Three-dimensional super-resolution microscopic imaging system and method

Abstract

The invention provides a three-dimensional super-resolution microscopic imaging system and method. The three-dimensional super-resolution microscopic imaging system comprises an illumination unit, a phase modulation unit, a light conduction unit and an imaging unit. Wherein the illumination unit is used for outputting uniform and parallel exciting light to the phase modulation unit, the phase modulation unit is used for generating multiple beams of structured light with differentiated axial phases, the light conduction unit is used for precise projection of the structured light and efficient convergence of fluorescence, and multi-axial depth information is integrated in combination with a super-resolution reconstruction algorithm. And finally, obtaining the total-volume optical super-resolution image of the sample to be detected. According to the invention, the collection efficiency of a single volume image can be greatly improved, three-dimensional super-resolution imaging with high transverse resolution, high axial resolution and high fidelity is realized, and the application scene and practical value of the structured illumination obvious micro-imaging technology in the fields of life science and the like are expanded.

Description

Technical Field

[0001] This application relates to the field of imaging technology, and in particular to a three-dimensional super-resolution microscopy system and method. Background Technology

[0002] The rapid development of super-resolution microscopy has broken the diffraction limit of traditional optical microscopes, making high-resolution observation of cellular components a reality. Among various super-resolution techniques, Structured Illumination Microscopy (SIM) is particularly suitable for long-term dynamic observation of live cell samples due to its use of low-power laser illumination, resulting in lower phototoxicity to live cells. Building upon this, 3D Structured Illumination Microscopy (3D-SIM) achieves three-dimensional super-resolution imaging by introducing structured light along the Z-axis, effectively improving isotropic resolution and capturing the full volumetric information of the sample. This makes it crucial in fields such as biomedical research.

[0003] However, traditional three-dimensional structured illumination microscopy (3D-SIM) technology mostly uses grating diffraction to generate structured light fringes. This method has inherent limitations, primarily that the phase of the fringes formed along the axial direction cannot be flexibly adjusted. This necessitates moving the sample along an axial displacement stage to perform layer-by-layer scanning to complete the acquisition of complete super-resolution information, directly resulting in slow imaging speed. The acquisition time for a single volume image can be as long as several seconds. This slow imaging efficiency not only makes it difficult to meet the real-time observation requirements of rapid three-dimensional biological processes but may also produce motion artifacts due to the sample's own movement, affecting the accuracy and reliability of the final image. Summary of the Invention

[0004] This application aims to at least partially address one of the technical problems in the related art.

[0005] Therefore, the first objective of this application is to propose a three-dimensional super-resolution microscopic imaging system and method that can equally separate the fluorescence signal excited by structured light illumination of the sample under test by a prism and obtain three-dimensional information through a single camera acquisition, which greatly improves the efficiency of volumetric imaging.

[0006] To achieve the above objectives, a first aspect of this application provides a three-dimensional super-resolution microscopic imaging system, comprising: An illumination unit for emitting excitation light of at least one wavelength in parallel; A phase modulation unit is disposed in the downstream optical path of the illumination unit for phase modulation of the incident excitation light and forming multiple beams of structured light with different axial phases. An optical transmission unit is disposed downstream of the phase modulation unit and is used to project and converge multiple incident beams of structured light onto the sample to be tested. An imaging unit, which shares a common path with the light transmission unit, is used to acquire and super-resolution reconstruct the fluorescence signal generated by the excitation of the sample under test, so as to obtain a three-dimensional super-resolution image of the sample under test.

[0007] Optionally, the multiple structured beams may include at least three structured beams with different axial phases.

[0008] Optionally, the phase modulation unit includes at least a spatial light modulator, on which a preset phase modulation pattern is loaded for phase modulation of the incident excitation light and forming multiple beams of structured light.

[0009] Optionally, the phase modulation pattern loaded on the spatial light modulator consists of multiple Fresnel zone plate patterns arranged in an array, and the center point coordinates of different Fresnel zone plate patterns on the spatial light modulator are different.

[0010] Optionally, the phase modulation pattern loaded on the spatial light modulator includes at least three Fresnel zone plate patterns arranged in an array.

[0011] Optionally, the phase modulation unit further includes a first half-wave plate, a polarization beam splitter, and a second half-wave plate; wherein, The first half-wave plate is disposed in the upstream optical path of the polarization beam splitter, and the second half-wave plate is disposed in the optical path between the polarization beam splitter and the spatial light modulator.

[0012] Optionally, the lighting unit includes a light source, a first lens, a pinhole, and a second lens; wherein, The first lens is disposed in the downstream optical path of the light source, the pinhole is disposed in the downstream optical path of the first lens, and the second lens is disposed in the downstream optical path of the pinhole. The focal points of the first lens and the second lens near the pinhole overlap and are both located within the aperture of the pinhole.

