Fluorescent dipole azimuth angle and orientation consistency analysis method, device and equipment

Abstract

The invention discloses a fluorescence dipole azimuth angle and orientation consistency analysis method, device and equipment, and relates to the technical field of fluorescence microscopic imaging, and the method comprises the steps: obtaining a polarization modulation image sequence with an optical layer cutting characteristic; in a plurality of preset polarization modulation directions, synchronously acquiring an original image sequence by a detector, and recording polarization modulation angle information corresponding to each frame of image; and carrying out demodulation operation on the original image sequence by using polarization modulation angle information, and extracting and obtaining fluorescence sample intensity information corresponding to a pixel level, a fluorescence dipole azimuth angle and fluorescence dipole orientation consistency information representing a molecular arrangement order degree. According to the invention, through combination of physical layer cutting and polarization demodulation, high-precision and high-signal-to-noise-ratio dipole orientation and order degree imaging is realized.

Description

Technical Field

[0001] This application relates to the field of fluorescence microscopy imaging technology, and in particular to a method, apparatus and equipment for resolving the azimuth angle and orientation consistency of a fluorescence dipole. Background Technology

[0002] Fluorescence polarization microscopy can reveal the orientation and orderliness of fluorescent molecules within biological samples, and has significant applications in cytoskeleton research, dynamic analysis of membrane proteins, and materials science. However, existing fluorescence polarization microscopy techniques are mostly based on wide-field imaging systems. When imaging thick samples (such as tissue sections, organoids, and suspended cells), the strong interference from defocused background signals and scattered light causes a sharp drop in the image signal-to-noise ratio, resulting in a significant decrease in the accuracy of fluorescent dipole orientation resolution and severe distortion of dipole orientation consistency calculation results. Although researchers have attempted to alleviate this problem through algorithmic deconvolution or background subtraction, they cannot fundamentally suppress the generation of defocused signals physically, making it difficult to meet the high-precision dipole orientation resolution requirements of three-dimensional tissue imaging. Summary of the Invention

[0003] The purpose of this application is to provide a method, apparatus, and device for resolving the azimuth angle and orientation consistency of fluorescent dipoles, which achieves high-precision, high signal-to-noise ratio imaging of dipole orientation and orderliness by combining physical tomography and polarization demodulation.

[0004] To achieve the above objectives, this application provides the following solution: In a first aspect, this application provides a method for analyzing the azimuth angle and orientation consistency of a fluorescent dipole, including: Obtaining a polarization modulation image sequence with optical tomographic characteristics: A rotating confocal system is used to image a fluorescent sample with polarization characteristics. Under multiple preset polarization modulation directions, the detector synchronously acquires the original image sequence and records the polarization modulation angle information corresponding to each frame of the image. The original image is an optical tomographic image obtained after spatial filtering by a rotating confocal pinhole array. The optical tomographic image contains only the fluorescence intensity distribution of the focal plane information by physically shielding the non-focal plane fluorescence signal through the pinhole array. The original image sequence is demodulated using polarization modulation angle information to extract and obtain pixel-level fluorescence sample intensity information, fluorescence dipole azimuth angle, and fluorescence dipole orientation consistency information characterizing the orderliness of molecular arrangement.

[0005] Optionally, in the step of acquiring a polarization-modulated image sequence with optical layer-cutting characteristics, polarization modulation is achieved by configuring polarization modulation elements in the excitation optical path, the emission optical path, or simultaneously in the excitation optical path and the emission optical path; the polarization modulation elements include polarizers, electro-optic modulators, or liquid crystal variable phase delayers.

[0006] Optionally, if the polarization modulation element is configured in the excitation optical path, the polarization state of the excitation light is changed by rotating the polarization axis or changing the phase delay, thereby selectively exciting the fluorescent dipoles in the sample; if the polarization modulation element is configured in the emission optical path, the intensity components of the sample emitted fluorescence in different polarization directions are detected by rotating the polarization orientation of the analyzer.

[0007] Optionally, in the step of acquiring a polarization-modulated image sequence with optical layer-cutting characteristics, the number of preset polarization modulation directions is greater than or equal to 3 when acquiring the original image.

[0008] Optionally, the demodulation operation is performed based on the following polarization modulation model: ; In the formula, For the first i Fluorescence sample intensity information under each polarization direction; The DC component represents the unmodulated fluorescence intensity. The amplitude is modulated to reflect the response intensity of the fluorescent dipole to polarization modulation. The azimuth angle of the fluorescent dipole; For the first i Angle of polarization modulation direction.

[0009] Optionally, in the demodulation operation, the DC component is solved by arranging the measurement results of N polarization directions into a matrix form. First intermediate variable Second intermediate variable : ; The modulation amplitude is then calculated using the following formula. and fluorescent dipole azimuth angle : ; .

[0010] Optionally, the fluorescence dipole orientation consistency information is calculated using the following formula: ; In the formula, It is the fluorescence dipole orientation consistency factor.

