A multi-color structured light illumination three-dimensional super-resolution imaging system and method

Abstract

The application discloses a kind of multi-color structured light illumination three-dimensional super-resolution imaging system and method, belong to microscopic imaging technical field.The system of the present application includes multi-band polarization multiplexing structured light illumination module and microscopic imaging module;Multi-band polarization multiplexing structured light illumination module contains metasurface lens, metasurface lens is configured to at least two different wavelengths of incident P polarization light, respectively in the same target face generates the sinusoidal distribution structured light field with same spatial frequency and same stripe orientation.It is different wavelength excitation light to generate consistent structured light illumination pattern by the metasurface lens with dispersion compensation characteristics, without separately calibrating structured light parameters to each wavelength channel, avoid the image artifact caused by parameter calibration error;While it can realize multi-wavelength synchronous illumination and synchronous imaging, significantly improve the imaging flux and system integration of multi-color structured light illumination super-resolution microscope.

Description

Technical Field

[0001] This invention relates to the field of microscopic imaging technology, and in particular to a three-dimensional super-resolution imaging system and method with multicolor structured light illumination. Background Technology

[0002] Structured illumination microscopy (SIM), a far-field super-resolution imaging technique that overcomes the optical diffraction limit, has been widely used in life sciences and biomedical research due to its advantages such as fast imaging speed, low phototoxicity, and no need for special fluorescent probes. SIM projects a structured light pattern of known spatial frequencies onto the sample, transferring the sample's high-frequency information into the passband of the optical system, and then reconstructing a super-resolution image that surpasses the diffraction limit through algorithms.

[0003] With the advancement of life science research, multicolor fluorescence imaging has become an indispensable tool for elucidating complex biological processes. By labeling different fluorescent probes, researchers can simultaneously observe the dynamic interactions of multiple biomolecules in time and space. However, existing SIM systems face the following technical bottlenecks in multicolor imaging: 1. Multicolor imaging is inefficient and struggles to capture dynamic processes. Traditional multicolor SIM systems typically employ a time-sequential switching mode. Specifically, the system sequentially switches between different wavelengths of excitation light, acquiring structured illumination images of each wavelength separately, and then performs multicolor super-resolution reconstruction through image registration and fusion. Because it requires time-divisional acquisition of image data from multiple wavelength channels, the total imaging time increases linearly with the number of channels. For example, Chinese patent ZL201710495353.7 discloses a high-speed multicolor multimodal structured illumination super-resolution microscopy system. Although it improves the switching speed through a high-speed gating module, it is essentially still a time-divisional acquisition mode. For three-color imaging, the total acquisition time is approximately three times that of monochromatic imaging. This significantly reduces the system's temporal resolution, making it difficult to capture rapid biological processes on the order of milliseconds. Furthermore, time-sequential switching introduces image registration errors between channels, which severely restrict the quality of reconstructed images, especially when the sample exhibits drift or deformation.

[0004] 2. Complex optical paths and poor system stability in multicolor illumination. To achieve multicolor structured light illumination, existing systems typically employ two approaches: First, using multiple independent laser sources and structured light generation modules, such as digital micromirror devices (DMDs) or spatial light modulators (SLMs), which are then combined using dichroic mirrors before being introduced into the microscope system. This approach results in a large system size, numerous optical components, complex assembly and adjustment, and long-term stability significantly affected by environmental factors. Second, using a supercontinuum laser in conjunction with an acousto-optic tunable filter (AOTF) for wavelength selection, but still requiring an SLM or DMD to generate structured light. These active devices are not only large and costly, but their light energy utilization rate is typically below 30%, with insufficient light power becoming even more pronounced in multicolor imaging.

[0005] 3. Inconsistent periods of structured light at different wavelengths affect reconstruction accuracy. The core of structured light illumination super-resolution reconstruction algorithms lies in accurately knowing the spatial frequency, direction, and phase of the structured light pattern. However, when using excitation light of different wavelengths, due to diffraction effects, the period of the structured light fringes generated by the same diffraction element is proportional to the wavelength. This means that the structured light periods generated by red, green, and blue light are different, requiring the reconstruction algorithm to calibrate the illumination parameters for each wavelength separately, increasing system complexity and algorithmic burden. More seriously, when axial displacement or chromatic aberration exists in the sample, the alignment accuracy of the structured light at different wavelengths on the sample surface is difficult to guarantee, directly affecting the fidelity of the reconstructed image.