[0013] Optionally, the light transmission unit includes a third lens, a field stop, a fourth lens, a dichroic mirror, a fifth lens, a barrel lens, and an objective lens, which are sequentially spaced apart in the downstream optical path of the phase modulation unit; wherein, The focal points of the third lens and the fourth lens near the field stop overlap and form a 4F optical system. The field stop is located on the middle focal plane of the 4F system formed by the third lens and the fourth lens. The fifth lens, the barrel lens, and the objective lens are arranged sequentially in the reflection or transmission light path of the dichroic mirror.

[0014] Optionally, the imaging unit includes a multifocal depth-of-field prism and at least one camera, and the multifocal depth-of-field prism and the camera are sequentially arranged in the transmission or reflection light path of the dichroic mirror.

[0015] To achieve the above objectives, a second aspect of this application provides a three-dimensional super-resolution microscopy imaging system, comprising: Acquire a dataset of multiple fluorescence images of the sample under test in multiple directions and multiple phases; Multiple acquired fluorescence image datasets were registered and corrected to obtain multiple calibrated fluorescence image datasets; A super-resolution reconstruction algorithm was used to perform full-spectrum separation, extraction, and frequency shifting on multiple calibrated fluorescence image datasets to obtain multiple super-resolution spectral components; Wiener filtering algorithm is used to perform noise reduction and signal enhancement on multiple spectral components to obtain multiple optimized super-resolution spectral components; A three-dimensional reconstruction algorithm is used to fuse and reconstruct multiple optimized super-resolution spectral components to obtain a full-volume optical super-resolution image of the sample under test. In this process, multiple fluorescence image datasets are acquired by using a three-dimensional super-resolution microscopy system as described above to collect the fluorescence signals generated by the excitation of the sample under test.

[0016] The three-dimensional super-resolution microscopy system and method provided in this application have at least the following beneficial effects: This application provides a three-dimensional super-resolution microscopy imaging system and method, including an illumination unit, a phase modulation unit, a light transmission unit, and an imaging unit. The illumination unit outputs uniform and parallel excitation light from the phase modulation unit. The phase modulation unit generates multiple beams of structured light with differentiated axial phases through flexible optical field phase modulation, covering the entire sample volume without the need for an axial displacement stage. The light transmission unit not only achieves precise projection and efficient convergence of the structured light, but its co-path design with the imaging unit simplifies the system structure, reduces light energy loss, and ensures efficient coordination between excitation and fluorescence signal acquisition. Combined with a super-resolution reconstruction algorithm, multi-axial depth information is integrated to ultimately achieve full-volume optical super-resolution image acquisition of the sample. This application can significantly improve the acquisition efficiency of single-volume images while possessing high lateral resolution, high axial resolution, and high fidelity three-dimensional super-resolution imaging. Furthermore, its multi-wavelength adaptability enhances compatibility with different fluorescently labeled samples, providing reliable technical support for real-time observation of rapid biological dynamic processes and precise analysis of microscopic three-dimensional structures. This expands the application scenarios and practical value of structured illumination microscopy imaging technology in life sciences and other fields.

[0017] Additional aspects and advantages of this application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of this application. Attached Figure Description

[0018] The above and / or additional aspects and advantages of this application will become apparent and readily understood from the following description of the embodiments taken in conjunction with the accompanying drawings, wherein: Figure 1 This is a structural block diagram of a three-dimensional super-resolution microscopy system according to an embodiment of this application.

[0019] Figure 2 This is a schematic diagram of the structure of a three-dimensional super-resolution microscopy system according to an embodiment of this application.

[0020] Figure 3 This is a schematic diagram of a phase modulation pattern loaded on a spatial light modulator according to an embodiment of this application. Detailed Implementation

[0021] The embodiments of this application are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain this application, and should not be construed as limiting this application.

[0022] The imaging mechanism of 3D-SIM relies on the interference effect of three structured beams, which, after interference, form a sinusoidal pattern of bright and dark fringes covering the entire volume of the sample. Fine details (high-frequency information) in the sample that are difficult to detect directly will undergo frequency aliasing through this fringe pattern, migrating to the mid-to-low frequency range detectable by the objective lens, thus forming moiré fringes. Subsequent calculations and analysis of these moiré fringes extract the super-resolution information contained within and recombine them to reconstruct a high-resolution image. According to SIM imaging theory, single-direction super-resolution imaging requires acquiring three original images of different phases, while traditional 3D-SIM requires acquiring five phase images, and information from different focal planes of the sample must be acquired by moving the sample layer by layer using an axial displacement stage.

