A microspectral imaging system and method of imaging

Abstract

The application provides a microscopic spectral imaging system and imaging method, comprising a non-uniform amplification module, a segmentation and convergence module and an intermediate image plane dispersion module, and the close cooperation of the three forms a complete spatial spectral decoupling and imaging system. The non-uniform amplification module is used for differentiating amplification of a fluorescence beam, so as to ensure the integrity of spatial information while expanding the spectral space; the segmentation and convergence module is used for segmenting the field of view after amplification into discrete sub-optical fields and converging to an intermediate image plane, so as to spatially isolate the spectral information of different regions and avoid aliasing; and the intermediate image plane dispersion module is used for spectral expansion and adaptive amplification of each sub-optical field, and converts the spectral information into a two-dimensional image signal that can be detected by a camera. The application effectively balances the spectral resolution, imaging speed and light utilization efficiency, and provides an innovative solution for fast and high-sensitivity spectral imaging.

Description

Technical Field

[0001] This application relates to the field of microscopic imaging technology, and in particular to a microscopic spectral imaging system and imaging method. Background Technology

[0002] In life science research, the efficient extraction and analysis of spectral information from fluorescence signals is crucial for distinguishing different molecular species, improving imaging throughput, and suppressing inter-channel crosstalk. Therefore, developing imaging technologies that can integrate spatial and spectral information acquisition has become a key direction in this field.

[0003] Currently, common methods for achieving multicolor fluorescence imaging rely on setting up 4 to 5 discrete filter imaging channels. Although this method can achieve a certain degree of signal differentiation, the signals of different types of fluorophores in the same channel are all represented as grayscale signals on the detector, making them impossible to directly separate and accurately identify, thus limiting its application in the simultaneous analysis of multiple components in complex biological samples.

[0004] To acquire continuous spectral information, multispectral or hyperspectral microscopy imaging techniques have been developed. However, these techniques typically face several bottlenecks: First, most schemes rely on scanning or spectral beam splitting mechanisms to acquire spectral information, which significantly reduces imaging speed or sacrifices spatial sampling range, limiting the system's temporal resolution and its ability to observe fast dynamic processes. Second, some schemes require using a portion of the signal light for spectral analysis, resulting in a weakened signal intensity used for imaging, directly reducing the image's signal-to-noise ratio and increasing the difficulty of subsequent data processing. Furthermore, hyperspectral imaging generates high-dimensional and massive amounts of data, putting pressure on storage systems, and its complex image reconstruction algorithms make real-time data processing and analysis difficult. In recent years, although imaging strategies combining compressed sensing, deep learning, and computational optics have emerged in an attempt to overcome traditional limitations, achieving a good balance between spectral resolution, imaging speed, and light utilization efficiency remains a core technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0005] This application aims to at least partially address one of the technical problems in the related art.

[0006] To achieve the above objectives, a first aspect of this application provides a microscopic spectral imaging system, including an excitation optical path and a detection optical path. The excitation optical path is used to transmit excitation light to a analyte, and the detection optical path is used to receive a fluorescence beam generated by the excitation of the analyte. The detection optical path includes:

[0007] The non-uniform amplification module includes at least one set of cylindrical lens groups composed of multiple cylindrical lenses with different curvature directions, used to differentially amplify the received fluorescence beam in different directions; A split-convergence module is disposed on the outgoing light path of the non-uniform amplification module, and is used to split the field of view of the differentially amplified fluorescent beam into multiple sub-light fields, and the light spots converged by the multiple sub-light fields are located on the same imaging plane. The intermediate image plane dispersion module is located in the output optical path of the segmented convergence module. It is used to disperse each converged light spot and adaptively amplify each dispersed beam before projecting it onto the detection surface of the camera to obtain the spatial spectral image of the object under test.

[0008] Optionally, the cylindrical lens group includes at least a pair of first cylindrical lenses and a pair of second cylindrical lenses, and the first cylindrical lenses and the second cylindrical lenses are alternately and spaced apart along the same optical axis; wherein the curvature directions of the first cylindrical lenses and the second cylindrical lenses are perpendicular to each other, and the pair of spaced-apart first cylindrical lenses and the first cylindrical lenses respectively constitute an independent 4f imaging system.

