An ultramicroscopic imaging device based on mask turntable modulation excitation light

Abstract

This invention discloses a super-resolution microscopic imaging device based on mask-driven rotating disk modulation of excitation light. It includes a laser source assembly, a rotating disk mask module, a synchronization control module, a microscopic imaging component, and an image processing unit. The rotating disk mask module comprises a rotating disk and a mask mounted on it, with several modulation units formed on the mask surface. The synchronization control module generates three phase-locked PWM signals via an FPGA chip to achieve microsecond-level timing synchronization of stepper motor drive, laser switching, and camera exposure. In the optical path design of this invention, the laser is incident on the mask plane and then forms a sub-diffraction-limited spot via the microscopic imaging component. This device, by combining the captured time-series images with fluorescence fluctuation super-resolution microscopic imaging technology, can achieve low-cost, high-fidelity, and high-resolution high-order fluorescence fluctuation super-resolution microscopic images, which is of great significance for observation and imaging in cell biology.

Description

Technical Field

[0001] This invention relates to the field of fluorescence microscopy, specifically to a super-resolution microscopy device based on mask turntable modulation of excitation light. Background Technology

[0002] Super-resolution microscopy based on fluorescence fluctuations is a newly developed technique that achieves super-resolution imaging by calculating the correlation cumulants of time-series fluorescence fluctuation signals. However, due to the inherently inappropriate fluorescence scintillation ratio of fluorescent dyes, artifacts can occur during high-order cumulant calculations, thus limiting further improvements in resolution and image quality. Currently, there are two main approaches to addressing this issue:

[0003] 1) Wave modulation is achieved by optimizing fluorescent dyes, with Pdots[1] proposed by Wu Changfeng's team at Southern University of Science and Technology being a prime example.

[0004] High-order imaging can be achieved by designing the fluorescence scintillation ratio of polymer dots, but this method has high requirements for sample preparation and is difficult to control the scintillation ratio.

[0005] 2) Active modulation of the fluctuation through illumination is mainly represented by the random illumination-active control fluorescence fluctuation super-resolution microscopy (AR-SOFI) developed by the Ma Jiong research group at Fudan University [2]. By using a liquid crystal spatial light modulator (SLM) or a digital microlens array (DMD), a random illumination source is generated. The scintillation ratio of the fluorescence fluctuation is controlled by performing time-series dynamic speckle modulation on the random illumination source. This method has relatively higher compatibility with sample preparation, but the cost of traditional liquid crystal spatial light modulators or digital microlens arrays is still relatively high, and the practical application prospects are still limited.

[0006] References

[0007] [1] Sun Z, Liu Z, Chen H, et al . Semiconducting Polymer Dots withModulated Photoblinking for High-Order Super-Resolution Optical FluctuationImaging[J]. Advanced Optical Materials , 2019, 7(9):1900007.1-1900007.9.DOI:10.1002 / adom.201900007;

[0008] [2] Wang B, Liu Z, Zhou L, et al . Active-modulated, random-illumination, super-resolution optical fluctuation imaging[J]. Nanoscale ,2020, 12.DOI:10.1039 / D0NR03255G. Utility Model Content

[0009] To address the shortcomings of existing fluorescence fluctuation super-resolution microscopy techniques, this invention aims to propose a super-resolution microscopy device based on mask-driven rotating disk-modulated excitation light. This device can achieve low-cost, high-fidelity, and high-resolution high-order fluorescence fluctuation super-resolution microscopic images, which is of great significance for observation and imaging in cell biology.

[0010] The technical solution of this utility model is described in detail below.

[0011] A super-resolution microscopic imaging device based on mask-driven rotating disk-modulated excitation light includes a laser source assembly, a rotating disk mask module, a microscopic imaging assembly, and an image processing unit; wherein:

[0012] The laser source assembly includes a laser source for generating an excitation beam and a beam expander and collimating lens for forming a parallel light field.

[0013] The turntable mask module includes a turntable and a mask. The mask is circular and consists of a base layer and a pattern layer. The mask is fixedly mounted on the turntable, which is driven by a stepper motor. Several randomly distributed modulation units are formed on the surface of the mask. The modulation units are used to modulate the excitation light field incident on the mask plane.

[0014] The microscopic imaging component is used to focus the modulated excitation light field onto the sample to excite the fluorescent spots in the sample to generate fluorescence signals;

[0015] The image processing unit is used to perform image analysis on the fluorescence time-series images acquired by the camera to achieve super-resolution reconstruction.

[0016] In this invention, modulation units of different sizes are non-periodicly distributed in the inner circle and different concentric rings within the mask.

[0017] In this invention, the number of concentric rings is 2-4 layers, and the size of the modulation unit increases in a gradient from the inside to the outside.