[0006] 4. Existing metasurface application schemes have not solved the core problem of multi-color SIM. In recent years, metasurfaces, as planar optical elements composed of subwavelength structure arrays, have provided new avenues for the miniaturization and multifunctionality of optical systems due to their ability to control the amplitude, phase, and polarization of the light field at the subwavelength scale. Existing research has attempted to apply metasurfaces to SIM systems or related imaging fields. For example, Chinese patent ZL202220071539.6 discloses a dual-function metalens and a super-resolution imaging device including it, which uses a metasurface lens to generate a ring-shaped hollow beam for a first wavelength and a focused spot for a second wavelength, for STED-type super-resolution imaging. Although this scheme achieves multi-wavelength control, its design goal is to generate beams with different functions for different wavelengths, rather than to generate structured light illumination patterns with the same parameters for different wavelengths. Another example is Chinese patent ZL202210777395.0, which discloses a super-resolution imaging system based on Bloch surface wave structured light illumination. It utilizes a one-dimensional photonic crystal and a periodic grating structure to generate structured light, but still fails to solve the problem of inconsistent periods for structured light at different wavelengths. Chinese patent application CN202280054215.4 relates to a multi-channel superlens color imaging device, but does not relate to structured light illumination super-resolution imaging, let alone multi-color structured light period consistency control.

[0007] In summary, existing SIM systems face key technical challenges in multicolor imaging applications, such as slow imaging speed, system complexity, and inconsistent structured light periods across wavelengths. Furthermore, the application directions of existing metasurface technology differ substantially from the technical concept of this invention.

[0008] Therefore, how to achieve efficient, compact, and independently tunable multicolor structured light illumination with varying wavelength structured light parameters has become an urgent need to drive the development of SIM technology. Summary of the Invention

[0009] To achieve the above-mentioned objectives and other advantages of the present invention, a first objective of the present invention is to provide a multicolor structured light illumination three-dimensional super-resolution imaging system, comprising: A multi-band polarization multiplexing structured light illumination module is used to generate at least two structured light illumination fields of different wavelengths and to realize phase translation, orientation rotation and polarization control of the structured light illumination fields. The microscopic imaging module is used to receive the fluorescence signal generated by the sample under the excitation of the structured light illumination field and form a multi-channel super-resolution image; The multi-band polarization multiplexing structured light illumination module includes a metasurface lens, which is configured to generate sinusoidal structured light fields with the same spatial frequency and the same fringe orientation on the same target surface for at least two incident P-polarized lights of different wavelengths.

[0010] Furthermore, the metasurface lens includes: Base; Subwavelength structure array disposed on the substrate; The subwavelength structure array is configured such that, for the first wavelength The incident P-polarized light generates a first illumination light field with a first structured light distribution on the target surface; For the second wavelength Incident P-polarized light, A second illumination field with a spatial frequency difference of less than 5% and a stripe orientation deviation of less than 1° is generated on the same target surface.

[0011] Furthermore, the unit structure of the subwavelength structure array is selected from one or more of nanopillars, nanofins, nanopores, and nanorings; The geometric parameters of the unit structure are configured to have dispersion compensation characteristics within the target wavelength range, so that the spatial frequency of the structured light field generated by incident light of different wavelengths is consistent.

[0012] Furthermore, the subwavelength structure array employs a polarization multiplexing design, assigning different phase distributions to incident P-polarized light of different wavelengths. These phase distributions, after Fourier transform, produce identical multi-beam interference fringes.

[0013] Furthermore, the spatial frequency of the sinusoidal structured light is adjustable, and the spatial frequency of the structured light can be continuously adjusted by changing the irradiation area or incident angle of the incident light on the metasurface lens.

[0014] Furthermore, the wavelength of the incident P-polarized light is selected from at least two of 405nm, 488nm, 532nm, 561nm, and 640nm.

[0015] Furthermore, the multi-band polarization multiplexing structured light illumination module also includes: A multi-band laser source module for emitting light in at least two bands; A collimating beam expander is used to convert the emitted light into collimated light rays; A polarizer is used to modulate the collimated light into linearly polarized light in the P direction; Fourier lens group, used to transform the light field modulated by the metasurface lens; The filter aperture is used to filter out light rays of the 0th and ±1st diffraction orders; A segmented polarization modulation half-wave plate is composed of sector waveplates with different fast axis directions, used to modulate diffracted light of each order into different polarization directions. The lens group and dichroic mirror are used to focus the modulated diffracted light onto the back focal plane of the microscope objective.