[0009] Optionally, the segmentation and convergence module includes a microlens array configured to segment the field of view of the differentially amplified fluorescence beam into multiple sub-fields and converge the multiple sub-fields to the front imaging plane of the intermediate image plane dispersion module.

[0010] Optionally, the intermediate image plane chromatic aberration module includes an object-side telecentric lens and an image-side telecentric lens, and a chromatic aberration prism group disposed at the intermediate image plane formed between the two; wherein, The object-side telecentric lens is used to project the light spots that converge on its front imaging surface onto the dispersive prism group. The dispersive prism group is used to uniformly disperse the incident light spots and form a dispersive beam. The image-side telecentric lens is used to magnify each of the dispersive beams and project them onto preset pixels on the camera's detection surface.

[0011] Optionally, the image-side telecentric lens is a variable magnification lens group, the magnification of which is configured to magnify the dispersive beam to an integer multiple of the camera pixel size.

[0012] Optionally, the dispersive prism includes at least one pair of wedge prisms, and each pair of wedge prisms is arranged in a symmetrical zero-bias configuration.

[0013] Optionally, it also includes a band beam splitting module, which is disposed between the segmentation and convergence module and the intermediate image plane dispersion module, and is used to split the incident sub-light fields according to wavelength and form multiple sub-band optical paths.

[0014] Optionally, the number of intermediate image plane dispersion modules may include multiple modules, and there is a one-to-one correspondence between the multiple intermediate image plane dispersion modules and the sub-band optical paths formed by the beam splitting of the band beam splitting module.

[0015] Optionally, the excitation optical path includes at least one of the following: single objective light plate, structured light illumination, confocal scanning, stimulated emission loss, random optical reconstruction, or photoactivated positioning microscope excitation optical path.

[0016] To achieve the above objectives, a first aspect of this application provides a microscopic spectral imaging method, comprising: The fluorescence beam generated by the excitation of the analyte is collected, and the collected fluorescence beam is amplified differentially in different directions; The field of view of the differentially amplified fluorescent beam is divided to form multiple uniformly divided sub-light fields, and the light spots of the multiple sub-light fields converge on the same imaging plane. The light spots that converge on the same imaging surface are uniformly dispersed to form a dispersive beam, and the dispersive beam is adaptively magnified and projected onto a preset position on the camera detection surface to obtain the spatial spectral image of the object under test. Spatial integration is performed on the spectral line data corresponding to each dispersive beam in the spatial spectral image, and a two-dimensional spatial image of the object under test is reconstructed and obtained based on the spatial integration result.

[0017] The microscopic spectral imaging system and imaging method provided in this application have at least the following beneficial effects: This application provides a microscopic spectral imaging system and method. Through the close collaboration of a non-uniform magnification module, a segmentation-convergence module, and an intermediate image plane dispersion module, a complete spatial spectral decoupling and imaging mechanism is formed. First, the non-uniform magnification module differentially amplifies the fluorescence beam, expanding the spectral space while ensuring the integrity of spatial information. Second, the segmentation-convergence module divides the magnified field of view into discrete sub-fields and converges them to the intermediate image plane, spatially isolating spectral information from different regions and avoiding aliasing. Finally, the intermediate image plane dispersion module performs spectral unfolding and adaptive amplification on each sub-field, converting the spectral information into a two-dimensional image signal detectable by the camera. The organic combination of these three components not only overcomes the performance bottlenecks of traditional spectral imaging systems but also improves the system's practicality and stability through a compact optical path design, providing a reliable technical solution for high-resolution spectral imaging applications in fields such as biomedicine and materials science.

[0018] 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

[0019] 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 first microscopic spectral imaging system according to an embodiment of this application.

[0020] Figure 2 This is a schematic diagram of a first type of microscopic spectral imaging system according to an embodiment of this application.

[0021] Figure 3 This is a schematic diagram of a second structure of a first microscopic spectral imaging system according to an embodiment of this application.

[0022] Figure 4 This is a schematic diagram illustrating the process of acquiring spatial spectral images using a microscopic spectral imaging system according to an embodiment of this application.