[0011] Optionally, the method for analyzing the azimuth and orientation consistency of fluorescent dipoles further includes: fusing and displaying the obtained fluorescence sample intensity information, fluorescent dipole azimuth, and fluorescent dipole orientation consistency information, specifically including: The image brightness of the sample is characterized by fluorescence sample intensity information, and pseudo-color coding is used to characterize the fluorescence dipole azimuth angle and fluorescence dipole orientation consistency information, so as to simultaneously present the morphological features and molecular orientation features of the sample in the same image.

[0012] Secondly, this application provides a device for resolving the azimuth angle and orientation consistency of a fluorescent dipole, comprising: An image acquisition unit is used to acquire a polarization modulation image sequence with optical tomographic characteristics: a rotating confocal system is used to image a fluorescent sample with polarization characteristics. Under multiple preset polarization modulation directions, the detector synchronously acquires the original image sequence and records the polarization modulation angle information corresponding to each frame of the image. The original image is an optical tomographic image obtained after spatial filtering by a rotating confocal pinhole array. The optical tomographic image contains only the fluorescence intensity distribution of the focal plane information by physically shielding the non-focal plane fluorescence signal through the pinhole array. The demodulation calculation unit uses polarization modulation angle information to perform demodulation operations on the original image sequence, extracts and obtains pixel-level fluorescence sample intensity information, fluorescence dipole azimuth angle, and fluorescence dipole orientation consistency information characterizing the orderliness of molecular arrangement.

[0013] Thirdly, this application provides a computer device, including: a memory, a processor, and a computer program stored in the memory and executable on the processor, wherein the processor executes the computer program to implement the steps of the fluorescence dipole azimuth and orientation consistency analysis method described in any one of the above.

[0014] Fourthly, this application provides a computer-readable storage medium having a computer program stored thereon, which, when executed by a processor, implements the steps of the fluorescence dipole azimuth and orientation consistency analysis method described in any one of the above descriptions.

[0015] Fifthly, this application provides a computer program product, including a computer program that, when executed by a processor, implements the steps of the fluorescence dipole azimuth and orientation consistency analysis method described in any one of the above descriptions.

[0016] According to the specific embodiments provided in this application, the following technical effects are disclosed: This application provides a method, apparatus, device, medium, and product for analyzing the azimuth angle and orientation consistency of fluorescence dipoles. When acquiring polarization-modulated image sequences with optical tomography characteristics, the physical tomography effect of a rotating confocal pinhole array filters out defocused fluorescence during signal acquisition, fundamentally solving the problem of defocused background interference in thick sample imaging. This provides a high signal-to-noise ratio original image sequence for subsequent polarization demodulation. Compared with traditional wide-field imaging, this application obtains a pure focal plane signal without complex deconvolution algorithms, avoiding artifacts introduced by algorithms. By using polarization modulation angle information to demodulate the original image sequence, pixel-level precise parameter extraction is achieved, obtaining core information such as sample morphology (fluorescence sample intensity information), molecular alignment direction (fluorescence dipole azimuth angle), and orderliness (fluorescence dipole orientation consistency factor). These three parameters are the final results analyzed by this method and can be directly used for subsequent statistical analysis and 3D reconstruction. In summary, this application solves the problem of low resolution caused by background interference from thick samples in existing technologies by combining physical layer cutting and polarization demodulation, providing a high-precision, high-signal-noise-ratio imaging tool for the study of three-dimensional anisotropy of biological tissues. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 A flowchart illustrating a method for analyzing the azimuth and orientation consistency of a fluorescent dipole according to an embodiment of this application; Figure 2 This is a schematic diagram of the structure of an optical system provided in an embodiment of this application; Figure 3 This is a polarization modulation timing control diagram provided in one embodiment of this application; Figure 4 A schematic diagram of the functional modules of a fluorescence dipole azimuth and orientation consistency analysis device provided in an embodiment of this application; Figure 5 This is a schematic diagram of the structure of a computer device provided in an embodiment of this application.

[0019] In the picture: 1. Excitation source, 2. First lens, 3. Homogenization device, 4. Excitation polarization modulation element, 5. Dichroic mirror, 6. Turntable, 7. Second lens, 8. Objective lens, 9. Emission polarization modulation element, 10. Imaging lens, 11. Detector. Detailed Implementation

[0020] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0021] To make the above-mentioned objectives, features and advantages of this application more apparent and understandable, the application will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0022] In one exemplary embodiment, such as Figure 1 As shown, a method for analyzing the azimuth angle and orientation consistency of a fluorescent dipole is provided. This method is executed by a computer device, specifically by a terminal or server alone, or by both a terminal and a server. In this embodiment, it includes the following steps 101 to 102. Wherein: Step 101: Obtain a polarization modulation image sequence with optical tomographic characteristics: A rotating confocal system is used to image a fluorescent sample with polarization characteristics. Under multiple preset polarization modulation directions, the detector synchronously acquires the original image sequence and records the polarization modulation angle information corresponding to each frame of the image. The original image is an optical tomographic image obtained after spatial filtering by a rotating confocal pinhole array. The optical tomographic image contains only the fluorescence intensity distribution of the focal plane information by physically shielding the non-focal plane fluorescence signal through the pinhole array.