[0016] Furthermore, the microscopic imaging module includes: Microscope objectives are used both to focus illumination light onto the sample surface and to collect the fluorescence emitted by the sample. The stage includes a three-dimensional electric displacement stage with sample clamps, used to drive the sample to make nanometer-scale step displacements relative to the structured light field. The spectral separation module is used to spatially separate fluorescence of different wavelengths into different optical paths. A tube lens is used to focus the separated single-band fluorescence onto the target surface of an image sensor. Image sensor used to acquire fluorescence images.

[0017] Furthermore, the spectral separation module can be implemented using one or more of the following methods: dichroic mirror spectral separation, metasurface spectral filter array, and tunable bandpass filter.

[0018] A second objective of this invention is to provide a three-dimensional super-resolution imaging method based on multicolor structured light illumination of the above-mentioned system, comprising the following steps: S1. The multi-band laser source module emits excitation light of at least two wavelengths simultaneously; S2. The excitation light is collimated, expanded, and polarized before being incident on the metasurface lens; S3. The metasurface lens imparts a preset phase distribution to incident light of different wavelengths, so that light of each wavelength produces a structured light field with consistent spatial frequency and consistent fringe orientation on the same target surface. S4. The generated structured light field is then subjected to Fourier transform, spatial filtering, and segmented polarization modulation, and focused onto the sample surface to excite the sample to generate multicolor fluorescence signals. S5. After the multicolor fluorescence signal is collected by the microscope objective, it is separated into different imaging channels according to wavelength by the spectral separation module; S6. Fluorescence images are acquired synchronously through each imaging channel, and multicolor super-resolution three-dimensional images are obtained after reconstruction by the algorithm.

[0019] Furthermore, in step S3, the metasurface lens ensures that the spatial frequency difference of the structured light field generated by incident light of different wavelengths is less than 5%, and the fringe orientation deviation is less than 1°, without the need to individually calibrate the structured light parameters for each wavelength channel.

[0020] Furthermore, it also includes: By changing the illumination area or incident angle of the incident light on the metasurface lens, the spatial frequency of the structured light field can be continuously adjusted to achieve super-resolution imaging at different resolutions.

[0021] Furthermore, it also includes: The sample is driven to perform nanometer-scale step displacement by a stage, and fluorescence image sequences under multi-phase structured light illumination are acquired for super-resolution image reconstruction.

[0022] Compared with the prior art, the beneficial effects of the present invention are: In traditional multicolor SIM, each wavelength channel requires individual calibration of structured light parameters. This involves acquiring multiple images using fluorescent microsphere samples and fitting the actual structured light frequency and phase using algorithms. Inaccurate parameter estimation can easily lead to artifacts, affecting image fidelity. This invention innovatively utilizes metasurfaces to achieve multi-band light field modulation, ensuring consistent structured light field parameters across different wavelengths. This eliminates the need for individual calibration of structured light frequency and phase for each channel in multicolor SIM imaging, reducing computational complexity and improving reconstruction accuracy and speed. The consistency error of structured light parameters in multi-channel reconstructed images is less than 5%, avoiding image artifacts caused by parameter calibration errors.

[0023] This invention enables simultaneous imaging of incident lasers at different wavelengths, eliminating the need for step-by-step laser band switching and repeated imaging processes, thus achieving rapid multi-channel super-resolution imaging. Furthermore, the metasurface device has no moving mechanical parts, resulting in a fast response speed and enabling video-level multi-wavelength super-resolution imaging.

[0024] This invention utilizes a single metasurface device to replace the SLM or DMD in a traditional SIM system, greatly simplifying the system structure and realizing a miniaturized, integrated structured light illumination module.

[0025] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it according to the contents of the specification, the preferred embodiments of the present invention are described in detail below with reference to the accompanying drawings. Specific embodiments of the present invention are given in detail below with reference to the accompanying drawings. Attached Figure Description

[0026] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this application, illustrate exemplary embodiments of the invention and, together with their description, serve to explain the invention and do not constitute an undue limitation thereof. In the drawings: Figure 1 Optical path diagram for a three-dimensional super-resolution imaging system with multicolor structured light illumination; Figure 2 Flowchart of a three-dimensional super-resolution imaging method using multicolor structured light illumination. Detailed Implementation

[0027] The present invention will now be further described with reference to the accompanying drawings and specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. It should be noted that, without conflict, the various embodiments or technical features described below can be arbitrarily combined to form new embodiments.

[0028] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention.

