample. Detailed Implementation

[0021] To make the objectives and advantages of this invention clearer, the invention will be specifically described below with reference to embodiments. It should be understood that the following text is merely used to describe one or more specific embodiments of the invention and does not strictly limit the scope of protection specifically claimed by the invention.

[0022] Example: Reference Figure 1 As shown, the variable curvature scanning illumination device for an inverted microscope includes: an inverted microscope 5, a flexible arc-shaped support 1, a curvature adjustment mechanism 3, and a rotation mechanism 4; wherein: The flexible arc support 1 is located above the inverted microscope 5, and multiple independently addressable LED light sources 2 are evenly arranged on the flexible arc support 1 along the arc direction. The curvature adjustment mechanism 3 is installed on one side of the flexible arc support 1 to adjust the curvature of the flexible arc support 1 and change the NA range of each LED light source 2. The rotation mechanism 4 is mounted above the flexible arc support 1 and is connected to the microscope stage via an adapter connector so that the center of rotation is approximately aligned with the optical axis of the microscope. The sample 6 (such as an organoid in a culture dish) is placed on the stage in the usual manner. The rotation mechanism 4 is used to control the flexible arc support 1 to rotate around the optical axis of the inverted microscope 5 at the current curvature.

[0023] In one specific embodiment, the flexible arc support 1 includes multiple linkage units 11, and adjacent linkage units 11 are connected by hinge joints 12. Each linkage unit 11 is equipped with an independently addressable LED light source 2. The curvature adjustment mechanism 3 is configured to control each linkage unit 11 to rotate around the hinge joint 12 to adjust the curvature of the flexible arc support 1 as a whole. refer to Figure 2 As shown, each linkage unit 11 is made by high-precision 3D printing, with a tiny wiring channel reserved inside; adjacent linkage units 11 are connected by stainless steel pins as hinge joints 12, forming a single-degree-of-freedom hinge chain; a miniature PCB board is fixed on the outer surface of each linkage unit 11, and a WS2812B type independently addressable RGB LED light source 2 is soldered on the board as LED light source 2. All LED light sources 2 are connected through a serial bus, and only one data line is needed to control the lighting, color and brightness of all LED light sources 2; Based on the above-mentioned flexible arc support 1, in a specific embodiment, the curvature adjustment mechanism 3 adopts a wire-driven method. The curvature adjustment mechanism 3 includes a winding structure and a drive wire 31 (e.g., a high-strength, low-elongation Kevlar fiber wire). The drive wire 31 passes through the through holes reserved inside all the linkage units 11. One end of the pull wire is fixed to the end of the flexible arc-shaped bracket 1, and the other end is wound around the winding structure. The winding structure is used to tighten or loosen the drive pull wire 31. Its structure can be a spool driven by a high-torque servo motor 32. When the servo motor 32 rotates forward to loosen the pull wire, the flexible arc-shaped bracket 1 bends and deforms away from the side where the pull wire is laid under the action of reduced tension of the pull wire, thereby forming an arc-shaped structure. By precisely controlling the rotation angle of the servo motor 32, the degree of bending of the flexible arc-shaped bracket 1, i.e., the radius of curvature, can be continuously and repeatedly changed. In this embodiment, an angle sensor, such as a miniature rotary potentiometer, a magnetic angle encoder, or an optical encoder, is installed at at least one hinge joint 12 to detect the actual bending angle of the bracket in real time. The curvature adjustment mechanism 3 adopts a closed-loop control mechanism. The closed-loop control mechanism is specifically: based on the real-time feedback value of the angle sensor and the target curvature, feedforward compensation control is performed to make the curvature of the flexible arc-shaped bracket 1 converge to the preset target and remain stable during the acquisition period, thereby accurately calibrating and controlling the NA range of the lighting.

[0024] In this embodiment, the curvature of the flexible arc-shaped bracket 1 refers to the degree of bending of the bracket along its arc direction, which can be expressed by its arc radius R or the equivalent lighting zenith angle. Characterization was performed; due to the difference between the illumination numerical aperture NA and the zenith angle... There is a fixed relationship: ; Where n is the refractive index of the medium, the illumination NA of the LED light source 2 relative to the sample 6 can be continuously changed by adjusting the curvature of the support.

