Combined imaging system based on single-rotating-disk microscopy and near-field enhancement of metamaterials

Abstract

The application relates to biomedical optical imaging technology and discloses a combined imaging system based on single-turntable microscopy and near-field enhancement of metamaterials, which comprises an excitation light beam light source modulation module for outputting uniform light spots; the excitation light beam is incident to a single-turntable platform through a polarization matching module and a dichroic mirror; a pinhole is uniformly distributed along a plurality of Archimedes spiral lines on the single-turntable platform; the laser beam transmitted through the pinhole on the single-turntable platform is sequentially converged on a sample table through a cylindrical mirror, a first galvanometer, a second galvanometer and an objective lens to excite a sample to generate fluorescence; the generated fluorescence returns to the dichroic mirror along the original path and is received by the imaging module; the single-turntable platform is arranged in the conjugate plane of the objective lens; and a layered double-hyperbolic metamaterial film is arranged on the surface of the sample table. The application can significantly improve the spatial resolution to about 80 nm while maintaining the millisecond-level high frame rate.

Description

Technical Field

[0001] This invention relates to the field of biomedical optical imaging technology, specifically a combined imaging system based on single-disc microscopy and metamaterial near-field enhancement. It combines high-speed single-disc scanning with near-field enhancement of hyperbolic metamaterials (HMM) to achieve dynamic live-cell imaging, and is particularly suitable for four-dimensional spatiotemporal analysis of immune synaptic dynamics and cell signal transduction mechanisms. Background Technology

[0002] In modern life science research, the dynamic spatiotemporal control of intracellular microscopic processes has become a focus, especially the immune synapse, a key structure for information transmission and effector execution. The formation and remodeling of immune synapses exhibit complex characteristics of "rapid dynamics, multi-scale spatial organization, and strong heterogeneity," which are closely related to the occurrence and development of diseases. Current microscopic imaging techniques generally suffer from a "dual bottleneck": in the temporal dimension, although techniques such as rotating disk confocal microscopy can achieve millisecond-level rapid scanning, they are limited by the diffraction limit (approximately 250 nm) and cannot resolve TCR nanoclusters at the 80-100 nm scale within the core region of the immune synapse; while super-resolution techniques such as STED and PALM / STORM can overcome the spatial limit, their high phototoxicity and low frame rate prevent them from capturing the instantaneous dynamics of the TCR-pMHC binding trigger signal within 50-200 ms.

[0003] With the development of metamaterials technology, it has been discovered that materials with negative refractive indices or surface plasmon resonances can overcome the diffraction limit. However, existing metamaterial imaging techniques (such as superlenses) are typically large in size, complex in fabrication processes, and difficult to achieve large-area scanning imaging. Hyperbolic metamaterials (HMMs) create high optical density states that do not exist in nature through the alternating stacking of metals and dielectrics at the nanoscale. Their isofrequency surfaces are hyperbolic in shape and can carry extremely high transverse wave vectors. When light passes through an HMM, the nanostructures inside the material scatter and form random speckle patterns with characteristic sizes of only 10-30 nm. This process successfully converts the subwavelength high-frequency information carried by the object into a signal that can be detected by a far-field optical system, breaking through the traditional optical diffraction limit.

[0004] Despite these challenges, current Hidden Markov Model (HMM) technology is primarily applied to static samples or simple fluorescence enhancement, and has not yet achieved seamless coupling with high-speed dynamic imaging techniques such as confocal scanning. The main challenges are: real-time signal capture, as traditional HMM imaging relies heavily on slow point scanning or fixed-angle observation, which is insufficient to meet the real-time capture requirements of millisecond-level immune synapse formation processes; and system integration, requiring solutions to the optical path matching and background suppression issues between the HMM film and the confocal microscopy system to ensure high frame rates even under low-light conditions. Despite these challenges, the unique advantages of HMM in signal amplification and spatial resolution breakthroughs make it a highly promising technology for addressing the "spatial blind spot" of immune synapses. Therefore, to simultaneously overcome the "dynamic capture failure" and the "contradiction of in vivo observation," improvements to existing technologies are urgently needed to achieve large-area, high-resolution imaging. Summary of the Invention