[0023] Traditional 3D structured illumination microscopy (3D-SIM) typically uses grating diffraction to generate structured light fringes. This approach has a significant inherent limitation: due to the physical characteristics of grating diffraction, the generated structured light fringes lack flexibility in axial phase adjustment, and dynamic control of the axial phase cannot be achieved directly through light field modulation. This core limitation directly necessitates that the system rely on an axial displacement stage to drive the sample to perform layer-by-layer scanning operations in order to complete the acquisition of full-volume super-resolution information. The layer-by-layer scanning operation mode inherently restricts the improvement of imaging efficiency, resulting in acquisition cycles for single-volume images often reaching several seconds.

[0024] From a practical application perspective, this relatively low imaging speed not only makes it difficult to match the real-time observation requirements of rapid three-dimensional biological dynamic processes (such as intracellular material transport and dynamic interactions of organelles), and fails to capture the instantaneous changes in biological processes; it also introduces motion artifacts due to the spontaneous movement of the sample during the acquisition cycle (such as cell drift and tissue contraction). These artifacts directly interfere with the accurate extraction of super-resolution signals, thereby affecting the accuracy and reliability of the final three-dimensional imaging results, and limiting the depth and breadth of application of traditional 3D-SIM technology in scenarios such as dynamic observation of living cells.

[0025] To address the aforementioned problems, the first aspect of this application provides a three-dimensional super-resolution microscopic imaging system, such as... Figure 1 and Figure 2 As shown, the system includes an illumination unit, a phase modulation unit, a light transmission unit, and an imaging unit. The illumination unit emits excitation light of at least one wavelength in parallel. The phase modulation unit, located downstream of the illumination unit, modulates the phase of the incident excitation light to form multiple beams of structured light with different axial phases. The light transmission unit, located downstream of the phase modulation unit, projects and converges the multiple beams of structured light onto the sample under test. The imaging unit, co-located with the light transmission unit, acquires and performs super-resolution reconstruction of the fluorescence signal generated by the excitation of the sample under test to obtain a three-dimensional super-resolution image of the sample.

[0026] Understandably, the illumination unit, as the core light source of the entire three-dimensional super-resolution microscopy system, has the core function of providing at least one wavelength of excitation light emitted in parallel. The output characteristics of parallel light ensure that the beam incident on the subsequent phase modulation unit has a uniform light intensity distribution and a stable propagation direction, laying the foundation for the accurate implementation of phase modulation. At the same time, the optional configuration of multiple wavelength excitation lights allows the system to adapt to the excitation requirements of different fluorescently labeled molecules, greatly improving the system's compatibility with various test samples and meeting diverse observation scenarios.

[0027] As a key functional unit for achieving super-resolution imaging, the phase modulation unit modulates the incident excitation light in the downstream optical path of the illumination unit. Essentially, it utilizes the flexibility of optical field phase control to replace the fixed mode of traditional grating diffraction, directly generating multiple beams of structured light with differentiated axial phases. Different axial phases allow the optical field formed by structured light interference to vary at different depths of the sample under test, thus eliminating the need for an axial displacement stage to drive layer-by-layer scanning of the sample. This fundamentally solves the core pain point of "slow imaging" in traditional techniques, providing crucial support for rapid imaging and reducing sample motion artifacts.

[0028] As the core carrier for optical path transmission and beam convergence, the optical transmission unit is located downstream of the phase modulation unit. It not only undertakes the function of accurately projecting multiple beams of structured light onto the sample under test, but its efficient convergence effect can also improve the excitation efficiency of fluorescence signals. More importantly, its shared-path design with the imaging unit simplifies the system structure, reduces light energy loss, and ensures that multiple beams of structured light act stably on the sample. On the other hand, it makes the propagation path of fluorescence signals highly compatible with the projection path of structured light, ensuring the efficiency from structure excitation to fluorescence signal acquisition, avoiding the signal delay and distortion caused by optical path switching in traditional scanning modes, and further enhancing the advantages of rapid imaging.

[0029] As the core of the system's signal processing and image output, the imaging unit shares a common path with the optical transmission unit, enabling it to simultaneously receive fluorescence signals generated by multiple beams of structured light from the sample under test. The imaging unit can simultaneously acquire fluorescence signals at multiple axial depths, directly replacing the traditional layer-by-layer scanning acquisition mode of 3D-SIM. This significantly shortens the acquisition cycle of a single volume image, avoids long acquisition times on the order of seconds, and fundamentally reduces motion artifacts caused by spontaneous sample movement. Simultaneously, the imaging unit integrates the synchronously acquired multi-axial depth signals through a super-resolution reconstruction algorithm, breaking through the diffraction limit of traditional optical imaging and restoring complete information about the sample's microscopic three-dimensional structure. Ultimately, it generates a three-dimensional image with high lateral resolution, high axial resolution, and high fidelity, achieving accurate representation of the sample's microscopic three-dimensional structure.