[0025] refer to Figure 3 The figure shows the variation of the numerical aperture (NA) of illumination under different curvature conditions (n=1.33) in this embodiment under the geometric conditions (arc length 150 mm, arc apex distance from sample 6100 mm). Figure 3 It can be seen that the illumination NA shows a significant monotonic increasing trend with the curvature of the support (expressed as 1 / mm); when the curvature approaches 0 (the support is nearly straight), the geometric deflection angle of the light source relative to sample 6 is small, and the calculated illumination NA is approximately 1.10; when the curvature increases to approximately 0.01 (1 / mm), the lateral offset of the arc-end LED light source 2 relative to sample 6 increases significantly, and the zenith angle... This increases the illumination NA to approximately 1.25.

[0026] The above results indicate that, under the conditions of fixed arc length and fixed apex height, the curvature of the flexible arc support 1 is the main adjustment variable determining the illumination NA; as the flexible arc support 1 gradually transitions from low curvature to high curvature, the effective incident direction of the LED light source 2 gradually changes from paraxial illumination to strong oblique illumination, thereby making the NA continuously adjustable in the range of 1.10–1.25; this range of NA variation covers the needs of cell imaging from low-angle enhanced contrast to high-angle deep-penetration illumination.

[0027] refer to Figure 4 The figure shows a schematic diagram of the application of the present invention in a three-dimensional hydrogel cell sample 6 imaging scenario (the hydrogel material is transparent or semi-transparent). The figure visually compares the lighting effects under two different curvature states. Figure 4 (a) demonstrates that the device is in a small radius of curvature (large curvature) state to achieve high numerical aperture (NA) oblique illumination of the deep structure of sample 6 (such as organoids or hydrogel spheres); Figure 4 (b) shows that the device is in a state with a large radius of curvature (small curvature), which is suitable for low numerical aperture (NA) illumination of the surface of sample 6.

[0028] In one specific embodiment, the rotating mechanism 4 includes a stepper motor 41 and a turntable bearing 42. The stepper motor 41 is connected to the turntable bearing 42, and the flexible arc-shaped bracket 1 is mounted on the turntable bearing 42. The stepper motor 41 controls the rotation of the turntable bearing 42, which in turn drives the flexible arc-shaped bracket 1 to rotate synchronously around the optical axis of the inverted microscope 5.

[0029] This device also includes a main controller for controlling the curvature adjustment mechanism 3, the rotation mechanism 4, and the LED light source 2, respectively, for curvature, rotation, and illumination. In a specific example, an STM32F4 series microcontroller is used as the main controller; specifically including: Host computer interface: The main controller communicates with a PC (host computer) via a USB virtual serial port; users can set scanning parameters (such as NA list, rotation step angle, exposure time, etc.) on the graphical user interface (GUI) software on the PC. Drive control: The main controller controls the drive through different I / O ports, including: Rotation control: Pulse (PUL) and direction (DIR) signals are sent to the stepper motor 41 driver to control the precise rotation of the rotary mechanism 4; Curvature control: Output PWM signal to servo motor 32 to control the tension of drive cable 31 and adjust the curvature of flexible arc bracket 1; at the same time, read the feedback voltage of rotary potentiometer through ADC to realize closed-loop control of curvature; Lighting control: Serial data is sent to the WS2812B LED light source 2 light chain via a single data cable to control the lighting status of any one or more LED light sources 2; The main controller sends a high-level pulse to the camera's external trigger port via a GPIO port to precisely synchronize image acquisition with the lighting of LED light source 2.