[0029] The drawing numbers in this application are only used to distinguish the steps in the scheme and are not used to limit the execution order of the steps. The specific execution order is as described in the specification.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0031] This invention provides a multicolor structured light illumination super-resolution imaging system and method based on a dispersive engineered metasurface, which is particularly suitable for applications requiring high spatiotemporal resolution multidimensional imaging, such as multicolor fluorescence microscopy, live cell dynamic observation, and biomedical detection. The specific solution is as follows: Example 1 See Figure 1This embodiment provides a three-dimensional super-resolution imaging system based on multicolor structured light illumination using a dispersive engineered metasurface, comprising: A multi-band polarization multiplexing structured light illumination module is used to generate at least two structured light illumination fields of different wavelengths and to realize phase translation, orientation rotation and polarization control of the structured light illumination fields. The microscopic imaging module is used to receive the fluorescence signal generated by the sample under the excitation of the structured light illumination field and form a multi-channel super-resolution image; The multi-band polarization multiplexing structured light illumination module includes a metasurface lens, which is configured to generate sinusoidal structured light fields with the same spatial frequency and the same fringe orientation on the same target surface for at least two incident P-polarized lights of different wavelengths.

[0032] In this embodiment, the multi-band polarization multiplexing structured light illumination module specifically includes a multi-band laser source module 1, a collimating beam expander 2, a polarizer 3, a metasurface lens 4, a Fourier lens group 5, a filter aperture 6, a segmented polarization modulation half-wave plate 7, a lens 8, a dichroic mirror 9, and a microscope objective 10.

[0033] The multi-band laser source module 1 can emit light in at least two bands. , In this embodiment, a dual-band laser source with wavelengths of 488nm and 561nm is used to excite the green and red fluorescent probes, respectively. It is understood that, depending on actual imaging requirements, other wavelengths such as 405nm, 532nm, and 640nm can also be used, or three or more multi-band light sources can be employed.

[0034] The collimating beam expander 2 is used to convert the emitted light from the multi-band laser source module 1 into collimated light, while expanding the beam diameter to meet the aperture requirements of subsequent optical components.

[0035] Polarizer 3 is used to modulate the collimated and expanded light into P-direction linearly polarized light. P-polarized light is chosen because the metasurface lens 4 of this invention is optimized for P-polarized incident light, resulting in higher modulation efficiency and better structured light quality.

[0036] The metasurface lens 4 is the core component of this invention. This metasurface lens 4 can modulate light from different specified wavelength bands into a structured light field with consistent spatial frequency, orientation, and phase. Specifically, the metasurface lens 4 includes a substrate and a subwavelength structure array disposed on the substrate. The unit structure of the subwavelength structure array is selected from one or more of nanopillars, nanofins, nanopores, and nanorings. In this embodiment, a nanopillar array is prepared using materials such as titanium dioxide, and precise control of the incident light phase is achieved by adjusting the diameter and rotation angle of the nanopillars.

[0037] The subwavelength structure array is configured such that, for the first wavelength The incident P-polarized light generates a first illumination field with a first structured light distribution on the target surface; for the second wavelength The incident P-polarized light produces a second illumination field on the same target surface with a spatial frequency difference of less than 5% and a fringe orientation deviation of less than 1° from the first illumination field. To achieve this function, the subwavelength structure array employs dispersion engineering and polarization multiplexing design to assign different phase distributions to incident P-light of different wavelengths. These phase distributions produce the same multi-beam interference fringes after Fourier transform.

[0038] By optimizing the geometric parameters of the subwavelength structure array (including the diameter, period, height, and rotation angle of the nanopillars), the metasurface lens 4 generates different phase distributions at two wavelengths of 488nm and 561nm, respectively, which meet the above functional requirements. This results in interference fringes with consistent spatial frequencies after Fourier transform.

[0039] The Fourier lens group 5 is used to perform Fourier transform on the light field modulated by the metasurface lens 4, converting the angular spectrum information into spatial position information, so that light of different diffraction orders is separated on the Fourier plane. After the light field transformation by the Fourier lens group 5, it becomes diffraction spots of various orders and is focused at the filter aperture 6.

[0040] The filter aperture 6 is positioned on the Fourier plane to filter out light rays of the 0th and ±1st order diffraction orders, while blocking other higher order diffraction light to improve the contrast of the structured light. The diameter of the filter aperture 6 can be adjusted according to actual needs.

[0041] The segmented polarization modulation half-wave plate 7 is composed of fan-shaped wave plates with different fast axis directions. Each order of diffracted light is modulated into different polarization directions by passing through different fan-shaped wave plates.