[0023] Figure 5 This is a structural block diagram of a second type of microscopic spectral imaging system according to an embodiment of this application.

[0024] Figure 6 This is a schematic diagram of a second type of microscopic spectral imaging system according to an embodiment of this application.

[0025] Figure 7 This is a schematic diagram of a second structure of a second type of microscopic spectral imaging system according to an embodiment of this application.

[0026] Figure 8 This is a schematic flowchart illustrating a microscopic spectral imaging method according to an embodiment of this application. Detailed Implementation

[0027] 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.

[0028] According to one aspect of this application, a microscopic spectral imaging system is provided, such as... Figures 1-3 As shown, the system includes an excitation optical path and a detection optical path. The excitation optical path is used to project excitation light onto the test object, causing the test object to be excited and generate fluorescence. The detection optical path is used to collect the fluorescence beam generated by the excitation of the test object, and after performing a series of optical processing on the collected fluorescence beam, it is projected onto the detection surface of the camera to obtain the spatial spectral image of the test object.

[0029] The detection optical path includes a non-uniform magnification module, a segmentation and convergence module, and an intermediate image plane dispersion module arranged sequentially. The non-uniform magnification module includes at least one set of cylindrical lenses composed of multiple cylindrical lenses with different curvature directions, used to differentially magnify the received fluorescence beam in different directions. The segmentation and convergence module is disposed in the output optical path of the non-uniform magnification module, used to segment the field of view of the differentially magnified fluorescence beam into multiple sub-fields, and the light spots formed by the convergence of these sub-fields are located on the same imaging plane. The intermediate image plane dispersion module is disposed in the output optical path of the segmentation and convergence module, used to disperse each converged light spot, and adaptively magnify the dispersed beams before projecting them onto the detection surface of the camera to acquire a spatial spectral image of the object under test.

[0030] Understandably, the microscopic spectral imaging system achieves the simultaneous acquisition of spatial and spectral information of the object under test through the synergistic effect of the excitation and detection optical paths. The non-uniform amplification module, segmentation and convergence module, and intermediate image plane dispersion module of the detection optical path constitute the core optical processing unit. Through functional complementarity and parameter matching, the three solve key problems in traditional spectral imaging systems, such as the mutual constraint between spatial and spectral resolution, redundant optical paths, and easy image aliasing, thus providing a structural foundation for obtaining high-quality spatial spectral images.

[0031] The non-uniform magnification module, as the front-end processing unit of the detection optical path, plays a core role in employing at least one set of cylindrical lens groups composed of multiple cylindrical lenses with different curvature directions. These cylindrical lenses are configured to be arranged alternately according to different curvature directions. By utilizing the orthogonality of their curvature directions, the magnification in the two directions can be decoupled, and the object-side field of view can be magnified at different magnifications in the dispersion direction and in the direction orthogonal to the dispersion direction without increasing the optical path length.

[0032] Specifically, in spectral imaging, it is typically necessary to expand spectral information in one direction (dispersion direction) while preserving the spatial information of the sample in another direction (spatial direction). Non-uniform magnification allows for appropriate magnification of the spatial image to match the spatial sampling rate, while simultaneously magnifying the dispersion image at a higher magnification, providing sufficient space for subsequent spectral dispersion and detection. This differentiated magnification design achieves independent magnification of the object-side field of view in two orthogonal directions without increasing the optical path length. This ensures that the system, within a limited camera detection surface size, can accommodate sufficient spectral information while maintaining the integrity of the spatial imaging. Thus, it achieves a balance between high spatial resolution and high spectral resolution within a limited camera detection surface, ultimately enabling isotropic sampling of the object-side image after reconstruction, laying the foundation for accurate image restoration.

[0033] As an example, the cylindrical lens group includes at least a pair of first cylindrical lenses and a pair of second cylindrical lenses, and the first cylindrical lenses and the second cylindrical lenses are alternately and spaced apart along the same optical axis; wherein the curvature directions of the first cylindrical lenses and the second cylindrical lenses are perpendicular to each other, and the pair of spaced-apart first cylindrical lenses and second cylindrical lenses respectively constitute an independent 4f imaging system.

