Dual-optical-path polarization-Fourier light field microscopic imaging system

Abstract

The invention discloses a dual-optical-path polarization-Fourier light field microscopic imaging system, which comprises a microscope, a polarization imaging module, a second semitransparent reflective mirror and a Fourier light field imaging module, and is characterized in that the microscope comprises an objective lens, a first semitransparent reflective mirror, an imaging lens and a first camera which are arranged along an imaging optical path; the polarization imaging module comprises a rotatable polaroid; the second semi-transparent reflective mirror is positioned between the imaging lens and the rotatable polarizing film; and the Fourier light field imaging module comprises a Fourier lens, a micro lens array and a second camera which are sequentially arranged along the light field branch. Through the double-optical-path design, the polarization branch provides sample surface polarization characteristics to achieve normal estimation and microstructure concave-convex characteristic judgment, the Fourier light field branch provides absolute depth information, and the reconstruction precision of the microstructure is greatly improved after two kinds of modal data are fused.

Description

Technical Field

[0001] This invention relates to the fields of image processing technology and three-dimensional reconstruction, and more specifically, to a dual-path polarization-Fourier light field microscopic imaging system. The invention may also be named a dual-path microscopic imaging system with a polarization light path and a Fourier light field light path. Background Technology

[0002] A light field is the collection of all light rays in space, used to describe the intensity distribution of light rays at any location and in any direction in space. In 1936, Gershun first proposed the concept of a "light field," defining it as the distribution of radiant energy carried by light rays propagating in a straight line in a homogeneous medium. In 1991, Adelson and Bergen further proposed the "holophoton function," using seven parameters (x, y, z, θ, φ, λ, t) to completely describe the light intensity information at any time, location, direction, and wavelength.

[0003] Because directly processing seven-dimensional information is extremely complex, and considering that most practical imaging systems involve static scenes, constant wavelengths, and sampling at a specific moment, Levoy and Hanrahan proposed a simplified "four-dimensional light field function" model, also known as the dual-plane parameterization model, in 1996. This model constructs two parallel planes, typically called the viewpoint plane and the pixel plane, and uses the coordinates of the intersection points of the light rays with these two planes to parameterize each ray in space. By using the sampling coordinates (s,t) on the viewpoint plane and the corresponding projection positions (u,v) on the pixel plane, the ray can be completely parameterized as a four-dimensional function:

[0004] L=L(u,v,s,t)

[0005] In this model, the light field can be viewed as the set of all rays originating from different points on the viewpoint plane (s,t) and passing through the pixel plane (u,v). Through the simplification and constraints of the two-plane model, the light field representation can be extracted from the complex description of the all-optical function, providing a more accurate and practical theoretical framework for optical imaging and computation, thus propelling light field imaging technology from theory to practical application. It not only supports functions such as image perspective synthesis, depth-of-field adjustment, and numerical refocusing, but also provides complete optical information support for further 3D structure reconstruction and inversion modeling, becoming one of the fundamental theories in modern computational optics.

[0006] Traditional microscopic imaging systems rely on objectives and tube lenses (TL) to focus the intensity distribution of the sample's two-dimensional plane onto an image sensor. Because only spatial information of light is recorded, its propagation direction cannot be preserved, thus lacking the ability to express depth structure and three-dimensional morphology. In traditional microscopes, the sensor can only acquire a clear image of the sample's focal plane, while light from other depth locations is blurred due to defocus and cannot form effective information. To reconstruct the three-dimensional structure, axial mechanical scanning (such as moving the sample or objective) is usually required to acquire an image stack layer by layer. This method is not only time-consuming and labor-intensive but also prone to registration errors due to sample drift or dynamic changes.