[0042] Lens 8 and dichroic mirror 9 constitute a relay optical system, focusing the polarized diffracted light of each order onto the back focal plane of microscope objective 10. Dichroic mirror 9 is used to separate the illumination optical path and the imaging optical path, and its coating characteristics are: high reflectivity for excitation wavelengths (488nm and 561nm) and high transmittance for fluorescence emission wavelengths (such as 520nm and 610nm).

[0043] The microscope objective 10 is a shared element for both the illumination and imaging optical paths. It is used to focus the illumination light onto the sample surface and to collect the fluorescence emitted by the sample. In this embodiment, an oil immersion objective with a numerical aperture NA of 1.4 is used to achieve high-resolution excitation and collection.

[0044] On the sample surface, the polarization-modulated 0th and ±1st order diffracted beams interfere to form sinusoidal striped structured light. Due to the dispersion compensation design of the metasurface lens 4, the structured light generated by the 488nm and 561nm excitation beams has the same spatial frequency and stripe orientation.

[0045] The spatial frequency of the sinusoidal structured light is adjustable. By changing the irradiation area or incident angle of the incident light on the metasurface lens 4, the effective phase distribution of the metasurface lens 4 can be changed, thereby achieving continuous adjustment of the spatial frequency of the structured light.

[0046] The microscopic imaging module is used to form fluorescence images based on the fluorescence acquired by the structured light illumination component. Specifically, it includes a microscope objective 10, a stage 11, a dichroic mirror 9, a spectral separation module 12, a first tube mirror 13 and a second tube mirror 15, a first image sensor 14 and a second image sensor 16.

[0047] The stage 11 includes a three-dimensional electric displacement stage in the XYZ direction with a sample clamp, which is used to drive the sample to make nanometer-scale step displacement relative to the structured light field in order to acquire fluorescence image sequences of multi-phase structured light illumination.

[0048] The microscope objective 10 collects the fluorescence emitted by the sample, and the fluorescence enters the spectral separation module 12 after being transmitted through the dichroic mirror 9.

[0049] The spectral separation module 12 is disposed in the imaging optical path, located between the sample and the image sensor. It is used to receive the signal light carrying the super-resolution information of the sample, which is excited by multicolor structured light illumination, and spatially separates it into different optical path channels according to the different wavelength components of the signal light, so as to eliminate spectral crosstalk between different wavelength signal lights and realize synchronous and independent acquisition of images of each wavelength channel. In this embodiment, the spectral separation module 12 adopts a dichroic mirror spectral separation method, setting a dichroic mirror with a center wavelength of 585nm to reflect green fluorescence with a wavelength less than 585nm (from 488nm excitation, emission peak about 520nm) to the first imaging channel, and transmit red fluorescence with a wavelength greater than 585nm (from 561nm excitation, emission peak about 610nm) to the second imaging channel.

[0050] The first tube mirror 13 and the first image sensor 14 constitute the first imaging channel. The first tube mirror 13 is used to focus the spectrally separated green fluorescence onto the target surface of the first image sensor 14 to form a green channel fluorescence image.

[0051] The second tube mirror 15 and the second image sensor 16 constitute the second imaging channel. The second tube mirror 15 is used to focus the spectrally separated red fluorescence onto the target surface of the second image sensor 16 to form a red channel fluorescence image.

[0052] The first image sensor 14 and the second image sensor 16 can be either a scientific-grade CMOS camera or an EMCCD camera.

[0053] The computer connects to and controls all electric components, including the switching and power adjustment of the multi-band laser source module 1, the displacement drive of the stage 11, and the acquisition triggering of the first image sensor 14 and the second image sensor 16. The computer performs algorithmic post-processing on all acquired raw images to reconstruct the super-resolution 3D structure. The reconstruction algorithm can adopt the classic structured light illumination super-resolution reconstruction algorithm, such as the spectrum separation algorithm proposed by Gustafsson. Since the spatial frequency and orientation of the structured light in the red and green channels are known and consistent in this embodiment, there is no need to calibrate the structured light parameters of each channel separately during the reconstruction process. The unified illumination parameters can be used directly for reconstruction, which greatly simplifies the calculation process and avoids artifacts introduced by parameter calibration errors.

[0054] In this invention, because the structured light field parameters are consistent across different wavelengths, the consistency error of structured light parameters in multi-channel reconstructed images is less than 5%, avoiding image artifacts caused by parameter calibration errors. Simultaneously, the metasurface lens has no moving mechanical parts, resulting in a fast response speed and enabling video-level multi-wavelength super-resolution imaging. Replacing the SLM or DMD in traditional SIM systems with a single metasurface lens significantly simplifies the system structure, achieving a miniaturized and integrated structured light illumination module.