[0034] Because the curvature directions of the first and second cylindrical lenses are orthogonal, this orthogonal design ensures that their effects on the light beam are completely independent in space, thus naturally decoupling the magnification of the two orthogonal directions. The alternating and spaced arrangement along the same optical axis ensures continuous action of the first and second cylindrical lenses on the light beam while avoiding optical interference from cylindrical lenses with different curvature directions through spatial separation. Furthermore, arranging these cylindrical lenses alternately according to different curvature directions reduces the optical path length, saves space occupied by the cylindrical lens group, and reduces aberrations introduced by the optical path length.

[0035] More importantly, a pair of spaced-apart first or second cylindrical lenses constitute an independent 4f imaging system, providing a distortion-free and precisely adjustable optical basis for differentiated magnification. In this example, a pair of first cylindrical lenses constitutes an independent 4f imaging subsystem perpendicular to the dispersion direction (spatial direction), while another pair of second cylindrical lenses constitutes a 4f imaging subsystem in the dispersion direction. Since the curvature directions of the two 4f subsystems are orthogonal and their optical paths are independent, their magnification can be differentiated by adjusting the focal length of the corresponding cylindrical lenses. For example, the magnification of the dispersion-direction 4f system can be set to a high magnification to expand the spatial occupancy of spectral information; the magnification of the spatial-direction 4f system can be set to a low magnification to compress the longitudinal dimension of spatial imaging and avoid the aliasing of spectral and spatial information.

[0036] For example, such as Figure 3 As shown, the cylindrical lens group consists of cylindrical lens CL1, cylindrical lens CL2, cylindrical lens CL3, and cylindrical lens CL4. The front focal planes of cylindrical lens CL1 and cylindrical lens CL2 coincide, and the rear focal planes of cylindrical lens CL3 and cylindrical lens CL4 coincide. Cylindrical lens CL1 and cylindrical lens CL3 are first cylindrical lenses with the same curvature direction and satisfy the 4f system relationship. Cylindrical lens CL2 and cylindrical lens CL4 are second cylindrical lenses with the same curvature direction and satisfy the 4f system relationship.

[0037] It should be noted that this application does not impose a specific limit on the number of cylindrical lens groups in the non-uniform magnification module. It can be one lens group as described in the above example, or multiple lens groups as described in the above example.

[0038] The split-convergence module is positioned on the output optical path of the non-uniform amplification module. Its core function is to divide the field of view of the non-uniformly amplified fluorescence beam into multiple sub-fields and converge these sub-fields to form light spots, with all spots located on the same imaging plane. This design can employ components such as microlens arrays or mirror arrays to achieve field-of-view segmentation. By segmenting the field of view, a large field of view can be decomposed into multiple smaller sub-fields, each corresponding to an independent detection channel. This approach improves the system's light throughput and imaging speed because each sub-field can be processed in parallel, ensuring that spectral information does not overlap while maintaining a consistent spatial sampling rate. Simultaneously, converging multiple sub-fields onto the same imaging plane facilitates uniform spectral dispersion processing of all sub-field spots by the subsequent dispersion module, ensuring that a complete spatial spectral image composed of stitched sub-spectral images is obtained on the camera's detection plane.

[0039] As an example, the segmentation and convergence module includes a microlens array (MCLA) configured to segment the field of view of the differentially amplified fluorescence beam into multiple sub-fields and converge the multiple sub-fields to the front imaging plane of the intermediate image plane dispersion module.

[0040] Because a microlens array (MCLA) is composed of a series of tiny lens units arranged in a specific pattern, when a fluorescent beam processed by a non-uniform amplification module is incident on the MCLA, each tiny lens unit is responsible for receiving and processing the light signal from a specific region of the field of view. These lens units divide the continuous field of view, which was originally significantly elongated in one direction, into multiple independent sub-fields arranged in another direction. This segmentation spatially isolates spectral information that might overlap during subsequent dispersion, thus fundamentally avoiding spectral aliasing at different spatial locations.

[0041] Furthermore, each microlens unit can also focus and image the light signal of its corresponding sub-field onto a common plane, namely the front imaging plane of the intermediate image plane dispersion module. On this plane, all the segmented sub-fields form an ordered array of light spots. This array of light spots preserves the spatial information of the original field of view and prepares for the next step of spectral dispersion. Converging all sub-fields onto the same plane ensures that the subsequent dispersion process can perform uniform and consistent spectral unfolding on all sub-fields.