[0023] In this embodiment of the application, step 101 specifically includes the following steps: Step 1011, optical path system construction.

[0024] In the embodiments of this application, the optical path system is as follows: Figure 2 As shown, this embodiment follows the optical path propagation sequence, for Figure 2 Detailed description of each component of the optical path system: Excitation source 1: Laser output is achieved using multimode fiber, but single-mode fiber can also be used. Multimode fiber provides a uniform illumination spot, while single-mode fiber, in conjunction with a microlens array, achieves flat-field illumination. The laser wavelength is selected based on the excitation spectrum of the fluorescent sample; for example, 488 nm or 514 nm can be used for YFP protein.

[0025] First lens 2: Located after the output end of the multimode fiber, it is used to collimate the diverging beam emitted from the fiber, converting it into a parallel beam. The focal length and aperture of the first lens 2 must be matched with subsequent optical elements to ensure that the beam diameter covers the entire field of view.

[0026] Homogenization device 3: Located after the first lens 2, it transforms the Gaussian-distributed beam into a flat-top beam, eliminating illumination inhomogeneities. Homogenization device 3 can employ a microlens array, diffractive optical elements, or achieve mode mixing via bent multimode fiber. This device redistributes the energy of the Gaussian beam emitted from the multimode fiber, converting it into a large-area uniform field, ensuring consistent illumination intensity across the entire field of view of the camera. This design eliminates the traditional Gaussian beam's "strong center, weak edge" distribution characteristic, providing a stable background reference for subsequent uniform resolution of fluorescence dipole orientation, significantly improving polarization resolution accuracy.

[0027] Excitation polarization modulation element 4: Optionally configured in the excitation optical path, located after the homogenizing device 3 and before the dichroic mirror 5. This element can be a polarizer, an electro-optic modulator, or a liquid crystal variable phase retarder, used to change the polarization state of the excitation light. For example, when using a liquid crystal variable phase retarder, a phase delay of 0° to λ / 2 can be achieved by changing the driving voltage, thereby rotating the polarization direction of the linearly polarized light to a preset angle.

[0028] Dichroic mirror 5: Located after the excitation polarization modulation element 4 (if excitation modulation is used), or directly after the homogenizing device 3 (if emission modulation is used). Dichroic mirror 5 has high reflectivity for excitation light and high transmittance for fluorescence, achieving spectral separation of excitation light and emitted fluorescence. The excitation light is reflected into the rotating confocal module, while the fluorescence is transmitted into the emission light path.

[0029] The rotating confocal module includes a high-speed rotating pinhole disk (rotary disk 6), a second lens 7, and an objective lens 8. The pinhole disk is densely covered with micron-sized pinholes, arranged in 32 equally spaced Archimedean spirals. The pinhole diameter can be optimized based on the magnification and numerical aperture of objective lens 8, typically ranging from 0.5 to 1 Airy unit. For example, for a 60× / 1.4 NA objective lens, a pinhole diameter of 30 μm (approximately 1 AU) can be selected. The pinhole spacing can be selected based on sample thickness and imaging requirements, with typical values ​​of 100 μm, 200 μm, and 400 μm. The pinhole disk rotation speed is adjustable, typically from 3000 to 5000 rpm. The pinhole disk is precisely positioned on the system's conjugate focal plane. The second lens 7 (sometimes called a tube lens) further collimates the excitation light passing through the pinholes and guides it to objective lens 8. Objective lens 8 focuses the excitation light onto the sample and collects the fluorescence excited by the sample.

[0030] Sample: A fluorescent sample with polarization characteristics located on the focal plane of objective lens 8 (such as biological tissue labeled with oriented fluorescent dyes).

[0031] The emission polarization modulation element 9 is optionally configured in the emission optical path, located after the dichroic mirror 5 and before the imaging lens 10. This element can be an electrically driven rotating polarizer or a liquid crystal variable phase retarder, used to detect the intensity components of the sample emitted fluorescence in different polarization directions. For example, a polarizer wheel driven by a stepper motor can be used to switch multiple preset angles (such as 0°, 45°, 90°, 135°, etc.).

[0032] Imaging lens 10: Located after the emission polarization modulation element 9 (if emission modulation is used), or directly after the dichroic mirror 5 (if excitation modulation is used), it is used to image the fluorescence signal onto the detector target surface.

[0033] Detector 11: Employs a high-sensitivity sCMOS camera, such as the Hamamatsu ORCA-Flash4.0, featuring low readout noise, high quantum efficiency, and high-speed readout. Detector 11 records optical tomographic images and transmits the data to a computer.