[0007] In 2006, Levoy et al. proposed Light Field Microscopy (LFM), introducing light field theory into microscopic imaging systems for the first time. LFM introduces a microlens array between the imaging lens and the camera, separating light rays from different incident angles at various points on the sample and projecting them onto the sensor's image plane. Therefore, the microlens array and the sensor's image plane constitute a dual-plane model, capable of recording spatial light field information. Each microlens acts as a small lens, forming a miniature image on the sensor, recording a two-dimensional image slice of the sample as seen from that microlens position. The entire array corresponds to scene information from different angles, essentially completing a spatial-angular joint sampling, thus achieving single-frame acquisition of a four-dimensional light field. Using this mechanism, LFM can acquire light field data containing three-dimensional structural information in a single exposure. Subsequent methods such as parallax analysis, digital refocusing, or wave optics modeling can be used to recover the sample's depth distribution, eliminating the dependence on axial scanning in traditional microscopic imaging. However, LFM also has significant limitations: because spatial and angular information must be allocated within a limited number of sensor pixels, spatial resolution is limited, and reconstructed images are often accompanied by artifacts and blurring.

[0008] In 2016, Llavador et al. proposed Fourier Light Field Microscopy (FLFM), which further improved the sampling method. The FLFM system adds a Fourier lens (FL) on the conjugate Fourier plane of the object plane to perform an optical Fourier transform on the image plane. Then, a microlens array (MLA) is placed on the back focal plane of the Fourier lens to achieve angular sampling of the frequency domain distribution of the objective exit pupil, thereby generating a multi-view image containing complete sample information on the sensor.

[0009] Specifically, the light emitted from the sample is first collected by the objective lens and approximately collimated into a beam, which is then focused by the imaging lens (TL) to form an intermediate image. To acquire angular information in different exit pupil directions, a Fourier lens (FL) is introduced after the original imaging plane to construct a 4f imaging system. A microlens array (MLA) is precisely placed on the back focal plane of the Fourier lens for spatial beam splitting sampling of angular information. Each microlens receives light from a specific direction and forms a sub-aperture image on its back focal plane, thereby enabling the encoding of multi-view images. The angle-encoded composite beam is received by a back-illuminated sCMOS image sensor, recording a two-dimensional Fourier light field image containing multiple sub-views. In this way, FLFM achieves simultaneous sampling of spatial and angular information in a single capture. Compared to traditional LFM, which places a microlens array on the native image plane (NIP) of the objective lens, FLFM has the following design advantages: (1) High sampling uniformity: Since the angle information is collected in the Fourier plane, FLFM avoids the artifacts caused by non-uniform angle sampling in traditional LFM; (2) Higher spatial resolution: The same pixel area is used to record the full-view image, maintaining better spatial resolution; (3) Unified reconstruction model: FLFM can use a unified three-dimensional point spread function for reconstruction modeling, with lower computation and faster efficiency; (4) Extended imaging depth of field: The effective imaging depth can be extended to several times that of LFM, which is suitable for imaging large-volume samples.

[0010] Traditional light field microscopy suffers from numerous inherent limitations. Its core problem stems from the trade-off between spatial and angular information sampling in a single exposure. This not only results in relatively low lateral resolution but also leads to an uneven decrease in axial resolution with increasing depth of field, making it difficult to simultaneously meet the demands of large-scale imaging and high-precision 3D reconstruction. Furthermore, its effective depth of field is narrow, only a fraction of that of subsequent Fourier light field microscopy, failing to meet the 3D observation requirements of large-volume microscopic samples. While Fourier light field microscopy has improved the sampling uniformity problem of traditional light field microscopy to some extent, it still hasn't overcome its core technological limitations. First, the fundamental conflict between spatial and angular sampling remains. The limited number of sensor pixels must simultaneously carry both types of information, resulting in a significant bottleneck in lateral resolution and difficulty in accurately reconstructing the fine textures of microstructures. Second, this technique can only acquire absolute depth information, lacking the ability to perceive the normal direction of the sample surface, making it unable to determine the concavity and convexity characteristics of microstructures, and resulting in severely insufficient reconstruction accuracy in edge contours and high-frequency texture areas.