[0018] In this invention, the base layer is made of transparent or semi-transparent quartz glass or polymer material, and the pattern layer is a metal plating layer, a photoresist layer, or a dye coating.

[0019] In this invention, the light transmittance of the base layer material is ≥90%; the surface roughness Ra is ≤0.1 μm; and the coefficient of thermal expansion is ≤5×10⁻⁶. -6 / ℃.

[0020] In this invention, the thickness of the base layer is 0.05-0.15mm; the size of the modulation unit is 50-70μm, and the edge is processed with a serrated anti-diffraction structure, with a light transmittance of 20%-30%.

[0021] In this invention, the mask is a film mask, the base layer is a polyester film base layer, the pattern layer is a silver halide emulsion layer, and the thickness of the silver halide emulsion layer is 3-7 μm.

[0022] This invention also includes a synchronization control module. The synchronization control module uses an embedded system to generate phase-locked control signals through an FPGA chip, which drive the stepper motor, laser light source and camera respectively, so as to control and realize the microsecond-level timing synchronization of stepper motor drive, laser switch and camera exposure.

[0023] In this invention, a displacement stage is provided below the stepper motor to adjust the position of the collimated laser beam irradiating the mask on the turntable, thereby enabling the switching of the modulation unit during operation.

[0024] Compared with the prior art, the beneficial effects of this utility model are as follows:

[0025] This invention achieves a significant breakthrough in cost, performance, and reliability through the synergistic innovation of a rotary mask design, a hardware-level synchronous control architecture, and an optimized optical path system. Compared to traditional DMD solutions, the total system cost is greatly reduced, with the film mask manufacturing cost less than 1% of that of traditional chromium masks. It also supports in-situ correction technology and, combined with commercially available optical components and ZYNQ embedded control modules, completely eliminates the reliance on imported high-value components. Attached Figure Description

[0026] Figure 1 Schematic diagram of a modulation illumination microscopic imaging system based on rotary control.

[0027] Figure 2 A schematic diagram of turntable control based on a circular mask.

[0028] Figure 3 Timing diagram for flash synchronization mode.

[0029] Figure 4 Alexa 488 tags BSC-1 microtubes in 1000 frames of average wide-field images with modulation.

[0030] The numbers in the diagram are: 1-Laser, 2-Beam expander and collimator system, 3-Turntable, 4-Stepper motor, 5-Modulation unit, 6-Fluorescent tube, 7-Microscope objective, 8-Sample, 9-Camera, 10-Control system. Detailed Implementation

[0031] The technical solution of this utility model will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0032] This invention provides a super-resolution microscopic imaging device based on mask-driven rotating disk-modulated excitation light, comprising a laser source assembly, a rotating disk mask module, a microscopic imaging assembly, and an image processing unit; wherein:

[0033] The laser source assembly includes a laser source for generating an excitation beam and a beam expander and collimating lens for forming a parallel light field.

[0034] The turntable mask module includes a turntable and a mask. The mask is circular and consists of a base layer and a pattern layer. The mask is fixedly mounted on the turntable, which is driven by a stepper motor. Several randomly distributed modulation units are formed on the surface of the mask. The modulation units are used to modulate the excitation light field incident on the mask plane.

[0035] The microscopic imaging component is used to focus the modulated excitation light field onto the sample to excite the fluorescent spots in the sample to generate fluorescence signals;

[0036] The image processing unit is used to perform image analysis on the fluorescence time-series images acquired by the camera to achieve super-resolution reconstruction;

[0037] like Figure 1 As shown, the laser source assembly includes a laser 1 and a beam expansion and collimation system 2. The laser 1 provides light source excitation, which is then irradiated onto the turntable 3 of the turntable mask module after passing through the beam expansion and collimation system 2. The turntable 3 is driven by a stepper motor 4. The surface of the turntable 3 forms speckle illumination through the modulation unit 5. After passing through the dichroic mirror in the fluorescence tube 6 of the microscopic imaging assembly and the microscope objective 7, the light is irradiated onto the sample surface. After the sample is excited to fluoresce, the fluorescence is collected by the dichroic mirror and imaged in the camera 9. The stepper motor 4 is controlled by the control system 10 to realize the continuous rotation of the turntable 3, thereby realizing the temporally continuous speckle illumination modulation and automatic shooting by the camera.

[0038] like Figure 2As shown, in a circular mask, modulation units of different sizes are distributed in concentric rings in a non-periodic manner. The size of several modulation units within the inner circle and different concentric rings is the same. When the light source is projected onto the surface of the rotating disk after beam expansion and collimation, it forms a speckle pattern due to the influence of the modulation units on the disk surface. As the disk rotates, the sample exhibits a temporally random speckle illumination effect. Because different concentric rings contain different modulation units, different types of speckle illumination can be formed, achieving various modulation effects. By adjusting the position of the collimated laser beam illuminating the mask on the rotating disk, the modulation units can be switched during operation. Figure 2 The left side displays a turntable and a mask mounted on it. The mask shows concentric circles containing modulation units of different sizes. Figure 2 The right side shows magnified views of different modulation units under different concentric circles from the inner ring to the outer ring.