[0030] In summary, this application, through the collaborative design of the illumination unit, phase modulation unit, light transmission unit, and imaging unit, uses parallel multi-wavelength excitation light as a basis, generates axially differentiated structured light through phase modulation, achieves precise excitation and efficient signal acquisition through common-path light transmission, and finally obtains three-dimensional super-resolution images through super-resolution reconstruction. It systematically solves the shortcomings of traditional microscopic imaging in three-dimensional resolution, while taking into account the system's compatibility and structural simplicity. It can provide a high-precision and high-reliability imaging solution for microstructure observation in fields such as biomedicine and materials science.

[0031] In some embodiments, the phase modulation unit includes at least a spatial light modulator loaded with a preset phase modulation pattern for phase modulation of the incident excitation light and forming multiple beams of structured light.

[0032] Understandably, the phase modulation unit uses a spatial light modulator as its core execution component. By loading a preset phase modulation pattern onto its surface, a precise and controllable phase modulation mechanism is constructed, providing an efficient path for generating multiple differentiated structured beams. The preset phase modulation pattern is designed based on the target axial phase distribution requirements of the multiple structured beams. Through precise encoding of the pixelated phase information of the pattern, the incident parallel excitation light, when illuminating the spatial light modulator, can be given a preset phase shift by different pixel regions. This partitioned phase modulation directly changes the wavefront propagation characteristics of the beam, causing the originally uniformly propagating excitation light to split into multiple beams of structured light with specific axial phase differences after modulation. Furthermore, the phase parameters of each beam of structured light can be precisely defined through the design of the phase modulation pattern, perfectly matching the axial coverage requirements of the three-dimensional imaging of the sample under test.

[0033] The use of a spatial light modulator further enhances the system's flexibility and adaptability. It supports dynamic editing and real-time updating of phase modulation patterns, allowing users to flexibly adjust the number, axial distribution interval, and focal length range of multiple structured beams according to the structural characteristics and imaging depth requirements of different samples, without requiring modifications to the hardware structure, thus significantly expanding the system's application scenarios. At the same time, the high-resolution pixel array of the spatial light modulator enables precise phase control of the excitation light, avoiding excitation signal distortion caused by modulation errors, and providing a high-quality light field foundation for subsequent fluorescence signal acquisition and super-resolution reconstruction.

[0034] This phase modulation scheme based on a spatial light modulator and a preset phase modulation pattern is well-matched with the parallel excitation light output by the illumination unit, ensuring the uniformity and stability of the phase modulation. At the same time, it works in synergy with the beam projection function of the light transmission unit, enabling multiple beams of structured light to be accurately and efficiently converged to different axial depths of the sample under test. This provides core light field modulation support for the imaging unit to achieve three-dimensional super-resolution reconstruction, further enhancing the system's advantages in three-dimensional imaging resolution and flexibility.

[0035] The configuration of multiple structured beams is precisely matched with the phase modulation pattern loaded on the spatial light modulator. Each structured beam can achieve precise phase control through the phase modulation pattern, ensuring the consistency and stability of the parameters of each beam and avoiding problems such as insufficient axial resolution or decreased imaging accuracy due to insufficient number of beams.

[0036] In some embodiments, such as Figure 3As shown, the phase modulation pattern loaded on the spatial light modulator consists of multiple Fresnel zone plate patterns arranged in an array, and the center point coordinates of different Fresnel zone plate patterns are different on the spatial light modulator.

[0037] It is understandable that loading a phase modulation pattern composed of multiple arrayed Fresnel zone plate patterns with different center point coordinates is a further optimization of the phase control mechanism. Through the synergistic design of the inherent optical properties of the Fresnel zone plates and the arrayed and differentiated layout, the precise generation and axial distribution control of multiple structured beams can be achieved.

[0038] Fresnel zone plates, as classic diffractive optical elements, possess the core characteristic of efficiently focusing incident light through phase modulation of their annular structure. Each Fresnel zone plate pattern can independently converge incident parallel excitation light into a beam with a specific phase. This characteristic perfectly matches the requirement of phase modulation units to generate multiple beams of structured light with different phases. The array-distributed design of multiple Fresnel zone plate patterns allows the spatial light modulator to simultaneously perform zoned synchronous modulation of the incident excitation light, generating multiple beams of structured light at once. This avoids the serial operation mode of single-pattern modulation, significantly improving the efficiency of light field manipulation and ensuring that multiple beams of structured light can act synchronously on the sample under test, providing continuous and comprehensive axial excitation data for three-dimensional imaging.

[0039] The different coordinates of the center point directly correspond to the differences in the incident angle and propagation path of each structured light beam. Combined with the preset parameters such as the ring radius and period of each Fresnel zone plate pattern, the structured light beams form a precise and controllable axial phase difference after modulation. This differentiated axial phase design allows multiple structured light beams to be focused on the same position of the sample under test and generate corresponding excitation fringes, thereby modulating the fluorescence signal and achieving a frequency shift with super-resolution effect without relying on an axial displacement stage to drive the sample to scan layer by layer. This enables layered excitation of the sample in three-dimensional space, and the phase information of each structured light beam can be accurately captured by the imaging unit, providing rich axial dimension data for subsequent super-resolution reconstruction, effectively breaking through the diffraction limit of traditional imaging in axial resolution.