[0030] In another embodiment, reference Figure 5 As shown, a variable curvature scanning imaging method for an inverted microscope 5 is provided, specifically including: The curvature adjustment mechanism 3 sequentially adjusts the flexible arc support 1 to multiple different predetermined curvatures; The angle scanning cycle is executed by controlling the flexible arc support 1 to rotate around the optical axis of the inverted microscope 5 under the corresponding predetermined curvature through the rotation mechanism 4, and positioning it in a number of discrete azimuth angles in sequence. At each azimuth angle position, the LED light source 2 is lit in sequence, and the corresponding image is acquired by the camera. All images acquired at all curvatures and all angular positions are input into an imaging algorithm (such as Fourier layered microscopy algorithm) for reconstruction to obtain the final image.

[0031] Take two different curvatures (curvature one and curvature two) as an example: The control system sends the target value of curvature one to the curvature adjustment mechanism 3. The curvature adjustment mechanism 3 retracts and extends the drive cable 31 at a preset speed, so that the flexible arc support 1 gradually approaches the target curvature. During the adjustment process, the angle sensor detects the bending state of the support in real time and feeds it back to the control system. The control system performs closed-loop adjustment of the driver according to the feedback error until the deviation between the actual curvature of the support and the target curvature one is less than the preset threshold. Then the system maintains the curvature and enters the curvature stabilization state, and begins the rotational scanning and LED light source 2 lighting sequence below the curvature. After acquiring data at curvature one, the control system issues the target value for curvature two. The curvature adjustment mechanism 3 smoothly tightens or loosens the drive cable 31 according to the pre-planned curvature transition trajectory, so that the flexible arc support 1 gradually transitions from curvature one to curvature two. During the transition, in order to avoid vibration caused by inertia or cable elasticity, a smooth trajectory that limits the maximum speed and maximum acceleration is adopted. When the actual curvature of the support stabilizes within the threshold range corresponding to curvature two and there is no significant change within the set stabilization time, the control system switches to the stable state of curvature two and starts the rotational scan and the second imaging sequence.

[0032] The curvature values ​​of curvature one and curvature two can change in the same direction (e.g., both bend inward, but to different degrees), or they can cross straight lines.

[0033] In order to reduce mechanical vibration and ensure the spatial position consistency of LED light source 2 during the process of switching from curvature one to curvature two, this embodiment adopts smooth trajectory planning in the curvature switching stage; the control system generates a cubic spline curve or S-shaped velocity curve according to the actual value of curvature one and the target value of curvature two, so that the winding spool maintains continuous speed and continuous acceleration throughout the switching process and avoids instantaneous "impact".

[0034] During curvature switching, the control system continuously reads the feedback signal from the angle sensor and performs fine-tuning of the curvature adjustment mechanism 3 when necessary to correct deviations caused by friction, wire slippage, or linkage clearance. Before executing the angle scanning cycle, a judgment mechanism is executed. The judgment mechanism is as follows: if the error between the actual curvature and the target curvature is always less than the set threshold within the set stable time, it is judged to be in a stable state and enters the next round of rotation scanning. This smooth transition method effectively improves the curvature consistency after dynamic switching and avoids illumination NA fluctuations caused by curvature jitter, thereby ensuring the availability of imaging data.

[0035] In a specific FPM imaging process: After initial use or lens replacement, a one-time calibration is performed. Multiple servo motor positions are set in segments within the range of minimum to maximum achievable curvature of the flexible arc-shaped support 1 by controlling servo motors 32. The potentiometer readings corresponding to each servo motor position are recorded, and the actual zenith angle or equivalent NA of the support is determined through geometric calculations or optical measurements, establishing a mapping relationship between potentiometer readings and zenith angle (or NA). Simultaneously, in a fully extended state, the precise position of each LED light source 2 in the spatial coordinate system is determined by rotation and sequential illumination. When the user inputs the target NA or target curvature, the control system locates the corresponding winding spool position based on the above mapping and performs the adjustment.