[0034] The working principle of the optical path system in this embodiment is as follows: Excitation light is output from a multimode fiber, collimated by the first lens 2, and homogenized by the homogenizing device 3. If excitation modulation is used, the polarization state is changed by the excitation polarization modulation element 4, and then reflected by the dichroic mirror 5 into the rotating confocal module. The excitation light passes through the pinhole of the rotating pinhole disk and is focused onto the sample by the second lens 7 and the objective lens 8. The fluorescence excited by the sample is collected by the objective lens 8 and then spatially filtered again by the same pinhole disk, so that only the fluorescence signal of the focal plane can pass through the pinhole. The transmitted fluorescence is transmitted through the dichroic mirror 5, and if emission modulation is used, it passes through the emission polarization modulation element 9, and finally is imaged onto the detector 11 by the imaging lens 10, forming an original polarization image with optical tomographic characteristics.

[0035] Step 1012, polarization modulation path selection.

[0036] In this embodiment, the polarization modulation element can be selectively configured in the excitation optical path, the emission optical path, or simultaneously in both the excitation and emission optical paths. Specifically, this includes: Excitation optical path modulation scheme: Only the excitation polarization modulation element 4 is configured, and the emission polarization modulation element 9 is omitted. This scheme achieves selective excitation of fluorescent dipoles with different orientations within the sample by changing the polarization state of the excitation light. Its advantage is that it avoids the influence of components such as dichroic mirrors in the emission optical path on the polarization state, but it is necessary to ensure that the excitation light itself has high polarization purity.

[0037] Emission optical path modulation scheme: Only the emission polarization modulation element 9 is configured, and the excitation polarization modulation element 4 is omitted. This scheme detects the projected intensity of the sample's emitted fluorescence in different polarization directions by rotating the analyzer. Its advantage is that it directly measures the polarization component of fluorescence emission, but factors such as fluorescence anisotropy attenuation must be considered.

[0038] Simultaneous excitation and emission modulation scheme: Polarization modulation elements are configured in both the excitation and emission optical paths. An exemplary implementation involves first setting the excitation polarization direction to 0°, then acquiring an image with the 0° polarization direction at the emission end; subsequently, the excitation polarization direction is switched to 60°, and images with the 60° polarization direction are acquired sequentially at the emission end; finally, the excitation polarization direction is set to 120°, and images with the 120° polarization direction are acquired at the emission end, resulting in a total of three images. Although this scheme reduces imaging speed (requiring more image acquisition), it improves system sensitivity and thus resolution accuracy through simultaneous excitation and detection polarization modulation. This scheme is suitable for scenarios requiring high accuracy and good sample quenching resistance.

[0039] This implementation method, by configuring polarization modulation elements in the excitation or emission optical path, allows for flexible alteration of the excitation light's polarization state or detection of fluorescence intensity in different polarization directions. This enables the acquisition of image sequences under multiple polarization modulation directions, providing necessary input data for subsequent demodulation operations. The selection of polarization modulation elements should comprehensively consider switching speed, accuracy, and system cost. Regardless of the element used (EOM, LCVR, or mechanical polarizer), the ultimate requirement is the ability to output linearly polarized light with a high extinction ratio. Specific parameters such as driving voltage and phase delay range can be determined based on the selected device model, as long as the requirement of adjusting the beam polarization state from 0° linear polarization to 90° linear polarization is met.

[0040] Step 1013, Hardware synchronization and sequence acquisition.

[0041] In this embodiment, a high-sensitivity sCMOS camera is used for image acquisition. The camera and polarization modulation element are triggered synchronously to acquire raw image sequences in N polarization directions. The system control terminal (such as a computer) outputs a TTL signal through a data acquisition card, one of which triggers the polarization modulation element driver to switch polarization states, and the other triggers the camera exposure. The timing design is as follows: Figure 3 As shown: In each scanning cycle, a control signal is first issued to switch the polarization modulation element to a preset angle. After waiting for a settling time (e.g., 10 ms), the camera is triggered to expose and read out the image, then the camera switches to the next angle. Repeat the above process until all N angles have been acquired. All images are saved in 16-bit TIFF format with a resolution of 1024×1024 pixels or higher. To reduce random noise, multiple frames can be acquired for each polarization direction and averaged, for example, averaging after acquiring 3-5 frames.

[0042] This implementation method, through hardware synchronization and sequential acquisition, enables sub-millisecond-level coordination between the polarization modulation element and the camera, rapidly acquiring a sequence of raw optical tomographic images at the same spatial location but under different polarization modulation states. This provides underlying data support for subsequent analysis of fluorescence dipole orientation and consistency. The high-speed acquisition capability allows this method to be used to observe dynamic processes, such as protein rearrangement in living cells.