[0055] Example 2 A three-dimensional super-resolution imaging method based on multicolor structured light illumination of the above system is described in detail in the corresponding descriptions of the system embodiments described above, and will not be repeated here. Figure 1 , Figure 2 As shown, the method includes the following steps: S1. The multi-band laser source module 1 emits excitation light of at least two wavelengths simultaneously, such as excitation light of 488nm and 561nm simultaneously. The two wavelengths of laser light are combined to form a common beam and enter the subsequent optical path.

[0056] S2. The excitation light is collimated, expanded, and polarized before being incident on the metasurface lens; Specifically, the combined excitation light is collimated and expanded by collimating and expanding lens 2 to become a parallel beam, increasing the beam diameter to meet the aperture requirements of metasurface lens 4. The collimated light is then modulated into P-direction linearly polarized light by polarizer 3.

[0057] S3. The metasurface lens imparts a preset phase distribution to incident light of different wavelengths, so that light of each wavelength produces a structured light field with consistent spatial frequency and consistent fringe orientation on the same target surface. Specifically, P-polarized collimated rays are incident on metasurface lens 4. Metasurface lens 4 assigns a preset phase distribution to incident light of different wavelengths: for a wavelength of 488 nm, it assigns a phase distribution φ1(x,y); for a wavelength of 561 nm, it assigns a phase distribution φ2(x,y). Although these two phase distributions are numerically different, after Fourier transform, the structured light field generated on the target surface has the same spatial frequency and the same fringe orientation.

[0058] S4. The generated structured light field is focused onto the sample surface after Fourier transform, spatial filtering, and segmented polarization modulation, exciting the sample to generate multicolor fluorescence signals. Specifically, the light field modulated by the metasurface lens 4 undergoes a Fourier transform via the Fourier lens group 5, forming diffraction spots of various orders on the Fourier plane. A filter aperture 6 is positioned on the Fourier plane to filter out the 0th and ±1st order diffraction light, blocking other higher order diffraction light.

[0059] S5. After the multicolor fluorescence signal is collected by the microscope objective, it is separated into different imaging channels according to wavelength by the spectral separation module; Specifically, the segmented polarization modulation half-wave plate 7 is composed of fan-shaped wave plates with different fast axis directions, and the diffracted light of each order is modulated into different polarization directions by passing through different fan-shaped wave plates.

[0060] S6. Fluorescence images are acquired synchronously through each imaging channel and reconstructed by an algorithm to obtain a multicolor super-resolution three-dimensional image.

[0061] Specifically, the polarization-modulated diffracted light of each order is focused onto the back focal plane of the microscope objective 10 through lens 8 and dichroic mirror 9. The microscope objective 10 re-collimates the light field at the back focal plane and projects it onto the sample surface, where interference occurs, forming a sinusoidal fringe-like structured light. For excitation light at 488 nm and 561 nm, the formed structured light has the same fringe period and fringe orientation. This step further includes: S61, the stage 11 drives the sample to perform nanometer-scale step displacement relative to the structured light field. This embodiment uses a three-step phase-shifting method, acquiring images of three phases in each direction, with a phase step of 120°, corresponding to a sample displacement of 1 / 3 of the fringe period, or approximately 93 nm. For each structured light direction, fluorescence images are acquired sequentially at phase shifts φ=0°, 120°, and 240°. Simultaneously, by rotating the polarization rotator in front of the metasurface lens 4, the orientation of the structured light fringes can be changed. This embodiment acquires structured light images in three directions: 0°, 60°, and 120°. Therefore, each wavelength channel acquires a total of 3 directions × 3 phases, equaling 9 original images. Because the two wavelengths are acquired simultaneously, the total number of frames is 9, with each frame containing information from both channels simultaneously, instead of the 18 frames of the traditional time-switching mode, doubling the imaging speed.

[0062] S62. The fluorescence generated by the sample under structured light excitation is collected by microscope objective 10. Of the fluorescence, green fluorescence (emission peak approximately 520 nm) and red fluorescence (emission peak approximately 610 nm) are transmitted through dichroic mirror 9 and then enter the spectral separation module 12. The 585 nm dichroic mirror in the spectral separation module 12 reflects the green fluorescence to the first imaging channel and transmits the red fluorescence to the second imaging channel.