[0039] The following describes the specific implementation process of the dynamic light field microscopy imaging system with reference to specific embodiments.

[0040] 1) Hardware Construction

[0041] First, a film mask turntable module was fabricated using a 0.1mm thick polyester film as the substrate material, with a transmittance ≥90% and tensile strength >200MPa. A 5µm thick silver halide emulsion layer was coated on the surface to form randomly distributed modulation units with a transmittance of 25%±2%. Using a laser direct-writing device, 55µm×55µm units were etched in the inner ring with a processing accuracy of ±0.5µm, and 70µm×70µm units were etched in the outer ring. A serrated anti-diffraction structure with a depth of 2µm and a spacing of 5µm was machined at the edge of each unit. The film mask was fixed to an 80mm diameter aluminum alloy turntable and mounted to the drive shaft of a stepper motor (model 17HS19-2004S1), ensuring a radial positioning accuracy of ±2µm. The synchronization control module uses the ZYNQ-7010 development board as its core, with an FPGA chip (XC7Z010) connected to 512MB of DDR3 memory. It outputs three phase-locked PWM signals: PWM1 connects to a stepper motor driver (TB6600, drive current 1.5A), PWM2 controls the TTL modulation port of the 488nm wavelength laser, and PWM3 triggers the flash synchronization mode of the EMCCD camera (Andori Xon Ultra888). During optical system setup, the laser beam is collimated into 8mm diameter parallel light by a collimating lens group (Thorlabs AC254-030-A), incident on the mask turntable plane, and magnified 420 times by an Olympus 150x / 1.45NA objective lens combined with a 200mm focal length tube lens, forming a 125nm×125nm modulation spot on the sample surface. A dichroic mirror (Semrock FF509-FDi01) separates the excitation light and fluorescence signal.

[0042] 2) Software control

[0043] A Linux system is deployed on the ZYNQ development board. An FPGA logic core is developed using Vivado 2020.1. A Python script runs on the ARM side to receive user parameter configurations. The FPGA generates three strictly synchronized PWM signals. The timing logic is as follows: after the motor stepper signal (S1) is triggered, the light source is started after a 40-second delay (S2), and then the camera exposure is triggered after another 90-second delay (S3). The signal parameters are stored in DDR3 memory and support offline operation. The host computer is developed based on LabVIEW 2021, integrating parameter setting, real-time monitoring, and data storage functions. Users can input parameters such as rotational speed (0-300 RPM), laser power (0-100 mW), and exposure time (0.1-100 ms). The system displays motor torque, light power, and frame rate data in real time. The original image sequence is stored in TIFF format and a timestamp log with ±1 second accuracy is recorded.

[0044] 3) Operating Procedures

[0045] The system was first initialized, with the film mask switched to the inner ring 55m element. Oil was added to the objective lens and the image was focused onto the sample. The laser was preheated for 10 minutes and then set to a power of 50mW. In the LabVIEW interface, the rotation speed was set to 120RPM, the exposure time to 2ms, and delay parameters t1=40s and t2=90s. The FPGA output an 800Hz pulse to drive the motor stepping. After each step, the laser was activated and the camera was exposed. The EMCCD acquired fluorescence images at 30fps, with a signal-to-noise ratio >60dB. Image post-processing used the ImageJ plugin for background subtraction and bleaching correction, and reconstruction was performed using a CUDA 11.0 accelerated algorithm, achieving a spatial resolution of 180nm. For multi-parameter experiments, the turntable was moved radially by 3mm via commands to switch to the outer ring 70m element. The mirror group was fine-tuned by <0.1° to achieve optical path collimation, taking <50ms. After calibrating the conjugate surface, a 210nm×210nm spot was formed, completing the imaging of nanoparticle distribution. Example 1

[0046] Alexa Fluor 488-labeled tubulin was excited using a 488nm laser. An active modulation module dynamically switched the film mask pattern at a frequency of 200Hz, inducing spatiotemporal random scintillation of fluorescent molecules. An EMCCD camera acquired 10,000 frames of raw images at 50fps. After reconstruction using a fourth-order SOFI algorithm, the spacing between adjacent microtubules in the white line region was improved from an indistinguishable state in low-order imaging (half-width at half maximum > 300nm) to 159nm. Combined with an expansion coefficient of 4.5, the actual physical resolution reached 35nm, clearly distinguishing adjacent microtubule structures with a spacing of 64nm. Figure 4In this process, the defects of the expanded sample are overcome through two mechanisms: first, the high spatiotemporal frequency characteristics of active modulation enhance the layering ability and effectively suppress the defocused background fluorescence caused by gel scattering (signal-to-noise ratio improved by 8.2dB); second, the statistical correlation of fluorescence fluctuation signals compensates for the effect of the decrease in label density, so that sparsely distributed probes can still reconstruct the continuous structure through high-order accumulation.