[0043] In this embodiment, the number N of preset polarization modulation directions can be selected according to actual needs, typically N≥3. The value of N needs to be balanced between imaging speed and resolution accuracy: N=3 is the minimum requirement, which can satisfy the solution of positive definite equations; as N increases, the overdeterminism of the equation system increases, which can suppress noise and improve accuracy through algorithms such as least squares, but will reduce imaging speed and increase sample exposure. Generally speaking, the optimal value of N is 3 or 4. When the sample has good anti-quenching performance and high accuracy requirements, N=6 or 8 can be used (the literature reports that up to 8 directions can be used). The polarization directions should preferably be uniformly distributed (e.g., 0°, 60°, 120° or 0°, 45°, 90°, 135°), but non-uniform distribution is also acceptable. It is only necessary to substitute the actual recorded polarization angle in the subsequent demodulation calculation. Small angle offsets caused by mechanical installation deviations can be corrected by the angle value in the correction formula. By implementing this method and setting N ≥ 3 preset polarization modulation directions, it is possible to ensure that the equation system is either overdetermined or just determined, providing a sufficient number of equations for subsequent least-squares solutions, thereby accurately decoupling fluorescence intensity, modulation amplitude, and fluorescence dipole azimuth angle. If N < 3, the equation system is underdetermined and cannot be uniquely solved.

[0044] Step 102: Demodulate the original image sequence using polarization modulation angle information to extract and obtain pixel-level fluorescence sample intensity information, fluorescence dipole azimuth angle, and fluorescence dipole orientation consistency information characterizing the molecular arrangement order.

[0045] As an optional implementation, according to fluorescence polarization theory, for a fluorescent dipole with a fixed orientation, the emission fluorescence intensity varies with the detection polarization direction according to a cosine square relationship. The demodulation operation is based on the following polarization modulation model: ; In the formula, For the first i Fluorescence sample intensity information under each polarization direction; The DC component represents the average fluorescence intensity (unmodulated fluorescence intensity) independent of polarization. The amplitude is modulated to reflect the response intensity of the fluorescent dipole to polarization modulation. The azimuth angle of the fluorescent dipole ranges from 0° to 180°. When the fluorescent dipoles are aligned parallel to the X-axis, it indicates that the arrangement of the fluorescent dipoles is parallel to the X-axis. The time indicates that the fluorescent dipoles are aligned parallel to the Y-axis; For the first i Angle of polarization modulation direction.

[0046] In this implementation method, a cosine square model is established based on the polarization characteristics of fluorescence dipole emission. This model accurately describes the variation of fluorescence intensity with the detection polarization direction, providing a theoretical basis for subsequent parameter extraction.

[0047] As an optional implementation, the demodulation operation solves for the DC component by arranging the measurement results of N polarization directions into a matrix form. First intermediate variable Second intermediate variable : ; The solution method for the above matrix depends on the value of N: When N=3 and the design matrix is ​​invertible, the positive definite equations can be solved directly to obtain the analytical solution. When N>3, the system of equations becomes overdetermined and can be solved using the least squares method, which effectively suppresses the influence of measurement noise. Alternatively, singular value decomposition (SVD) can be used to solve for the generalized inverse of the matrix to obtain an approximate solution.

[0048] In this implementation method, the multi-polarization angle measurement results are arranged into a matrix form and solved using an appropriate linear algebra method to obtain... , and By identifying three unknowns, the fluorescence sample intensity, modulation amplitude, and fluorescence dipole azimuth angle are decoupled. This method fully utilizes multiple measurements, improving the robustness of parameter estimation.

[0049] In this embodiment, the modulation amplitude is calculated using the following formula. and fluorescent dipole azimuth angle : ; .

[0050] This implementation method, through square root operations and arctangent functions, enables the recovery of modulation amplitude and fluorescence dipole azimuth angles from intermediate variables, achieving pixel-level fluorescence dipole orientation resolution. This calculation process can be parallelized across all pixels, making it suitable for GPU acceleration.

[0051] As an optional implementation, the fluorescence dipole orientation consistency information is calculated using the following formula: ; In the formula, This is the fluorescence dipole orientation uniformity factor, ranging from 0 to 1. When the fluorescence dipoles are completely randomly oriented... , When all fluorescent dipoles are perfectly parallel, = , OUF quantifies the degree of order in molecular arrangement within a local region. In practical applications, OUF < 0.2 is generally considered to indicate disordered molecular arrangement, OUF > 0.6 indicates highly ordered molecular arrangement, and values ​​between 0.2 and 0.6 indicate partially ordered molecular arrangement. This quantification standard can be used as a reference for researchers, and the specific threshold can be adjusted appropriately according to the characteristics of the sample.

[0052] By implementing this method, the degree of orderliness of fluorescent dipole alignment in a local region can be quantified by constructing a fluorescent dipole orientation consistency factor (OUF), providing a quantitative indicator for studying the anisotropy of biological structures. For example, in the cell membrane, lipid molecules are usually aligned perpendicular to the membrane plane, resulting in a high OUF; while in the cytoplasm, molecular orientation is random, resulting in a low OUF.