[0063] S63. In the first imaging channel, green fluorescence is focused onto the target surface of image sensor 14 via tubular lens 13, recording the green channel image; in the second imaging channel, red fluorescence is focused onto the target surface of image sensor 16 via tubular lens 15, recording the red channel image. The image sensors of the two channels are synchronously triggered by the computer, achieving true simultaneous acquisition and avoiding time delays and registration errors introduced by timing switching.

[0064] S64. The computer performs algorithmic post-processing on all acquired raw images to reconstruct the super-resolution 3D structure. The reconstruction process is as follows: For each channel's 9 original frames of images, a spectrum separation algorithm is used to extract the amplitude and phase of each frequency component, separating the 0-frequency, +1-frequency, and -1-frequency components; The separated frequency components are shifted and weighted in the frequency domain to expand the spectral range to about twice the original objective lens passband; Perform an inverse Fourier transform on the expanded spectrum to obtain the super-resolution image of that channel; Pixel-level registration and fusion of the two channels' super-resolution images are performed to obtain a multicolor super-resolution image; For three-dimensional imaging, the spatial frequency of structured light is changed by altering the illumination area of ​​the incident light on the metasurface lens 4, or by performing axial layer-by-layer scanning through the stage 11, and the above steps are repeated to reconstruct a three-dimensional super-resolution image stack.

[0065] Since the spatial frequency and orientation of the structured light in the red and green channels are known and consistent in this embodiment, there is no need to calibrate the structured light parameters of each channel separately during the reconstruction process. The unified illumination parameters can be used directly for reconstruction.

[0066] Existing structured illumination (SIM) systems face key technical challenges in multicolor imaging applications, including slow imaging speed, system complexity, and inconsistent structured light periods across wavelengths. To address these shortcomings, this invention provides a multicolor structured light illumination super-resolution imaging system and method based on a dispersive engineered metasurface. This invention utilizes a metasurface with specific dispersive properties to generate structured light patterns with pre-defined periodic matching relationships after incident light of different wavelengths passes through the metasurface, achieving synchronous illumination of multicolor structured light. Combined with a spectral separation module, it enables the synchronous acquisition and reconstruction of multi-channel super-resolution images. This invention significantly improves the imaging throughput and system integration of multicolor SIM, providing a new technical solution for multicolor super-resolution observation of dynamic biological processes, and making structured light illumination super-resolution imaging a more promising in vivo multi-channel rapid imaging technology in the life sciences.

[0067] The number of devices and processing scale described herein are for the purpose of simplifying the description of the invention. Applications, modifications, and variations of the invention will be readily apparent to those skilled in the art.

[0068] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

[0069] It should also be noted that the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0070] The various embodiments in this specification are described in a progressive manner. Similar or identical parts between embodiments can be referred to interchangeably. Each embodiment focuses on describing the differences from other embodiments. In particular, the system embodiments are basically similar to the method embodiments, so the description is relatively simple; relevant parts can be referred to the descriptions in the method embodiments.

[0071] The above description is merely an embodiment of this specification and is not intended to limit the scope of one or more embodiments of this specification. Various modifications and variations can be made to one or more embodiments of this specification by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principle of one or more embodiments of this specification should be included within the scope of the claims of one or more embodiments of this specification.

Claims

1. A three-dimensional super-resolution imaging system with multicolor structured light illumination, characterized in that, include: A multi-band polarization multiplexing structured light illumination module is used to generate at least two structured light illumination fields of different wavelengths and to realize phase translation, orientation rotation and polarization control of the structured light illumination fields. The microscopic imaging module is used to receive the fluorescence signal generated by the sample under the excitation of the structured light illumination field and form a multi-channel super-resolution image; The multi-band polarization multiplexing structured light illumination module includes a metasurface lens, which is configured to generate sinusoidal structured light fields with the same spatial frequency and the same fringe orientation on the same target surface for at least two incident P-polarized lights of different wavelengths.

2. The three-dimensional super-resolution imaging system with multicolor structured light illumination as described in claim 1, characterized in that, The metasurface lens includes: Base; Subwavelength structure array disposed on the substrate; The subwavelength structure array is configured such that, for the first wavelength The incident P-polarized light generates a first illumination light field with a first structured light distribution on the target surface; For the second wavelength Incident P-polarized light, A second illumination field with a spatial frequency difference of less than 5% and a stripe orientation deviation of less than 1° is generated on the same target surface.

3. The three-dimensional super-resolution imaging system with multicolor structured light illumination as described in claim 2, characterized in that, The unit structure of the subwavelength structure array is selected from one or more of nanopillars, nanofins, nanopores, and nanorings. The geometric parameters of the unit structure are configured to have dispersion compensation characteristics within the target wavelength range, so that the spatial frequency of the structured light field generated by incident light of different wavelengths is consistent.