[0036] This embodiment performs a rapid calibration before scanning imaging to correct deviations caused by wear or temperature changes, further ensuring the consistency of the illumination geometry. The rapid calibration includes: With the flexible arc support 1 under a preset reference curvature, a small number of LED light sources 2 are selected from the flexible arc support 1 for lighting, and the selected LED light sources 2 are symmetrically distributed in the arc direction of the flexible arc support 1; for example, LED light source points located in the middle of the flexible arc support and at both ends of the arc can be selected. The selected LED light sources are lit sequentially, and the corresponding imaging data is collected using a camera; Based on a pre-established LED spatial position model or illumination NA mapping relationship, the theoretical incident angle or equivalent NA value is calculated and compared with the illumination response characteristics reflected in actual imaging to obtain the geometric deviation of the current system. The geometric deviation can be characterized as a small shift in the overall curvature of the flexible arc support, the overall shift of the equivalent illumination NA, or a proportional change. The control system corrects the curvature-NA mapping relationship or the control parameters of the servo motor 32 based on the deviation, thereby completing the rapid calibration. Since this calibration process only involves lighting up a small number of LED light source points and acquiring a small number of images, the calibration time is significantly shortened compared to the first full calibration. It can effectively correct minor geometric deviations caused by wear, wire slippage, or changes in ambient temperature, without affecting the overall imaging efficiency of the system.

[0037] In the host computer software, the operator sets parameters according to the characteristics of sample 6 and imaging requirements; in this embodiment, for an organoid with a diameter of about 300 μm, three NA ranges are set for scanning; NA range 1: 0.1-0.3 (corresponding to radius of curvature R1); NA range 2: 0.3-0.5 (corresponding to radius of curvature R2) NA range 3: 0.5-0.7 (corresponding to radius of curvature R3) and the azimuth rotation step is set to 10 degrees (i.e., 36 angles are collected for each NA circle). Set the first curvature: The main controller drives the servo motor 32 to the position corresponding to the curvature radius R1; Perform the first round of rotational scanning: Stepper motor 41 rotates to 0 degrees; The main controller sequentially lights up 15 LED light sources 2 on the flexible arc bracket 1. After each LED light source 2 is lit, a 20ms delay is set to wait for it to stabilize before sending a 5ms trigger pulse to the camera. The camera then exposes and captures an image. Stepper motor 41 rotates to 10 degrees and repeats the scanning process of LED light source 2. The first round of rotational scanning is completed when stepper motor 41 rotates to 350 degrees (a total of 36×15 = 540 images are acquired). Set the second curvature and scan: The main controller drives the servo motor 32 to the position corresponding to the curvature radius R2, and then repeats the entire rotation scanning process described above to acquire 540 images again.

[0038] Set the third curvature and scan: The main controller drives the servo motor 32 to the position corresponding to the curvature radius R3, and repeats the entire rotation scanning process described above to acquire another 540 images; After the scan was completed, a total of 540×3 =1620 low-resolution images were obtained. These images, along with their corresponding LED light source 2 position information (calculated based on calibration data and real-time scanning parameters), were transmitted to the PC. The FPM reconstruction algorithm on the PC used this data, which covered different NA rings and all azimuth angles, to perform iterative optimization and finally reconstruct a final image that has high resolution, a large field of view, and good depth information for the three-dimensional sample 6.

[0039] The embodiments of the present invention have been described in detail above with reference to the examples. However, the present invention is not limited to the above embodiments. For those skilled in the art, after learning the contents described in the present invention, several equivalent changes and substitutions can be made without departing from the principle of the present invention. These equivalent changes and substitutions should also be considered to fall within the protection scope of the present invention.

Claims

1. A variable curvature scanning illumination device for an inverted microscope, characterized in that: It includes an inverted microscope and a flexible arc-shaped support located above the inverted microscope. Multiple independently addressable LED light sources are evenly arranged on the flexible arc-shaped support along the arc direction. And, also includes: Curvature adjustment mechanism, used to adjust the curvature of the flexible arc support to change the NA range of each LED light source; The rotation mechanism is configured to control the flexible arcuate support to rotate around the optical axis of the inverted microscope at the current curvature.

2. The variable curvature scanning illumination device for an inverted microscope according to claim 1, characterized in that: The flexible arc-shaped support includes multiple linkage units, and adjacent linkage units are connected by hinge joints. Each linkage unit is equipped with an independently addressable LED light source. The curvature adjustment mechanism is configured to control the rotation of each linkage unit around the hinge joint to adjust the curvature of the flexible arc-shaped support as a whole.