[0053] In practical calculations, weighted least squares or regularization terms can be used to improve noise resistance. For example, when some pixels have low intensity and poor signal-to-noise ratio, they can be assigned smaller weights. Furthermore, since the arctangent function is sensitive to noise, the calculated β may exhibit abrupt changes, requiring phase unwrapping. A simple unwrapping method is: for adjacent pixels, if the angle difference is greater than 90°, add or subtract 180° to make them continuous.

[0054] The fluorescence sample intensity information of each pixel was obtained through the demodulation operation in step 102. The fluorescence dipole orientation angle β and the fluorescence dipole orientation consistency factor OUF are the three parameters that this method ultimately aims to analyze, representing the sample's morphological structure, molecular alignment direction, and molecular alignment order, respectively.

[0055] As an optional implementation, these three parameters can be fused for easier observation and analysis. For example, the HSV color space can be used for fusion: the fluorescence dipole azimuth angle β is mapped to hue H, the fluorescence dipole orientation consistency factor OUF is mapped to saturation S, and the fluorescence sample intensity information is mapped to brightness V, generating a pseudo-color fused image. In this image, different colors represent different fluorescence dipole orientations, color saturation represents order, and color brightness represents intensity, allowing researchers to intuitively correlate the morphological structure and molecular arrangement characteristics of the sample. This fusion display step is a preferred presentation method and is not essential to the method described in this application.

[0056] For 3D imaging applications, different focal planes can be scanned step by step by moving the objective lens or stage along the axis. The above steps are repeated at each focal plane to obtain the orientation result in 3D space. (x,y,z), β(x,y,z), and OUF(x,y,z). The 3D data stack can be imported into software such as ImageJ and Imaris for 3D reconstruction, intuitively displaying the spatial distribution of anisotropic structures within the sample.

[0057] Existing wide-field polarization microscopy techniques lack physical tomography capabilities, leading to the superposition of defocused signals from thick samples onto the focal plane signal. This contaminates the polarization modulation curve, causing deviations in the demodulated β and OUF. For example, randomly oriented fluorescence dipoles in defocused regions contribute isotropic background, reducing the effective modulation depth and underestimating OUF. The method in this application, however, physically filters out defocused signals using a pinhole array via a rotating confocal microscope, preserving pure focal plane polarization information. Therefore, it achieves higher resolution, making it particularly suitable for thick samples such as tissue sections. Furthermore, wide-field methods often require complex deconvolution algorithms to improve image quality, but these algorithms themselves may introduce artifacts. This method suppresses defocused background at its source, simplifying subsequent processing.

[0058] By implementing steps 101 and 102 above, this application utilizes the physical tomography effect of a rotating confocal pinhole array to filter out off-focus fluorescence during the signal acquisition stage, fundamentally solving the problem of defocus background interference in thick sample imaging and providing a high signal-to-noise ratio original image sequence for subsequent polarization demodulation. Compared with traditional wide-field imaging, this application can obtain a pure focal plane signal without complex deconvolution algorithms, avoiding artifacts introduced by algorithms. By using polarization modulation angle information to demodulate the original image sequence, pixel-level precise parameter extraction is achieved, obtaining core information such as sample morphology (fluorescence sample intensity information), molecular alignment direction (fluorescence dipole azimuth angle), and orderliness (fluorescence dipole orientation consistency factor). These three parameters are the final results to be analyzed by this method and can be directly used for subsequent statistical analysis and 3D reconstruction. In summary, this application, through the deep integration of physical tomography and polarization demodulation, solves the problem of low analytical accuracy caused by off-focus background interference in thick samples in existing technologies, providing a high-precision, high signal-to-noise ratio imaging tool for the study of 3D anisotropy of biological tissues.

[0059] Based on the same inventive concept, this application also provides a fluorescence dipole azimuth and orientation consistency analysis device for implementing the fluorescence dipole azimuth and orientation consistency analysis method described above. The solution provided by this device is similar to the solution described in the above method; therefore, the specific limitations in one or more embodiments of the fluorescence dipole azimuth and orientation consistency analysis device provided below can be found in the limitations of the fluorescence dipole azimuth and orientation consistency analysis method described above, and will not be repeated here.

[0060] In one exemplary embodiment, such as Figure 4 As shown, a device for resolving the azimuth angle and orientation consistency of a fluorescent dipole is provided, comprising: Image acquisition unit 201 is used to acquire a polarization modulation image sequence with optical tomographic characteristics: a rotating confocal system is used to image a fluorescent sample with polarization characteristics. Under multiple preset polarization modulation directions, the detector synchronously acquires the original image sequence and records the polarization modulation angle information corresponding to each frame of the image; the original image is an optical tomographic image obtained after spatial filtering by a rotating confocal pinhole array. The optical tomographic image contains only the fluorescence intensity distribution of the focal plane information by physically shielding the non-focal plane fluorescence signal through the pinhole array. The demodulation calculation unit 202 uses polarization modulation angle information to perform demodulation operation on the original image sequence, and extracts and obtains pixel-level fluorescence sample intensity information, fluorescence dipole azimuth angle, and fluorescence dipole orientation consistency information that characterizes the orderliness of molecular arrangement.