[0047] In summary, this invention achieves a significant breakthrough in cost, performance, and reliability through the synergistic innovation of a rotary film mask design, a hardware-level synchronous control architecture, and an optimized optical path system. In terms of technical performance, based on sub-microsecond synchronous control (timing error ≤0.1), the image ghosting rate is reduced from 5.2% in traditional solutions to 0.3%, and the light energy utilization rate is increased to 85% (compared to only 33% in the DMD system). After 420x magnification using the objective lens and tube lens combination, a high-resolution modulation of 125nm×125nm is formed on the sample surface. The light spot size meets the requirements of super-resolution imaging; the system reliability is significantly enhanced, the anti-static properties of the film mask reduce the contamination rate by 87%, the polyester substrate can withstand temperature changes from -20℃ to 60℃, the continuous working life is >2000 hours, and the automatic optical path calibration technology controls the conjugate deviation within 0.5, improving debugging efficiency by 12 times; in terms of application scalability, it supports multi-wavelength switching of 405 / 488 / 561nm, recall of 6 sets of pre-stored parameters and integration of third-party algorithms, and the mode switching time is <50ms; it is suitable for dynamic observation of live cells.

Claims

1. A super-resolution microscopic imaging device based on mask-driven rotating disk-modulated excitation light, characterized in that, It includes a laser source assembly, a rotating mask module, a microscopic imaging assembly, and an image processing unit; wherein: The laser source assembly includes a laser source for generating an excitation beam and a beam expander and collimating lens for forming a parallel light field. The turntable mask module includes a turntable and a mask. The mask is circular and consists of a base layer and a pattern layer. The mask is fixedly mounted on the turntable, which is driven by a stepper motor. Several randomly distributed modulation units are formed on the surface of the mask. The modulation units are used to modulate the excitation light field incident on the mask plane. The microscopic imaging component is used to focus the modulated excitation light field onto the sample to excite the fluorescent spots in the sample to generate fluorescence signals; The image processing unit is used to perform image analysis on the fluorescence time-series images acquired by the camera to achieve super-resolution reconstruction.

2. The super-resolution microscopic imaging apparatus based on mask-rotating disk modulated excitation light according to claim 1, wherein, In the mask, modulation units of different sizes are distributed non-periodically in the inner circle and in different concentric rings.

3. The super-resolution microscopic imaging apparatus based on mask-turing-disc modulated excitation light according to claim 2, wherein, The number of concentric rings is 2-4 layers, and the size of the modulation unit increases in a gradient from the inside to the outside.

4. The super-resolution microscopic imaging apparatus based on mask-rotating disk modulated excitation light according to claim 1, wherein, The base layer is made of transparent or semi-transparent quartz glass or polymer material, and the pattern layer is a metal plating layer, photoresist layer or dye coating.

5. The mask-based turntable modulated excitation light super-resolution microscopic imaging device according to claim 1, wherein, The base layer material has a light transmittance of ≥ 90%; a surface roughness Ra of ≤ 0.1 m; a thermal expansion coefficient of ≤ 5 x 10 -6 / °C.

6. The mask-based turntable modulated excitation light super-resolution microscopic imaging device according to claim 1, wherein, The substrate thickness is 0.05-0.15mm; the modulation unit size is 50-70μm, and the edges are processed with a serrated anti-diffraction structure, with a light transmittance of 20%-30%.

7. The mask-based turntable-modulated excitation light super-resolution microscopic imaging apparatus according to claim 1, wherein, The mask is a film mask, its base layer is a polyester film base layer, and the pattern layer is a silver halide emulsion layer with a thickness of 3-7 μm.

8. The mask-based turntable-modulated excitation light super-resolution microscopic imaging apparatus according to claim 1, wherein, It also includes a synchronization control module, which uses an embedded system to generate phase-locked control signals through an FPGA chip to drive the stepper motor, laser light source and camera respectively, so as to achieve microsecond-level timing synchronization of stepper motor drive, laser switch and camera exposure.

9. The mask-based turntable-modulated excitation light super-resolution microscopic imaging apparatus according to claim 1, wherein, A displacement stage is set below the stepper motor to adjust the position of the collimated laser beam illuminating the mask on the turntable, thereby enabling the switching of the modulation unit during operation.

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