[0005] To address the core technical bottleneck of the trade-off between "high speed" and "super resolution" in dynamic cell imaging, this invention provides a combined imaging system based on single-disc microscopy and metamaterial near-field enhancement to achieve millisecond-level full-field high-resolution imaging.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: a combined imaging system based on single-disk microscopy and metamaterial near-field enhancement, including a light source modulation module, a polarization beam splitting module, a polarization matching module, a dichroic mirror, a single-disk platform, a tube mirror, a first galvanometer, a second galvanometer, an objective lens, a sample stage, and an imaging module. The light source modulation module is used to output a uniform excitation beam. The excitation beam is incident on a single rotating platform after passing through a polarization matching module and a dichroic mirror. The single rotating platform is provided with pinholes evenly distributed along multiple Archimedean spirals. The laser beam transmitted through the pinholes on the single rotating platform passes sequentially through a cylindrical lens, a first galvanometer, a second galvanometer, and an objective lens before converging on the sample stage to excite the sample to generate fluorescence. The generated fluorescence returns along the original path to the dichroic mirror and is received by the imaging module. The single-rotor platform is positioned within the conjugate plane of the objective lens; the sample stage surface is covered with a layered hyperbolic metamaterial thin film.

[0007] The layered hyperbolic metamaterial thin film consists of alternating stacked metal layers and dielectric layers.

[0008] The light source modulation module includes a light source, a microlens array, and a reference lens. The microlens array is used to homogenize the excitation beam, and the reference lens is used to collimate the homogenized beam.

[0009] The imaging module includes a first cemented lens, a filter, a second cemented lens, and an sCMOS. The generated fluorescence returns along the original path to the dichroic mirror and separates from the excitation beam. It then passes through the first cemented lens, the filter, and the second cemented lens in sequence before entering the sCMOS.

[0010] The aforementioned combined imaging system based on single-disc microscopy and metamaterial near-field enhancement further includes a drive motor, a turntable control console, and a drive circuit. The single-disc platform is mounted inside the turntable control console via the drive motor, and the drive circuit controls the rotation of the drive motor, thereby driving the single-disc platform to rotate, so as to realize the scanning of the excitation beam on the single-disc platform.

[0011] The drive circuit includes a main controller, a speed control knob, a power inverter, a positioning circuit, a DC power supply, and a display screen. The output of the main controller is connected to the drive motor through the power inverter. The output of the positioning circuit of the drive motor is connected to the main controller. The speed control knob is connected to the main controller.

[0012] The drive motor is a hydrodynamic bearing motor.

[0013] The aforementioned imaging system based on single-disc microscopy and metamaterial near-field enhancement further includes an encapsulation base and an encapsulation cover. The single-disc platform, drive motor, turntable control console, and drive circuit are disposed inside the encapsulation base and sealed by the encapsulation cover. Light-transmitting holes are provided on the encapsulation cover and the encapsulation base.

[0014] The aforementioned imaging system based on single-disk microscopy and metamaterial near-field enhancement further includes a six-degree-of-freedom precision positioning stage. The sample stage is set on the six-degree-of-freedom precision positioning stage, which is used to adjust the tilt angle and lateral displacement of the sample stage, thereby changing the wave vector direction of the incident excitation beam or emitted light to satisfy the phase matching condition of the layered hyperbolic metamaterial thin film.

[0015] The single turntable platform includes a transparent quartz substrate and a thin film layer. The thin film layer has multiple pinholes distributed along multiple Archimedean spirals; the number of spirals is 30-80.