[0040] Furthermore, this design based on arrayed Fresnel zone plate patterns offers high flexibility and scalability. By adjusting the array density, center point coordinate interval, and ring parameters of the Fresnel zone plate pattern, the number of multiple structured beams, axial coverage, and focusing accuracy can be flexibly controlled to adapt to samples of different thicknesses and structural characteristics. Simultaneously, the digital characteristics of the Fresnel zone plate pattern, combined with the dynamic loading function of the spatial light modulator, allow the phase modulation pattern to be updated in real time according to imaging requirements, further expanding the system's application scenarios. This design is well-matched with the parallel excitation light output from the illumination unit, ensuring uniform phase modulation of each structured beam during modulation. Simultaneously, it works synergistically with the beam converging and projection functions of the light transmission unit, enabling multiple structured beams to accurately and efficiently act on the target area of ​​the sample, laying a solid optical field foundation for the imaging unit to achieve high-quality three-dimensional super-resolution reconstruction.

[0041] For example, such as Figure 3 As shown, the phase modulation pattern loaded on the spatial light modulator includes at least three Fresnel zone plate patterns arranged in an array, and the three Fresnel zone plate patterns in each phase modulation pattern can be arbitrarily arranged to form different array distributions.

[0042] The phase modulation pattern loaded on the spatial light modulator includes at least three Fresnel zone plate patterns arranged in an array. This design is precisely adapted to the requirement that multiple structured beams consist of at least three differentiated beams. Through the one-to-one correspondence between the number of patterns and the number of beams, it ensures that each structured beam can obtain independent and precise phase modulation, providing core support for the axial coverage integrity and data richness of three-dimensional super-resolution imaging.

[0043] This design further enhances the synergy between the phase modulation unit and other system components. It not only achieves excellent compatibility with the parallel excitation light output from the illumination unit, ensuring that each Fresnel zone plate pattern provides uniform and stable phase modulation of the parallel light; it also works in conjunction with the beam projection function of the light transmission unit to ensure that three or more structured beams can be accurately and efficiently converged to the target axial depth of the sample; simultaneously, it works closely with the super-resolution reconstruction algorithm of the imaging unit, providing the algorithm with sufficiently rich axial phase information and fluorescence signal data, resulting in a significant improvement in the axial resolution and detail reproduction of the reconstructed 3D image. Furthermore, the design of three or more Fresnel zone plate patterns reserves space for system performance expansion. Future improvements in axial coverage and imaging accuracy can be achieved by increasing the number of patterns and optimizing the array layout, meeting the observation needs of more complex scenarios.

[0044] In some embodiments, the phase modulation unit further includes a first half-wave plate, a polarization beam splitter, and a second half-wave plate. The first half-wave plate is disposed upstream of the polarization beam splitter, and the second half-wave plate is disposed in the optical path between the polarization beam splitter and the spatial light modulator.

[0045] Understandably, the parallel light emitted from the illumination unit first enters the first half-wave plate HWP1. The optical axis angle of the first half-wave plate HWP1 is pre-calibrated, and its core function is to adjust the polarization state of the incident light to match the reflection characteristics of the polarization beam splitter PBS (such as converting it to s-polarization), ensuring that the light is efficiently reflected by the polarization beam splitter PBS to the subsequent optical path and avoiding energy loss caused by light passing through the polarization beam splitter PBS. The linearly polarized light reflected by the polarization beam splitter PBS then enters the second half-wave plate HWP2. The optical axis angle of the second half-wave plate HWP2 is set to rotate the polarization direction of the incident light by π / 8, adjusting it to match the initial polarization state of the spatial light modulator SLM liquid crystal pixel response, laying the foundation for subsequent pixelated polarization control.

[0046] A spatial light modulator (SLM) consists of a liquid crystal panel composed of multiple liquid crystal pixels. Each pixel can switch between "on" and "off" states under computer program control. When a pixel is in the "on" state, it rotates the polarization direction of the incident light counterclockwise by π / 4; when a pixel is in the "off" state, it rotates the polarization direction of the incident light clockwise by π / 4. By controlling the state distribution of each pixel, the SLM can modulate the polarization direction of the incident light at the pixel level, providing a basis for the subsequent formation of phase difference.