[0011] Chinese patent document (application number: 202510668118.X, application date: 2025.05.22) discloses a light field microscopy imaging system and its operating method, including an imaging module, a viewing angle shifting module, and a detector. The imaging module includes a microlens array and is configured to acquire multiple imaging views from different perspectives of the object to be observed through the microlens array. The viewing angle shifting module is configured to separate the imaging views from the different perspectives and project the separated views onto the target surface of the detector. The detector is configured to receive the imaging views processed by the viewing angle shifting module. The imaging module includes an objective lens and a tube lens, configured to image the fluorescence signal of the object to be observed onto the original image plane; a Fourier lens located at a predetermined distance behind the original image plane, with the microlens array disposed at the back focal plane of the Fourier lens; the microlens array is configured to focus the light field signal from the back focal plane of the Fourier lens into multiple imaging views from different perspectives. This scheme only includes Fourier imaging, and still has the problems of using only Fourier imaging in microscopy.

[0012] The existing technology has the following problems:

[0013] 1. Traditional light field microscopy can only acquire spatial-angular information and cannot perceive the normal direction of the sample surface. Fourier light field microscopy can only acquire absolute depth information and lacks the ability to perceive the normal direction of the sample surface. It cannot determine the concave and convex characteristics of the microstructure and has seriously insufficient reconstruction accuracy in edge contours and high-frequency texture areas.

[0014] 2. Traditional light field microscopy, due to the trade-off between spatial and angular information sampling, not only results in relatively low lateral resolution, but also exhibits uneven axial resolution degradation with increasing depth of field, making it difficult to simultaneously meet the needs of large-scale imaging and high-precision 3D reconstruction. In Fourier light field microscopy, the inherent conflict between spatial and angular sampling is not eliminated, and the limited sensor pixels need to carry both types of information simultaneously, resulting in a significant bottleneck in lateral resolution and making it difficult to accurately reproduce the fine texture of microstructures.

[0015] Therefore, how to solve the problems existing in the traditional light field microscopy and Fourier light field microscopy has become an urgent technical problem to be solved in this field. Summary of the Invention

[0016] In view of this, the present invention provides a dual-path microscopic imaging system with a polarization optical path and a Fourier optical field optical path to solve the above-mentioned technical problems: 1. Traditional optical field microscopic imaging technology cannot sense the normal direction of the sample surface, while Fourier optical field microscopic imaging technology cannot determine the concavity and convexity characteristics of microstructures, resulting in serious deficiencies in the reconstruction accuracy of edge contours and high-frequency texture areas; 2. Traditional optical field microscopic imaging technology struggles to simultaneously meet the needs of large-scale imaging and high-precision 3D reconstruction, while Fourier optical field microscopic imaging technology suffers from a significant bottleneck in lateral resolution, making it difficult to accurately reproduce the fine texture of microstructures. The dual-path microscopic imaging system of the present invention consists of a polarization imaging module and a Fourier optical field imaging module. The polarization branch is used to acquire a high-resolution polarization image of the sample surface to support subsequent fine normal estimation and topography reconstruction; the optical field branch, based on the Fourier optical field microscopic imaging principle, acquires a Fourier optical field image with spatial-angular information and can estimate the absolute depth information of the sample surface through algorithms. The two optical paths are structurally independent but acquire data synchronously. Through system calibration and spatial registration, the two modal data are unified in the temporal and spatial dimensions.

[0017] This application provides a dual-path microscopic imaging system with a polarization optical path and a Fourier optical field optical path, comprising: a microscope, a polarization imaging module, a second semi-transparent mirror, and a Fourier optical field imaging module, wherein...

[0018] The microscope includes an objective lens, a first semi-transparent mirror, an imaging lens, and a first camera arranged along the imaging optical path;

[0019] The polarization imaging module includes a rotatable polarizer for acquiring the degree of polarization and polarization angle distribution of the sample surface; the rotatable polarizer is located between the imaging lens and the first camera;

[0020] The second semi-transparent mirror is located between the imaging lens and the rotatable polarizer, and is used to divide the imaging optical path into a polarization branch and an optical field branch that are perpendicular to each other. The polarization imaging module is located on the polarization branch. The first semi-transparent mirror and the second semi-transparent mirror have adjustable degrees of freedom in two dimensions: pitch and deflection. The imaging optical path is transmitted along the horizontal optical axis.