[0016] Compared with the prior art, the present invention has the following advantages: This invention provides a combined imaging system based on single-disc microscopy and metamaterial near-field enhancement. Through high-momentum-state excitation of layered hyperbolic metamaterial (HMM) films, combined with rotating scanning on a single-disc platform featuring pinholes arranged in an Archimedean spiral pattern and high-resolution imaging by an sCMOS camera, the spatial resolution is significantly improved to approximately 80 nm while maintaining a high frame rate in milliseconds (≥100 fps). Furthermore, due to the use of a low-power light source and an efficient fluorescence enhancement mechanism, phototoxicity is significantly reduced, making it suitable for long-term imaging of live cells. This physical coupling not only overcomes the limitation of traditional optical imaging's inability to simultaneously achieve both speed and accuracy, but also provides a key tool for simultaneously capturing the causal chain of nanoscale structure assembly (such as TCR nanocluster formation) and signal molecule recruitment (such as ZAP70 activation) in immune synapses. This invention can be widely applied in fields such as four-dimensional immune synaptic dynamics analysis, disease mechanism research, and drug screening and evaluation. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the optical path structure of a combined imaging system based on single-disc microscopy and metamaterial near-field enhancement provided in Embodiment 1 of the present invention. Figure 2 Here are schematic diagrams of pinholes set on a single turntable platform in Embodiment 1 of the present invention: (a) is a schematic diagram of the spiral arrangement of pinholes on the turntable; (b) is a schematic diagram of the pinhole layout on the turntable; (c) is an enlarged view of the local ROI region in (b); Figure 3 This is a schematic diagram of the HMM thin film structure in an embodiment of the present invention; Figure 4 This is a system design framework diagram of the turntable module in an embodiment of the present invention; Figure 5 This is a circuit diagram of the main controller in an embodiment of the present invention; Figure 6 This is a circuit diagram of the power inverter in an embodiment of the present invention; Figure 7 This is a circuit diagram of the positioning circuit in an embodiment of the present invention; Figure 8 This is a circuit diagram of the DC power supply circuit in an embodiment of the present invention; Figure 9 This is a circuit diagram of the speed control circuit in an embodiment of the present invention; Figure 10 This is a schematic diagram of the overall encapsulation of the turntable assembly in an embodiment of the present invention.

[0018] Explanation of reference numerals in the attached figures: 1-Excitation source, 2-Microlens array, 3-Reference lens, 4-Polarization matching module, 5-Dichroic mirror, 6-Single turntable platform, 7-Turntable control console, 8-Bullet lens, 9-First galvanometer, 10-Second galvanometer, 11-Objective lens, 12-Sample, 13-HMM substrate, 14-Six-DOF precision positioning stage, 15-First cemented lens, 16-Filter, 17-Second cemented lens, 18-sCMOS, 19-Packaging base, 20-Packaging cover. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are some embodiments of the present invention, but not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] like Figure 1 As shown, this embodiment of the invention provides a combined imaging system based on single-disk microscopy and metamaterial near-field enhancement, including a light source modulation module, a polarization beam splitting module, a single-disk platform 6, a tube mirror 8, a first galvanometer 9, a second galvanometer 10, an objective lens 11, a sample stage 13, and an imaging module.

[0021] The light source modulation module is used to output a uniform excitation beam. The excitation beam is incident on the single rotating platform 6 after passing through the polarization matching module 4 and the dichroic mirror 5. The single rotating platform 6 is provided with pinholes distributed in an Archimedean spiral pattern. The laser beam transmitted through the pinholes on the single rotating platform passes sequentially through the tube lens 8, the first galvanometer 9, the second galvanometer 10, and the objective lens 11 before converging on the sample stage 13 to excite the sample to generate fluorescence. The generated fluorescence returns along the original path to the dichroic mirror 5 and is received by the imaging module. The single rotating platform 6 is located in the conjugate plane of the objective lens 11. The surface of the sample stage 13 is provided with a layered hyperbolic metamaterial (HMM) thin film.

[0022] Specifically, in this embodiment, the light source modulation module includes a light source 1, a microlens array 2, and a reference lens 3. The microlens array 2 is used to homogenize the excitation beam and perform preliminary wavefront shaping. The reference lens 3 is used to collimate the homogenized beam. The microlens array can be a random phase plate or a Gaussian beam shaper, and its parameters are optimized through ZEMAX simulation. The light source 1 can be a tunable (wavelength range 400-720 nm) or broadband light-emitting diode (LED, emission wavelength covering 400-700 nm) to adapt to the excitation requirements of different fluorescent dyes.