[0042] Thus, by introducing the microlens array (MCLA) as a core optical element, the segmentation and convergence module achieves efficient processing of the non-uniformly amplified fluorescence beam. Specifically, the MCLA is used to segment the differentially amplified continuous field of view into multiple independent sub-fields, and these sub-fields are precisely converged onto the front imaging plane of the subsequent intermediate image plane dispersion module.

[0043] The intermediate image plane dispersion module is located in the output optical path of the split-converging module. Its main function is to perform spectral dispersion and adaptive amplification on the separately converged light spots, that is, to spatially separate the light of different wavelengths in each spot to form a spectrum. After dispersion, the light spot of each sub-field of view is unfolded into a spectral line. In order for these spectral lines to be effectively received by the camera's detection surface, the intermediate image plane dispersion module also needs to adaptively amplify the dispersed beams formed by dispersion. Here, adaptive amplification means that the magnification ratio needs to be adjusted according to the length of the dispersed spectral line and the pixel size of the camera's detection surface to ensure that the spectral information can be completely projected onto the preset area on the camera's detection surface, and that the spectral lines corresponding to different sub-fields have different pixel positions and appropriate spacing to avoid overlap and ensure high-resolution sampling of spectral information. Through the function of the intermediate image plane dispersion module, a spatial spectral image of the test object can finally be obtained on the camera's detection surface, where each pixel corresponds to the light intensity information of a specific wavelength at a specific location on the sample, thereby realizing simultaneous imaging of the spatial distribution and spectral characteristics of the sample.

[0044] As an example, the intermediate image plane aberration module includes an object-side telecentric lens TCL1 and an image-side telecentric lens TCL2, as well as a group of aberration prisms disposed at the intermediate image plane formed between the two.

[0045] Because the object-side telecentric lens TCL1 is positioned on the outgoing optical path of the split-converging module, its core function is to receive the sub-field light spot array from the split-converging module, enabling it to project each sub-field light spot into the dispersive prism assembly as parallel light. A key characteristic of the telecentric lens is that its entrance pupil is located at infinity. This means that only principal rays parallel to the optical axis can pass through the lens, ensuring that sub-fields at different positions in the object-side field of view, regardless of their off-axis angle, are incident on the dispersive prism assembly at the same angle. This design effectively avoids the magnification differences and image distortion caused by different field of view angles in traditional non-telecentric optical paths, providing a uniform and consistent incident condition for subsequent spectral dispersion, ensuring that the spectral information of each sub-field can be accurately and without deviation.

[0046] Because the dispersive prism assembly is positioned at the intermediate image plane formed between the object-side telecentric lens TCL1 and the image-side telecentric lens TCL2, its core function is to perform spectral decomposition on the sub-light field collimated by the object-side telecentric lens TCL1. Thus, when the polychromatic sub-light field spot is incident on the prism assembly, the refraction angles differ due to the different refractive indices of different wavelengths within the dispersive prism, thereby decomposing the polychromatic light into spectral lines arranged in wavelength order (i.e., spectral unwrapping). Since the object-side telecentric lens TCL1 provides a uniform incident angle, the dispersive prism assembly can perform uniform and consistent spectral unwrapping on each sub-light field, ensuring that the spectral lines corresponding to different sub-light fields are spatially separated and neatly arranged, avoiding spectral aliasing or overlap.

[0047] For example, the dispersive prism PA1 includes at least one pair of wedge prisms, and each pair of wedge prisms is arranged in a symmetrical zero-bias configuration. This arrangement ensures that the central axis of the outgoing beam remains parallel to the central axis of the incident beam while achieving spectral dispersion. Specifically, the light is deflected in one direction in the first wedge prism and in the opposite direction in the second symmetrically placed wedge prism. The final effect is that the spectrum is expanded, but the overall propagation direction of the beam remains essentially unchanged. This arrangement effectively avoids image plane shift or distortion caused by beam deflection, ensuring stable and accurate imaging of the spectral image on the camera's detection surface.