[0061] In this implementation, the present application utilizes the physical tomography effect of a rotating confocal pinhole array to filter out defocused fluorescence during signal acquisition when acquiring polarization-modulated image sequences with optical tomography characteristics. This fundamentally solves the problem of defocused background interference in thick sample imaging, providing a high signal-to-noise ratio original image sequence for subsequent polarization demodulation. Compared with traditional wide-field imaging, this application obtains a pure focal plane signal without complex deconvolution algorithms, avoiding artifacts introduced by algorithms. Furthermore, pixel-level precise parameter extraction is achieved, obtaining core information such as sample morphology (fluorescence sample intensity information), molecular alignment direction (fluorescence dipole azimuth angle), and orderliness (fluorescence dipole orientation consistency factor). These three parameters are the final results to be analyzed by this method and can be directly used for subsequent statistical analysis and 3D reconstruction. In summary, this application, through the deep integration of physical tomography and polarization demodulation, solves the problem of low analytical accuracy caused by defocused background interference in thick samples in existing technologies, providing a high-precision, high signal-to-noise ratio imaging tool for the study of 3D anisotropy in biological tissues.

[0062] In one exemplary embodiment, a computer device is provided, which may be a server or a terminal, and its internal structure diagram may be as follows. Figure 5 As shown, the computer device includes a processor, memory, input / output (I / O) interfaces, and a communication interface. The processor, memory, and I / O interfaces are connected via a system bus, and the communication interface is also connected to the system bus via the I / O interfaces. The processor provides computational and control capabilities. The memory includes non-volatile storage media and internal memory. The non-volatile storage media stores the operating system, computer programs, and a database. The internal memory provides the environment for the operating system and computer programs in the non-volatile storage media to run. The database stores fluorescence dipole azimuth and orientation consistency analysis data. The I / O interfaces are used for information exchange between the processor and external devices. The communication interface is used for communication with external terminals via a network connection. When the computer program is executed by the processor, it implements a method for analyzing the azimuth and orientation consistency of fluorescence dipoles.

[0063] Those skilled in the art will understand that Figure 5 The structure shown is merely a block diagram of a portion of the structure related to the present application and does not constitute a limitation on the computer device to which the present application is applied. Specific computer devices may include more or fewer components than those shown in the figure, or combine certain components, or have different component arrangements.

[0064] In one exemplary embodiment, a computer device is also provided, including a memory and a processor, wherein the memory stores a computer program, and the processor executes the computer program to implement the steps in the above-described method embodiments.

[0065] In one exemplary embodiment, a computer-readable storage medium is provided storing a computer program that, when executed by a processor, implements the steps in the above-described method embodiments.

[0066] In one exemplary embodiment, a computer program product is provided, including a computer program that, when executed by a processor, implements the steps in the above-described method embodiments.

[0067] It should be noted that the user information (including but not limited to user device information, user personal information, etc.) and data (including but not limited to data used for analysis, data stored, data displayed, etc.) involved in this application are all information and data authorized by the user or fully authorized by all parties, and the collection, use and processing of the relevant data must comply with relevant regulations.

[0068] Those skilled in the art will understand that all or part of the processes in the above embodiments can be implemented by a computer program instructing related hardware. The computer program can be stored in a non-volatile computer-readable storage medium. When executed, the computer program can include the processes of the embodiments described above. Any references to memory, databases, or other media used in the embodiments provided in this application can include at least one of non-volatile and volatile memory. Non-volatile memory can include read-only memory (ROM), magnetic tape, floppy disk, flash memory, optical memory, high-density embedded non-volatile memory, resistive random access memory (ReRAM), magnetic random access memory (MRAM), ferroelectric random access memory (FRAM), phase change memory (PCM), graphene memory, etc. Volatile memory can include random access memory (RAM) or external cache memory, etc. By way of illustration and not limitation, RAM can take many forms, such as Static Random Access Memory (SRAM) or Dynamic Random Access Memory (DRAM).

[0069] The databases involved in the embodiments provided in this application may include at least one type of relational database and non-relational database. Non-relational databases may include, but are not limited to, blockchain-based distributed databases. The processors involved in the embodiments provided in this application may be general-purpose processors, central processing units, graphics processing units, digital signal processors, programmable logic devices, quantum computing-based data processing logic devices, etc., and are not limited to these.

[0070] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0071] This document uses specific examples to illustrate the principles and implementation methods of this application. The descriptions of the above embodiments are only for the purpose of helping to understand the methods and core ideas of this application. Furthermore, those skilled in the art will recognize that, based on the ideas of this application, there will be changes in the specific implementation methods and application scope. Therefore, the content of this specification should not be construed as a limitation of this application.