[0023] In this embodiment, the polarization matching module 4 can employ a λ / 2 waveplate. By rotating the angle of the λ / 2 waveplate, the polarization of the excitation beam can be adjusted to ensure that the polarization state of the incident light on the sample surface matches the excitation polarization direction of the plasmons on the HMM thin film surface, thereby optimizing the coupling efficiency. The dichroic mirror 5 can either transmit the excitation beam and reflect the emitted light, or reflect the excitation beam and transmit the emitted light.

[0024] like Figure 2 As shown, in this embodiment, the single-disc platform 6 includes a transparent quartz substrate layer and a thin film layer, wherein the thin film layer is an opaque chromium metal layer; pinholes are uniformly distributed along multiple Archimedean spirals on the thin film layer, with a constant radial spacing and uniform angular distribution of the pinholes. In this embodiment, the number of spirals corresponding to the pinholes is 20-80, which can improve imaging uniformity and speed. Preferably, the number of spirals is 60, and the thin film layer is prepared on the quartz substrate using a lift-off process to form the single-disc platform 6.

[0025] Specifically, the starting points of each Archimedean spiral are evenly distributed on a circle centered on the center of the single-disc platform 6, with the distance between the starting point and the center of the single-disc platform 6 being 10-15 mm. In this embodiment, the distance between the starting point of the Archimedean spiral and the center of the single-disc platform 6 is 12.7 mm. For example... Figure 2 As shown, the area formed by pinholes distributed along multiple spiral lines is a circular pinhole region. The spot size of the excitation beam on the single rotating platform 6 is larger than the width of the annular pinhole region, thus completely covering it. In this embodiment, the annular width is set to 10~20mm, and the radius of the pinholes is 10~40μm. The distance between pinholes on the same spiral line is 50~150μm, and the distance between different spiral lines is 50~150μm.

[0026] Furthermore, this embodiment of the combined imaging system based on single-disc microscopy and metamaterial near-field enhancement also includes a drive motor, a turntable control console 7, and a drive circuit. The single-disc platform 6 is mounted within the turntable control console 7 via the drive motor. The drive circuit controls the rotation of the drive motor, thereby driving the single-disc platform 6 to rotate, thus enabling the excitation beam to scan on the single-disc platform 6. When the single-disc platform 6 rotates, the pinholes within the annular pinhole region can transmit fluorescence from different locations on the sample, thereby achieving scanning imaging of the sample. The spiral arrangement of the pinholes ensures uniform scanning of the sample plane without missing points. Moreover, using a spiral arrangement maintains a consistent scanning interval, improving imaging quality.

[0027] The drive motor is preferably a hydrodynamic bearing motor, which can solve the problem of noise and vibration of traditional high-speed motors affecting imaging.

[0028] Specifically, in this embodiment, the layered hyperbolic metamaterial thin film can be deposited on a quartz or glass substrate to form an HMM substrate, and then the prepared HMM substrate is placed on the sample stage 13. Figure 3 As shown, the layered hyperbolic metamaterial thin film consists of alternating stacked metal and dielectric layers, such as a periodic Ag / SiO2 structure. A Si3N4 protective layer is finally deposited on the surface of the layered hyperbolic metamaterial thin film to prevent oxidation. The thicknesses of the metal and dielectric layers are set between 5-30 nm and 10-50 nm, respectively. Specific layer thickness parameters can be designed using deep reinforcement learning (DRL) or Bayesian optimization models, with the design goal of achieving extremely low loss and high k-state density distribution. Specifically, firstly, a search space is constructed, setting the metal layer thickness to 5-30 nm and the dielectric layer thickness to 10-50 nm. Next, using existing FDTD simulation data or literature data, a deep neural network (DNN) surrogate model is trained to quickly predict the effective dielectric tensors ϵ_∥ and ϵ_⊥ of the HMM. Subsequently, a reinforcement learning algorithm (such as Deep Q-Network) is introduced to use hyperbolic dispersion at a specific wavelength as the reward function to automatically search for the optimal material combination and thickness parameters.