[0047] The light modulated by the spatial light modulator (SLM) is reflected and re-incidentally incident on the second half-wave plate (HWP2), where its polarization state is rotated twice by π / 8. At this point, the polarization directions corresponding to the SLM's "on" state and "off" state form angles of +45° and -45° respectively with the outgoing polarization direction of the polarization beam splitter (PBS). When these two polarized beams are incident on the PBS, an optical path difference arises due to the difference in polarization direction. After passing through the PBS, the modulated light at the corresponding pixel points in the SLM's "on" and "off" states will form a phase difference of π. Therefore, the SLM, HWP2, and PBS together constitute a controllable phase grating, and a preset phase pattern can be loaded by programmatically controlling the pixel states of the SLM.

[0048] Therefore, based on the phase grating function formed by the combination of polarization beam splitter PBS, second half-wave plate HWP2, and spatial light modulator SLM, an algorithm loads an array of Fresnel zone plate patterns onto the spatial light modulator SLM. For example, at the coordinates (0, Y), (0, 0), and (0, - Y) of the spatial light modulator SLM, three Fresnel zone plate patterns are loaded with these as the center points. The pixel state of the spatial light modulator SLM is adjusted according to the distribution of the zone plate patterns, modulating the incident parallel light into three phase-controllable structured light beams. That is, each Fresnel zone plate pattern modulates a beam of structured light, which is focused at a specific distance to form a light spot. These three light spots propagate through the light field of the subsequent lens and converge and interfere at the same position on the sample under test, finally forming sine / cosine structured light stripes with specific direction and phase, providing the required light field basis for three-dimensional super-resolution imaging.

[0049] It should be noted that the phase modulation unit also includes a light-shielding mask disposed on the light-emitting side of the spatial light modulator (SLM), which is used to filter the high-order diffraction light generated by the SLM diffraction, improve the coherence of the excitation fringes, and also filter out other background stray light that affects the imaging effect, thereby achieving better super-resolution effect.

[0050] In some embodiments, the illumination unit includes a light source, a first lens, a pinhole, and a second lens; wherein the first lens is disposed in the downstream optical path of the light source, the pinhole is disposed in the downstream optical path of the first lens, the second lens is disposed in the downstream optical path of the pinhole, and the focal points of the first lens and the second lens near the pinhole overlap and are both located within the aperture of the pinhole.

[0051] Understandably, the diverging light emitted from the light source first enters the first lens L1. As a converging lens, the focal point of the first lens L1 near the pinhole is precisely set within the aperture of the pinhole to perform spatial filtering, allowing only the central principal spot converged by the first lens L1 to pass through, while filtering out stray light, higher-order diffraction light, and other interfering light from the light source, significantly improving the coherence and uniformity of the emitted light. Subsequently, the light passing through the pinhole enters the second lens L2. The focal point of the second lens L2 near the pinhole completely overlaps with the focal point of the first lens L1, and both are located within the pinhole. This allows the second lens L2 to reconvert the converged light at the pinhole back into parallel light for outward emission.

[0052] As an example, the light source may include one or more lasers, and the lasers available may include, but are not limited to, lasers with wavelengths of 405nm, 445nm, 488nm, 560nm, and 642nm. Exemplarily, the light source may include lasers Laser1, Laser2, and Laser3. These three lasers, Laser1, Laser2, and Laser3, can emit laser light of different wavelengths. Each of these three lasers, Laser1, Laser2, and Laser3, is equipped with a reflector M1, a dichroic mirror (or beam splitter) DM1, and DM2, respectively, to combine parallel beams of equal diameter from different lasers into a single laser beam.

[0053] In some embodiments, the light transmission unit includes a third lens, a field stop, a fourth lens, a dichroic mirror, a fifth lens, a barrel lens, and an objective lens arranged sequentially at intervals in the downstream optical path of the phase modulation unit.

[0054] It is understandable that the third lens L3 and the fourth lens L4 constitute a standard 4F optical system, used for multiple beams of structured light output from the distortion-free conjugate transmission phase modulation unit. The field stop is located between L3 and L4 and is situated in the central focal plane region of the 4F system. The dichroic mirror DM3 is located downstream of the fourth lens L4. The fifth lens L5, the barrel lens, and the objective lens are sequentially and coaxially arranged in the reflection or transmission path of DM3, forming a high-precision focusing optical system.

[0055] Among them, the dichroic mirror DM3 utilizes selective spectral characteristics to precisely match the wavelengths of excitation light and fluorescence signal, so as to efficiently transmit phase-modulated structured light in the forward optical path and let it enter the subsequent focusing optical path; and in the reverse optical path, the fluorescence signal generated by the total internal reflection of the sample is coupled to the imaging unit.

[0056] In some embodiments, the imaging unit includes a multifocal depth-of-field prism and at least one camera; wherein the multifocal depth-of-field prism is disposed in the transmission light path of the dichroic mirror DM3, and the camera is disposed in the downstream light path of the multifocal depth-of-field prism.