4. The three-dimensional super-resolution imaging system with multicolor structured light illumination as described in claim 2, characterized in that, The subwavelength structure array employs a polarization multiplexing design, assigning different phase distributions to incident P-polarized light of different wavelengths. These phase distributions, after Fourier transform, produce identical multi-beam interference fringes.

5. A three-dimensional super-resolution imaging system with multicolor structured light illumination as described in claim 1, characterized in that, The spatial frequency of the sinusoidal structured light is adjustable. By changing the irradiation area or incident angle of the incident light on the metasurface lens, the spatial frequency of the structured light can be continuously adjusted.

6. The three-dimensional super-resolution imaging system with multicolor structured light illumination as described in claim 1, characterized in that, The wavelength of the incident P-polarized light is selected from at least two of 405nm, 488nm, 532nm, 561nm, and 640nm.

7. The three-dimensional super-resolution imaging system with multicolor structured light illumination as described in claim 1, characterized in that, The multi-band polarization multiplexing structured light illumination module also includes: A multi-band laser source module for emitting light in at least two bands; A collimating beam expander is used to convert the emitted light into collimated light rays; A polarizer is used to modulate the collimated light into linearly polarized light in the P direction; Fourier lens group, used to transform the light field modulated by the metasurface lens; The filter aperture is used to filter out light rays of the 0th and ±1st diffraction orders; A segmented polarization modulation half-wave plate is composed of sector waveplates with different fast axis directions, used to modulate diffracted light of each order into different polarization directions. The lens group and dichroic mirror are used to focus the modulated diffracted light onto the back focal plane of the microscope objective.

8. A three-dimensional super-resolution imaging system with multicolor structured light illumination as described in claim 1, characterized in that, The microscopic imaging module includes: Microscope objectives are used both to focus illumination light onto the sample surface and to collect the fluorescence emitted by the sample. The stage includes a three-dimensional electric displacement stage with sample clamps, used to drive the sample to make nanometer-scale step displacements relative to the structured light field. The spectral separation module is used to spatially separate fluorescence of different wavelengths into different optical paths. A tube lens is used to focus the separated single-band fluorescence onto the target surface of an image sensor. Image sensor used to acquire fluorescence images.

9. A three-dimensional super-resolution imaging system with multicolor structured light illumination as described in claim 8, characterized in that, The spectral separation module is implemented using one or more of the following methods: dichroic mirror spectral separation, metasurface spectral filter array, and tunable bandpass filter.

10. A method for three-dimensional super-resolution imaging with multicolor structured light illumination based on the system described in any one of claims 1 to 9, characterized in that, Includes the following steps: S1. The multi-band laser source module emits excitation light of at least two wavelengths simultaneously; S2. The excitation light is collimated, expanded, and polarized before being incident on the metasurface lens; S3. The metasurface lens imparts a preset phase distribution to incident light of different wavelengths, so that light of each wavelength produces a structured light field with consistent spatial frequency and consistent fringe orientation on the same target surface. S4. The generated structured light field is then subjected to Fourier transform, spatial filtering, and segmented polarization modulation, and focused onto the sample surface to excite the sample to generate multicolor fluorescence signals. S5. After the multicolor fluorescence signal is collected by the microscope objective, it is separated into different imaging channels according to wavelength by the spectral separation module; S6. Fluorescence images are acquired synchronously through each imaging channel, and multicolor super-resolution three-dimensional images are obtained after reconstruction by the algorithm.

11. The three-dimensional super-resolution imaging method with multicolor structured light illumination as described in claim 10, characterized in that, In step S3, the metasurface lens ensures that the spatial frequency difference of the structured light field generated by incident light of different wavelengths is less than 5% and the fringe orientation deviation is less than 1°, without the need to individually calibrate the structured light parameters for each wavelength channel.

12. The three-dimensional super-resolution imaging method with multicolor structured light illumination as described in claim 10, characterized in that, Also includes: By changing the illumination area or incident angle of the incident light on the metasurface lens, the spatial frequency of the structured light field can be continuously adjusted to achieve super-resolution imaging at different resolutions.

13. The three-dimensional super-resolution imaging method with multicolor structured light illumination as described in claim 10, characterized in that, Also includes: The sample is driven to perform nanometer-scale step displacement by a stage, and fluorescence image sequences under multi-phase structured light illumination are acquired for super-resolution image reconstruction.

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