[0029] like Figure 4 As shown in this embodiment, the drive circuit includes a main controller, a speed control knob, a power inverter, a positioning circuit, a DC power supply, and a display screen. The output terminal of the main controller is connected to the drive motor through the power inverter. The output terminal of the positioning circuit of the drive motor is connected to the main controller. The speed control knob is connected to the main controller.

[0030] like Figures 5-9 The diagram shown illustrates the circuit schematics of the main controller, power inverter, positioning circuit, DC power supply circuit, and speed control circuit in this embodiment. The positioning circuit includes a back electromotive force sampling circuit and a current sampling circuit, achieving positioning through signal sampling. The speed control knob connects to the speed control circuit to regulate the motor speed.

[0031] In this embodiment, the first galvanometer 9 and the second galvanometer 10 are used to achieve precise scanning of the incident angle. By using the first galvanometer 9 and the second galvanometer 10, the angle of the excitation beam can be scanned, thereby achieving precise scanning of the incident angle of the sample surface. In this embodiment, by strictly optimizing the parameters (focal length, diameter) of the tube lens 8, it can be ensured that the incident angle adjustment range covers 0° to 80°, and the radial resolution of the light spot at the focal plane is maintained below 80 nm.

[0032] Specifically, in this embodiment, the imaging module includes a first cemented lens 15, a filter 16, a second cemented lens 17, and an sCMOS 18. The generated fluorescence returns along the original path to the dichroic mirror 5 and separates from the excitation beam. It then passes through the first cemented lens 15, the filter 16, and the second cemented lens 17 in sequence before entering the sCMOS 18.

[0033] Specifically, in this embodiment, the objective lens 11 is a high numerical aperture (NA>0.8) objective lens, which includes a first cemented lens 15 and a second cemented lens 17 to form a cemented doublet lens group. It is optimized by ZEMAX sequence mode simulation to eliminate aberrations and chromatic aberrations. The filter 16 is used to filter out the excitation light. The high-frequency information after HMM conversion is collected by the objective lens 11, and then returns to the dichroic mirror 5 after passing through the second galvanometer 10, the first galvanometer 9 and the tube lens 8. The collected light path after the dichroic mirror 5 passes through the cemented doublet lens group and the filter 16, and is finally projected onto the back-illuminated sCMOS camera 18 for high-speed capture. The single-frame exposure of the sCMOS camera 18 is ≤10 ms and the light poison power is ≤10 mW.

[0034] Furthermore, such as Figure 10 As shown, this embodiment of an imaging system based on single-disc microscopy and metamaterial near-field enhancement also includes a packaging base 19 and a packaging cover 20. The single-disc platform 6, drive motor, turntable control console 7 and drive circuit are disposed in the packaging base 19 and sealed by the packaging cover 20. The packaging cover 20 and the packaging base 19 are provided with light-transmitting holes.

[0035] Furthermore, the combined imaging system based on single-disk microscopy and metamaterial near-field enhancement in this embodiment also includes a six-degree-of-freedom precision positioning stage 14. The sample stage 13 is disposed on the six-degree-of-freedom precision positioning stage 14. The six-degree-of-freedom precision positioning stage 14 is used to adjust the tilt angle (θX, θY) and lateral displacement (X, Y) of the sample stage 13 to change the wave vector direction of the incident light or emitted light so that its wave vector direction satisfies the phase matching condition of the layered hyperbolic metamaterial thin film.

[0036] In summary, this invention provides a combined imaging system based on single-disk microscopy and metamaterial near-field enhancement. Through high-momentum-state excitation of a layered hyperbolic metamaterial thin film, combined with the rotational scanning of a single-disk platform 6 with pinholes arranged in an Archimedean spiral pattern, and the scanning cooperation of a first galvanometer 9 and a second galvanometer, and high-resolution imaging by an sCMOS camera 18, the spatial resolution is significantly improved to approximately 80 nm while maintaining a high frame rate in milliseconds (≥100 fps). Furthermore, due to the use of a low-power light source and an efficient fluorescence enhancement mechanism, phototoxicity is significantly reduced, making it suitable for long-term imaging of live cells.