[0057] Understandably, the core design goal of the imaging unit is to cooperate with the scanless excitation of the front-end unit to achieve rapid and high-fidelity acquisition of fluorescence signals across the entire depth of the sample, shortening the imaging cycle and reducing sample motion artifacts from the acquisition end. Specifically, the multi-focal depth-of-field extension prism is coaxially positioned in the transmission path of the dichroic mirror DM3. Its optical structure is specially designed to perform axial beam splitting and multi-focal synchronous imaging processing of the fluorescence signal transmitted by the dichroic mirror DM3. This allows for the simultaneous decomposition of fluorescence signals generated at different axial depths of the sample into optical signals corresponding to multiple focal planes, effectively extending the axial detection range of the imaging unit. This enables simultaneous acquisition of multi-axial information from the sample, improving the efficiency of full-volume fluorescence detection and providing full-depth fluorescence signal support for 3D super-resolution reconstruction.

[0058] At least one camera is positioned downstream of the multifocal depth-of-field prism, and the camera's photosensitive surface is precisely conjugate to the multifocal optical signal output by the multifocal depth-of-field prism. The camera is configured to synchronously acquire fluorescence signals from each focal plane after processing by the multifocal depth-of-field prism, convert the optical signals into digital electrical signals, and transmit them to the back-end processing module. The camera's pixel resolution, frame rate, and photosensitivity are all adapted to the intensity characteristics of the fluorescence signal and the imaging speed requirements to ensure the fidelity of the acquisition of each focal plane signal and avoid affecting the accuracy of 3D reconstruction due to signal attenuation or distortion.

[0059] In addition, the imaging unit also includes a filter wheel and a sixth lens L6, which are arranged sequentially between the dichroic mirror DM3 and the multifocal depth-of-field extension prism.

[0060] The filter wheel is positioned between the dichroic mirror DM3 and the sixth lens L6. It is equipped with at least one set of narrowband filters. The center wavelength of the filter is precisely matched with the characteristic wavelength of the fluorescence signal generated by the sample under test. It is used to perform secondary wavelength screening on the fluorescence signal transmitted by the dichroic mirror DM3 to efficiently filter out interference signals such as residual excitation light, ambient stray light and sample autofluorescence, and only allow the target fluorescence signal to pass through, which greatly improves the signal-to-noise ratio and purity of the fluorescence signal, and provides a pure signal source for subsequent signal acquisition and reconstruction.

[0061] For example, the filter wheel is an electrically switched filter assembly, which can adapt to different fluorescence signal acquisition needs in multi-wavelength excitation scenarios, thereby improving the system's compatibility and flexibility.

[0062] It should be noted that the above-described configuration of the dichroic mirror DM3 is only an example and should not be construed as a limitation of this application. That is to say, in other examples, the configuration of the dichroic mirror DM3 can also be adjusted so that the fifth lens L5, the barrel lens, and the objective lens are coaxially arranged in sequence on the transmitted light path of the dichroic mirror DM3, and the multifocal depth-of-field prism is arranged on the reflected light path of the dichroic mirror DM3.

[0063] According to a second aspect of this application, a super-resolution microscopy imaging method is also provided, comprising: Acquire a dataset of multiple fluorescence images of the sample under test in multiple directions and phases; Multiple acquired fluorescence image datasets were registered and corrected to obtain multiple calibrated fluorescence image datasets; A super-resolution reconstruction algorithm was used to perform full-spectrum separation, extraction, and frequency shifting on multiple calibrated fluorescence image datasets to obtain multiple super-resolution spectral components; Wiener filtering algorithm is used to perform noise reduction and signal enhancement on multiple spectral components to obtain multiple optimized super-resolution spectral components; A three-dimensional reconstruction algorithm is used to fuse and reconstruct multiple optimized super-resolution spectral components to obtain a full-volume optical super-resolution image of the sample under test. In this process, multiple fluorescence image datasets are acquired by using the three-dimensional super-resolution microscopy imaging system described in any of the embodiments of the first aspect above to collect the fluorescence signals generated by the excitation of the sample under test.

[0064] In summary, this application provides a three-dimensional super-resolution microscopy imaging system and method, including an illumination unit, a phase modulation unit, a light transmission unit, and an imaging unit. The illumination unit outputs uniform and parallel excitation light from the phase modulation unit. The phase modulation unit generates multiple beams of structured light with differentiated axial phases through flexible optical field phase modulation, covering the entire sample volume without the need for an axial displacement stage. The light transmission unit not only achieves precise projection and efficient convergence of the structured light, but its co-path design with the imaging unit simplifies the system structure, reduces light energy loss, and ensures efficient coordination between excitation and fluorescence signal acquisition. Combined with a super-resolution reconstruction algorithm, multi-axial depth information is integrated to ultimately achieve full-volume optical super-resolution image acquisition of the sample. This application can significantly improve the acquisition efficiency of single-volume images while possessing high lateral resolution, high axial resolution, and high fidelity three-dimensional super-resolution imaging. Furthermore, its multi-wavelength adaptability enhances compatibility with different fluorescently labeled samples, providing reliable technical support for real-time observation of rapid biological dynamic processes and precise analysis of microscopic three-dimensional structures. This expands the application scenarios and practical value of structured illumination microscopy imaging technology in life sciences and other fields.