[0048] For example, in one specific embodiment, to adapt to the detection requirements of a wide spectral range (e.g., 300 nm) while ensuring the integrity of the object-side field of view, the dispersive prism assembly PA1 can adopt a small wedge angle design. This design achieves low-resolution extraction of the entire spectral range by reducing the spectral spread angle, suitable for scenarios such as rapid scanning or spectral overview. In another specific embodiment, if it is necessary to focus on a specific narrow band (e.g., 50 nm) for detailed analysis, the dispersive prism assembly PA1 can adopt a large wedge angle design. A larger wedge angle can enhance the spectral dispersion capability, fully spread the target band in space, thereby achieving higher resolution spectral extraction and meeting the application requirements such as accurate identification of material components. In addition, by using a time-division multiplexing strategy, the above two implementation methods can be flexibly switched or combined to achieve high-resolution spectral imaging of at least two bands, further expanding the application scenarios of the system.

[0049] It should be noted that regardless of the wedge angle design used, the dispersive prism assembly PA1 is configured to provide uniform dispersion of the imaging beam across the entire field of view. Its unique optical structure minimizes optical path deflection, effectively reducing the generation of higher-order aberrations, thereby achieving the ideal effect of near-pure dispersion and ensuring the accuracy of spectral information and imaging quality.

[0050] Since the image-side telecentric lens TCL2 is positioned in the exit light path of the dispersive prism group PA1, its core function is to image the spectral lines expanded by the dispersive prism group PA1 onto the camera's detection surface. Similar to the object-side telecentric lens TCL1, the exit pupil of the image-side telecentric lens TCL2 is located at infinity, meaning that its emitted principal ray is parallel to the optical axis. This design ensures that the magnification of the spectral image remains stable regardless of the slight shift in the position of the camera's detection surface, thereby obtaining a high-quality spectral image with sharp edges and minimal distortion. Furthermore, the image-side telecentric lens TCL2 can effectively suppress image brightness unevenness caused by uneven light source intensity distribution or optical element defects, further improving the quality of the spectral image.

[0051] As an example, the TCL2 telecentric lens is a variable magnification lens group whose magnification is configured to amplify the dispersive beam to an integer multiple of the camera pixel size. This ensures that the spectral line distance of the dispersive beam projected onto the camera's detection surface is equal to or slightly less than an integer multiple of the side length PS of the camera's detection surface pixel, thus guaranteeing that each pixel of the camera accurately corresponds to a specific wavelength range. This maximizes the use of the camera's resolution, avoids the waste or aliasing of spectral information, and provides high-quality raw data for subsequent spectral data processing and analysis. For example, if the side length of the camera's detection surface pixel is set to 'a', and the spectral line distance of the dispersive beam projected onto the camera's detection surface is 4.3a~4.5a, then the magnification of the TCL2 telecentric lens group can be set to twice, so that the amplified spectral line distance is equal to or slightly less than 9a, that is, an integer multiple of the side length PS of the camera's detection surface pixel, thereby maximizing the use of the camera's resolution.

[0052] like Figure 4 As shown, after the dispersive beam is processed, it is adapted, amplified and calibrated by the image-side telecentric lens TCL2. This results in a clear, non-aliased two-dimensional spatial spectral image on the camera detection surface. The spatial spectral image simultaneously carries the spatial distribution information of the object under test and the spectral information of the corresponding region.

[0053] Specifically, based on the spatial periodicity of the segmentation and convergence module (i.e., the arrangement pattern of the sub-light fields), the spatial spectral image can be divided into several independent spectral lines, each corresponding to a local spatial region of the object under test. The grayscale distribution of each spectral line corresponds to the spectral response of its local region, which is the spectral curve shown in the figure (the figure illustrates the spectral curves corresponding to multiple spectral lines, such as Spectra1-4). By performing a simple spatial integration operation along the spectral dimension on each spectral line of the spatial spectral image, the information in the spectral dimension can be compressed, the spatial distribution characteristics of the object under test can be preserved, and finally a spatial domain image that meets the requirements of spatial isotropic sampling can be obtained.