Claims

1. A method for analyzing the azimuth angle and orientation consistency of a fluorescent dipole, characterized in that, The method for analyzing the azimuth and orientation consistency of fluorescent dipoles includes: Obtaining a polarization modulation image sequence with optical tomographic characteristics: A rotating confocal system is used to image a fluorescent sample with polarization characteristics. Under multiple preset polarization modulation directions, the detector synchronously acquires the original image sequence and records the polarization modulation angle information corresponding to each frame of the image. The original image is an optical tomographic image obtained after spatial filtering by a rotating confocal pinhole array. The optical tomographic image contains only the fluorescence intensity distribution of the focal plane information by physically shielding the non-focal plane fluorescence signal through the pinhole array. The original image sequence is demodulated using polarization modulation angle information to extract and obtain pixel-level fluorescence sample intensity information, fluorescence dipole azimuth angle, and fluorescence dipole orientation consistency information characterizing the orderliness of molecular arrangement.

2. The method for analyzing the azimuth angle and orientation consistency of a fluorescent dipole according to claim 1, characterized in that, In the step of acquiring a polarization-modulated image sequence with optical layer-cutting characteristics, polarization modulation is achieved by configuring polarization modulation elements in the excitation optical path, the emission optical path, or simultaneously in the excitation optical path and the emission optical path; the polarization modulation elements include polarizers, electro-optic modulators, or liquid crystal variable phase delayers.

3. The method for analyzing the azimuth angle and orientation consistency of a fluorescent dipole according to claim 2, characterized in that, If the polarization modulation element is configured in the excitation optical path, the polarization state of the excitation light can be changed by rotating the polarization axis or changing the phase delay, thereby selectively exciting the fluorescent dipoles in the sample; if the polarization modulation element is configured in the emission optical path, the intensity components of the sample emitted fluorescence in different polarization directions can be detected by rotating the polarization orientation of the analyzer.

4. The method for analyzing the azimuth angle and orientation consistency of a fluorescent dipole according to claim 1, characterized in that, In the step of acquiring a polarization modulation image sequence with optical layer cutting characteristics, the number of preset polarization modulation directions is greater than or equal to 3 when acquiring the original image.

5. The method for analyzing the azimuth angle and orientation consistency of a fluorescent dipole according to claim 1, characterized in that, The demodulation operation is based on the following polarization modulation model: ； In the formula, For the first i Fluorescence sample intensity information under each polarization direction; The DC component represents the unmodulated fluorescence intensity. The amplitude is modulated to reflect the response intensity of the fluorescent dipole to polarization modulation. The azimuth angle of the fluorescent dipole; For the first i Angle of polarization modulation direction.

6. The method for analyzing the azimuth angle and orientation consistency of a fluorescent dipole according to claim 5, characterized in that, In the demodulation operation, the DC component is solved by arranging the measurement results of N polarization directions into a matrix form. First intermediate variable Second intermediate variable : ； The modulation amplitude is then calculated using the following formula. and fluorescent dipole azimuth angle : ； 。 7. The method for analyzing the azimuth angle and orientation consistency of a fluorescent dipole according to claim 5 or 6, characterized in that, The fluorescence dipole orientation consistency information is calculated using the following formula: ； In the formula, It is the fluorescence dipole orientation consistency factor.

8. The method for analyzing the azimuth angle and orientation consistency of a fluorescent dipole according to claim 1, characterized in that, The method for analyzing the azimuth and orientation consistency of fluorescent dipoles further includes: fusing and displaying the obtained fluorescence sample intensity information, fluorescent dipole azimuth, and fluorescent dipole orientation consistency information, specifically including: The image brightness of the sample is characterized by fluorescence sample intensity information, and pseudo-color coding is used to characterize the fluorescence dipole azimuth angle and fluorescence dipole orientation consistency information, so as to simultaneously present the morphological features and molecular orientation features of the sample in the same image.

9. A device for analyzing the azimuth angle and orientation consistency of a fluorescent dipole, characterized in that, The fluorescence dipole azimuth and orientation consistency analysis device includes: An image acquisition unit is used to acquire a polarization modulation image sequence with optical tomographic characteristics: a rotating confocal system is used to image a fluorescent sample with polarization characteristics. Under multiple preset polarization modulation directions, the detector synchronously acquires the original image sequence and records the polarization modulation angle information corresponding to each frame of the image. The original image is an optical tomographic image obtained after spatial filtering by a rotating confocal pinhole array. The optical tomographic image contains only the fluorescence intensity distribution of the focal plane information by physically shielding the non-focal plane fluorescence signal through the pinhole array. The demodulation calculation unit uses polarization modulation angle information to perform demodulation operations on the original image sequence, extracts and obtains pixel-level fluorescence sample intensity information, fluorescence dipole azimuth angle, and fluorescence dipole orientation consistency information characterizing the orderliness of molecular arrangement.

10. A computer device, comprising: A memory, a processor, and a computer program stored in the memory and executable on the processor, characterized in that the processor executes the computer program to implement the steps of the fluorescence dipole azimuth and orientation consistency analysis method according to any one of claims 1-8.

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