[0037] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A combined imaging system based on single-disc microscopy and metamaterial near-field enhancement, characterized in that, It includes a light source modulation module, a polarization beam splitting module, a polarization matching module (4), a dichroic mirror (5), a single turntable platform (6), a tube mirror (8), a first galvanometer (9), a second galvanometer (10), an objective lens (11), a sample stage (13), and an imaging module; The light source modulation module is used to output a uniform excitation beam. The excitation beam is incident on a single rotating platform (6) after passing through a polarization matching module (4) and a dichroic mirror (5). The single rotating platform (6) is provided with pinholes evenly distributed along multiple Archimedean spirals. The laser beam transmitted through the pinholes on the single rotating platform passes sequentially through a tube lens (8), a first galvanometer (9), a second galvanometer (10), and an objective lens (11) and then converges on the sample stage (13) to excite the sample to generate fluorescence. The generated fluorescence returns along the original path to the dichroic mirror (5) and is received by the imaging module. The single turntable platform (6) is set in the conjugate plane of the objective lens (11); the sample stage (13) is provided with a layered hyperbolic metamaterial film on its surface.

2. The combined imaging system based on single-disc microscopy and metamaterial near-field enhancement according to claim 1, characterized in that, The layered hyperbolic metamaterial thin film consists of alternating stacked metal layers and dielectric layers.

3. The combined imaging system based on single-disc microscopy and metamaterial near-field enhancement according to claim 1, characterized in that, The light source modulation module includes a light source (1), a microlens array (2), and a reference lens (3). The microlens array (2) is used to homogenize the excitation beam spot, and the reference lens (3) is used to collimate the homogenized spot.

4. The combined imaging system based on single-disc microscopy and metamaterial near-field enhancement according to claim 1, characterized in that, The imaging module includes a first cemented lens (15), a filter (16), a second cemented lens (17), and an sCMOS (18). The generated fluorescence returns along the original path to the dichroic mirror (5) and separates from the excitation beam. It then passes through the first cemented lens (15), the filter (16), and the second cemented lens (17) in sequence before entering the sCMOS (18).

5. The combined imaging system based on single-disc microscopy and metamaterial near-field enhancement according to claim 1, characterized in that, It also includes a drive motor, a turntable control console (7) and a drive circuit. The single turntable platform (6) is set inside the turntable control console (7) by the drive motor. The drive circuit is used to control the rotation of the drive motor, thereby driving the single turntable platform (6) to rotate, so as to realize the scanning of the excitation beam on the single turntable platform (6).

6. The combined imaging system based on single-disc microscopy and metamaterial near-field enhancement according to claim 5, characterized in that, The drive circuit includes a main controller, a speed control knob, a power inverter, a positioning circuit, a DC power supply, and a display screen. The output of the main controller is connected to the drive motor through the power inverter. The output of the positioning circuit of the drive motor is connected to the main controller. The speed control knob is connected to the main controller.

7. The combined imaging system based on single-disc microscopy and metamaterial near-field enhancement according to claim 5, characterized in that, The drive motor is a hydrodynamic bearing motor.

8. The combined imaging system based on single-disc microscopy and metamaterial near-field enhancement according to claim 5, characterized in that, It also includes a packaging base and a packaging cover. The single turntable platform (6), drive motor, turntable control console (7) and drive circuit are set inside the packaging base and sealed by the packaging cover. The packaging cover and the packaging base are provided with light-transmitting holes.

9. The combined imaging system based on single-disc microscopy and metamaterial near-field enhancement according to claim 1, characterized in that, It also includes a six-degree-of-freedom precision positioning stage (14), on which the sample stage (13) is set. The six-degree-of-freedom precision positioning stage (14) is used to adjust the tilt angle and lateral displacement of the sample stage (13), thereby changing the direction of the wave vector of the incident excitation beam or emitted light so that it meets the phase matching condition of the layered hyperbolic metamaterial thin film.

10. The combined imaging system based on single-disc microscopy and metamaterial near-field enhancement according to claim 1, characterized in that, The single turntable platform (6) includes a transparent quartz substrate layer and a thin film layer. The thin film layer is provided with multiple pinholes distributed along multiple Archimedean spirals; the number of spirals is 30-80.

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