[0054] In some embodiments, such as Figures 5-7As shown, the system also includes a band beam splitting module, which is located between the split-converging module and the intermediate image plane dispersion module. It is used to split the incident sub-light fields according to wavelength and form multiple sub-band optical paths.

[0055] Understandably, due to the limitations of the camera's detection surface size, the spectral line length under a single channel cannot be extended indefinitely. If a wide band is directly covered, insufficient spectral line expansion will lead to a decrease in resolution. However, the band splitting module can achieve high-resolution detection with a limited spectral line length through band-specific fine processing.

[0056] Specifically, the band splitting module employs a dichroic mirror DM2, which divides the visible light range covered by the incident sub-light field into two independent sub-bands, using a preset intermediate wavelength λ as the boundary. This allows light signals from different wavelength ranges to enter their respective sub-band optical paths. Correspondingly, multiple intermediate image plane dispersion modules are also included, with a one-to-one correspondence between these modules and the sub-band optical paths formed by the band splitting module. Furthermore, each sub-band optical path incorporates a time-division multiplexing strategy to further subdivide its sub-band into two narrower sub-bands, achieving high-resolution dispersion imaging of each narrow band through time-series switching.

[0057] This design can increase the overall spectral resolution of the system to four times that of single-channel imaging without increasing the length of the spectral lines or expanding the camera's detection surface. It achieves complete coverage of the wide band while ensuring that the spectral information in each narrow band can be fully unfolded, avoiding the spectral aliasing problem in single-channel wide-band imaging. At the same time, it maintains the compactness of the optical path, taking into account both the detection performance and practicality of the system.

[0058] Furthermore, the band-splitting module may include multiple dichroic mirrors to achieve spectral detection in three or more bands. The transmission characteristics of the multiple dichroic mirrors differ, ensuring that the beam bands ultimately entering the corresponding intermediate image plane dispersive modules are distinct. Maintaining a constant spectral line length, as the number of dichroic mirrors increases, the wavelength range of each intermediate image plane dispersive module gradually decreases, thus gradually improving the accuracy of each spectral curve and consequently, the spectral resolution.

[0059] It should be noted that the system provided in this application does not impose specific restrictions on the excitation optical path, and may include, but is not limited to, one of the excitation optical paths of a single objective light plate, structured light illumination, confocal scanning, stimulated emission loss, random optical reconstruction, or photoactivated positioning microscope.

[0060] According to another aspect of this application, a microscopic spectral imaging method is also provided, used with the microscopic spectral imaging system described in any of the above embodiments to acquire spatial spectral images and two-dimensional spatial images of the analyte, such as... Figure 8 As shown, the method specifically includes the following steps: S1, collect the fluorescence beam generated by the excitation of the analyte, and amplify the collected fluorescence beam in different directions in a differentiated manner; S2, the field of view of the differentially amplified fluorescent beam is divided to form multiple uniformly divided sub-light fields, and the light spots of the multiple sub-light fields converge on the same imaging plane. S3, uniformly disperses the light spots that converge on the same imaging surface to form a dispersive beam, and adaptively amplifies each dispersive beam before projecting it onto a preset position on the camera detection surface to obtain the spatial spectral image of the object under test; S4, perform spatial integration on the spectral line data corresponding to each dispersive beam in the spatial spectral image, and reconstruct and obtain a two-dimensional spatial image of the object under test based on the spatial integration result.

[0061] In summary, this application establishes a complete spatial spectral decoupling and imaging mechanism through the close collaboration of a non-uniform magnification module, a segmentation-convergence module, and an intermediate image plane dispersion module. First, the non-uniform magnification module differentially amplifies the fluorescence beam, expanding the spectral space while ensuring the integrity of spatial information. Second, the segmentation-convergence module divides the amplified field of view into discrete sub-fields and converges them to the intermediate image plane, spatially isolating spectral information from different regions and preventing aliasing. Finally, the intermediate image plane dispersion module performs spectral unfolding and adaptive amplification on each sub-field, converting the spectral information into a two-dimensional image signal detectable by the camera. This organic combination not only overcomes the performance bottlenecks of traditional spectral imaging systems but also enhances the system's practicality and stability through a compact optical path design, providing a reliable technical solution for high-resolution spectral imaging applications in fields such as biomedicine and materials science.