[0065] In the foregoing descriptions of the embodiments, the terms "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0066] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this application, "multiple" means at least two, such as two, three, etc., unless otherwise explicitly specified.

Claims

1. A three-dimensional super-resolution microscopic imaging system, characterized in that, include: An illumination unit for emitting excitation light of at least one wavelength in parallel; A phase modulation unit is disposed in the downstream optical path of the illumination unit for phase modulation of the incident excitation light and forming multiple beams of structured light with different axial phases. An optical transmission unit is disposed downstream of the phase modulation unit and is used to project and converge multiple incident beams of structured light onto the sample to be tested. An imaging unit, which shares a common path with the light transmission unit, is used to acquire and super-resolution reconstruct the fluorescence signal generated by the excitation of the sample under test, so as to obtain a three-dimensional super-resolution image of the sample under test.

2. The three-dimensional super-resolution microscopic imaging system according to claim 1, characterized in that, The structured light beams comprise at least three beams with different axial phases.

3. The three-dimensional super-resolution microscopy imaging system according to claim 2, characterized in that, The phase modulation unit includes at least a spatial light modulator, on which a preset phase modulation pattern is loaded for phase modulation of the incident excitation light and forming multiple beams of structured light.

4. The three-dimensional super-resolution microscopy imaging system according to claim 3, characterized in that, The phase modulation pattern loaded on the spatial light modulator consists of multiple Fresnel zone plate patterns arranged in an array, and the center point coordinates of different Fresnel zone plate patterns on the spatial light modulator are different.

5. The three-dimensional super-resolution microscopy imaging system according to claim 4, characterized in that, The phase modulation pattern loaded on the spatial light modulator includes at least three Fresnel zone plate patterns arranged in an array.

6. The three-dimensional super-resolution microscopy imaging system according to claim 3, characterized in that, The phase modulation unit further includes a first half-wave plate, a polarization beam splitter, and a second half-wave plate; wherein... The first half-wave plate is disposed in the upstream optical path of the polarization beam splitter, and the second half-wave plate is disposed in the optical path between the polarization beam splitter and the spatial light modulator.

7. The three-dimensional super-resolution microscopic imaging system according to claim 1, characterized in that, The lighting unit includes a light source, a first lens, a pinhole, and a second lens; wherein... The first lens is disposed in the downstream optical path of the light source, the pinhole is disposed in the downstream optical path of the first lens, and the second lens is disposed in the downstream optical path of the pinhole. The focal points of the first lens and the second lens near the pinhole overlap and are both located within the aperture of the pinhole.

8. The three-dimensional super-resolution microscopy imaging system according to claim 1, characterized in that, The optical transmission unit includes a third lens, a field stop, a fourth lens, a dichroic mirror, a fifth lens, a barrel lens, and an objective lens, which are sequentially spaced apart in the downstream optical path of the phase modulation unit; wherein, The focal points of the third lens and the fourth lens near the field stop overlap and form a 4F optical system. The field stop is located on the middle focal plane of the 4F system formed by the third lens and the fourth lens. The fifth lens, the barrel lens, and the objective lens are arranged sequentially in the reflection or transmission light path of the dichroic mirror.

9. The three-dimensional super-resolution microscopic imaging system according to claim 8, characterized in that, The imaging unit includes a multifocal depth-of-field prism and at least one camera, and the multifocal depth-of-field prism and the camera are sequentially arranged in the transmission or reflection light path of the dichroic mirror.

10. A three-dimensional super-resolution microscopic imaging method, characterized in that, include: Acquire a dataset of multiple fluorescence images of the sample under test in multiple directions and multiple phases; Multiple acquired fluorescence image datasets were registered and corrected to obtain multiple calibrated fluorescence image datasets; A super-resolution reconstruction algorithm was used to perform full-spectrum separation, extraction, and frequency shifting on multiple calibrated fluorescence image datasets to obtain multiple super-resolution spectral components; Wiener filtering algorithm is used to perform noise reduction and signal enhancement on multiple spectral components to obtain multiple optimized super-resolution spectral components; A three-dimensional reconstruction algorithm is used to fuse and reconstruct multiple optimized super-resolution spectral components to obtain a full-volume optical super-resolution image of the sample under test. In this process, multiple fluorescence image datasets are acquired by using the three-dimensional super-resolution microscopy imaging system as described in any one of claims 1 to 9 to collect the fluorescence signals generated by the excitation of the sample under test.

Images