3. The variable curvature scanning illumination device for an inverted microscope according to claim 2, characterized in that: The curvature adjustment mechanism includes a winding structure and a drive cable. The winding structure is installed at one end of the flexible arc-shaped bracket, and one end of the drive cable is connected to the other end of the flexible arc-shaped bracket. The other end of the drive cable is connected to the winding structure. The winding structure is configured to cause the flexible arc-shaped bracket to bend and change its curvature by tightening or loosening the drive cable.

4. The variable curvature scanning illumination device for an inverted microscope according to claim 2, characterized in that: An angle sensor is installed at at least one hinge joint. The curvature adjustment mechanism adopts a closed-loop control mechanism. Specifically, the closed-loop control mechanism is as follows: feedforward compensation control is performed based on the real-time feedback value of the angle sensor and the target curvature, so that the curvature of the flexible arc support converges to the preset target and remains stable during the acquisition.

5. The variable curvature scanning illumination device for an inverted microscope according to claim 1, characterized in that: The rotating mechanism is used to support the flexible arc-shaped support and the curvature adjustment mechanism, and to drive the flexible arc-shaped support and the curvature adjustment mechanism to rotate as a whole around the optical axis of the inverted microscope.

6. The variable curvature scanning illumination device for an inverted microscope according to claim 1, characterized in that: The rotating mechanism includes a stepper motor and a turntable bearing. The stepper motor is connected to the turntable bearing, and a flexible arc-shaped bracket is mounted on the turntable bearing. The stepper motor controls the rotation of the turntable bearing, which in turn drives the flexible arc-shaped bracket to rotate synchronously around the optical axis of the inverted microscope.

7. A variable curvature scanning imaging method for an inverted microscope, comprising using the variable curvature scanning illumination device for an inverted microscope as described in any one of claims 1-6 for scanning imaging, characterized in that: Includes the following steps: The curvature adjustment mechanism sequentially adjusts the flexible arc-shaped support to multiple different predetermined curvatures; The angle scanning cycle includes controlling the flexible arc support to rotate around the optical axis of the inverted microscope at a corresponding predetermined curvature through a rotating mechanism, and sequentially positioning it at several discrete azimuth angles. At each azimuth angle, the LED light source is lit sequentially, and the corresponding image is acquired by the camera. All images acquired at all curvatures and all angular positions are input into the imaging algorithm for reconstruction to obtain the final image.

8. The variable curvature scanning imaging method for an inverted microscope according to claim 7, characterized in that: When the curvature adjustment mechanism controls the curvature adjustment mechanism to switch curvature, a cubic spline curve or S-shaped velocity curve is generated based on the actual value of the previous curvature and the target value of the curvature to be switched, so that continuous speed and continuous acceleration are maintained throughout the switching process.

9. The variable curvature scanning imaging method for an inverted microscope according to claim 8, characterized in that: Before executing the angle scanning cycle, a determination mechanism is performed. The determination mechanism is as follows: if the error between the actual curvature and the target curvature is always less than a set threshold within a set stable time, then it is determined to be a stable state, and the angle scanning cycle is started.

10. The variable curvature scanning imaging method for an inverted microscope according to claim 7, characterized in that: A rapid calibration is performed before scanning imaging to correct for deviations caused by wear or temperature variations. This rapid calibration includes: Under the preset reference curvature of the flexible arc support, several LED light sources are selected from the flexible arc support for lighting, and the selected LED light sources are symmetrically distributed in the arc direction of the flexible arc support. The selected LED light sources are lit sequentially, and the corresponding imaging data is collected using a camera; Based on a pre-established LED spatial position model or illumination NA mapping relationship, the theoretical incident angle or equivalent NA value is calculated and compared with the illumination response characteristics reflected in actual imaging to obtain the geometric deviation of the current system. The curvature-NA mapping relationship or servo control parameters are corrected based on the geometric deviation.

Images