[0062] 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.

[0063] 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 universal microscopic spectroscopic imaging system, comprising an excitation optical path and a detection optical path, wherein the excitation optical path is used to transmit excitation light to a analyte, and the detection optical path is used to receive a fluorescence beam generated by the excitation of the analyte; characterized in that, The detection optical path includes: The non-uniform amplification module includes at least one set of cylindrical lens groups composed of multiple cylindrical lenses with different curvature directions, used to differentially amplify the received fluorescence beam in different directions; A split-convergence module is disposed on the outgoing light path of the non-uniform amplification module, and is used to split the field of view of the differentially amplified fluorescent beam into multiple sub-light fields, and the light spots converged by the multiple sub-light fields are located on the same imaging plane. The intermediate image plane dispersion module is located in the output optical path of the segmented convergence module. It is used to disperse each converged light spot and adaptively amplify each dispersed beam before projecting it onto the detection surface of the camera to obtain the spatial spectral image of the object under test.

2. The system according to claim 1, characterized in that, The cylindrical lens group includes at least a pair of first cylindrical lenses and a pair of second cylindrical lenses, and the first cylindrical lenses and the second cylindrical lenses are alternately and spaced apart along the same optical axis; wherein the curvature directions of the first cylindrical lenses and the second cylindrical lenses are perpendicular to each other, and the pair of spaced-apart first cylindrical lenses and the first cylindrical lenses respectively constitute an independent 4f imaging system.

3. The system according to claim 1, characterized in that, The segmentation and convergence module includes a microlens array configured to segment the field of view of the differentially amplified fluorescence beam into multiple sub-fields and converge the multiple sub-fields to the front imaging plane of the intermediate image plane dispersion module.

4. The system according to claim 1, characterized in that, The intermediate image plane chromatic aberration module includes an object-side telecentric lens and an image-side telecentric lens, as well as a chromatic aberration prism assembly disposed at the intermediate image plane formed between the two; wherein, The object-side telecentric lens is used to project the light spots that converge on its front imaging surface onto the dispersive prism group. The dispersive prism group is used to uniformly disperse the incident light spots and form a dispersive beam. The image-side telecentric lens is used to magnify each of the dispersive beams and project them onto preset pixels on the camera's detection surface.

5. The system according to claim 4, characterized in that, The image-side telecentric lens is a variable magnification lens group, and its magnification is configured to magnify the dispersive beam to an integer multiple of the camera pixel size.

6. The system according to claim 4 or 5, characterized in that, The dispersive prism includes at least one pair of wedge prisms, and each pair of wedge prisms is arranged in a symmetrical zero-bias configuration.

7. The system according to claim 1, characterized in that, It also includes a band beam splitting module, which is disposed between the segmentation and convergence module and the intermediate image plane dispersion module, and is used to split the incident sub-light fields according to wavelength and form multiple sub-band light paths.

8. The system according to claim 7, characterized in that, The number of intermediate image plane dispersion modules includes multiple modules, and there is a one-to-one correspondence between the multiple intermediate image plane dispersion modules and the sub-band optical paths formed by the beam splitting of the band beam splitting module.

9. The system according to claim 1, characterized in that, The excitation optical path includes at least one of the following: single objective light plate, structured light illumination, confocal scanning, stimulated emission loss, random optical reconstruction, or photoactivated positioning microscope excitation optical path.

10. A microscopic spectral imaging method, characterized in that, include: The fluorescence beam generated by the excitation of the analyte is collected, and the collected fluorescence beam is amplified differentially in different directions; The field of view of the differentially amplified fluorescent beam is segmented to form multiple uniformly segmented sub-light fields, and the light spots of the multiple sub-light fields converge on the same imaging plane. The light spots that converge on the same imaging surface are uniformly dispersed to form a dispersive beam, and the dispersive beam is adaptively magnified and projected onto a preset position on the camera detection surface to obtain the spatial spectral image of the object under test. Spatial integration is performed on the spectral line data corresponding to each dispersive beam in the spatial spectral image, and a two-dimensional spatial image of the object under test is reconstructed and obtained based on the spatial integration result.

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