[0021] The Fourier light field imaging module, located on the light field branch, is used to acquire a Fourier light field image with spatial-angular information. The Fourier light field imaging module includes a Fourier lens, a microlens array, and a second camera arranged sequentially along the light field branch. The target surface of the second camera is in a conjugate position with the imaging surface of the microscope. The Fourier lens is located on the conjugate Fourier surface of the object plane, and the microlens array is located on the back focal plane of the Fourier lens, used to collect light beams at least two angles mapped from the exit pupil of the objective lens.

[0022] The focal length f of the Fourier lens FL According to Equation 1:

[0023]

[0024] Where M is the amplification factor of the system, and NA obj D is the numerical aperture of the objective lens. cam D is the width of the detection surface of the second camera; pupil The diameter of the beam exit pupil;

[0025] The microlens array has a hexagonal arrangement structure composed of microlens units;

[0026] The number of microlens units on the diagonal of the microlens array is n 对角线 To satisfy Equation 2:

[0027]

[0028] Where, d MLA The aperture of the microlens unit;

[0029] The sampling ratio N of the microlens array satisfies the requirement of Equation 3:

[0030]

[0031] Among them, R xy Let λ be the target lateral resolution of the system, and λ be the wavelength of the light source;

[0032] The numerical aperture NA of the microlens unit ml Satisfy the requirements of equations 4-5;

[0033]

[0034] Among them, M T S is the overall system magnification factor; P is the pixel size of the second camera, S r This refers to the system sampling rate parameter;

[0035] The aperture d of the microlens unit MLA Satisfy the requirements of Equation 6:

[0036] d MLA ≥2·f FL ·NA ml Formula 6;

[0037] The focal length f of the microlens unit MLA Satisfy the requirements of Equation 7:

[0038]

[0039] The effective target area of ​​the second camera is 13.3mm × 13.3mm;

[0040] The second camera achieves a quantum efficiency of 95% at a wavelength of 600nm.

[0041] Optionally, the dual-optical-path microscopic imaging system further includes a Z-axis piezoelectric nano-displacement stage, used to achieve nanoscale displacement of the microscopic stage in the Z-axis direction;

[0042] The Z-axis piezoelectric nanostage has a displacement range of 100 μm.

[0043] Optionally, the spectral splitting ratio of the first semi-transparent mirror and the second semi-transparent mirror is 50:50.

[0044] Optionally, the dual-light-path microscopic imaging system further includes a light source, which emits light that shines on the reflective surface of the first semi-transparent mirror and then deflects the light to illuminate the sample, forming an illumination path;

[0045] A narrow-band filter assembly is also provided in the lighting path, and the narrow-band filter assembly is located between the light source and the first semi-transparent reflector.

[0046] Optionally, the objective lens includes a 10× objective lens, a 20× objective lens, a 50× objective lens, and a 100× objective lens.

[0047] Optionally, the second camera is a back-illuminated camera.

[0048] Optionally, the microscope is an Olympus BX53M microscope.

[0049] Compared with existing technologies, the dual-path microscopic imaging system with polarization and Fourier light field provided by this invention achieves at least the following beneficial effects:

[0050] Compared to traditional light field microscopy and Fourier light field microscopy, this invention overcomes the limitations of single-modal imaging and achieves complementary multi-dimensional information. Specifically, the effects are as follows:

[0051] First, existing traditional light field microscopes can only acquire spatial-angular information and cannot perceive the normal direction of the sample surface. However, this invention uses a dual-light path design, where the polarization branch provides polarization characteristics of the sample surface to achieve normal estimation and judgment of the concavity and convexity characteristics of the microstructure, and the Fourier light field branch provides absolute depth information. The fusion of the two modal data greatly improves the reconstruction accuracy of the microstructure and effectively solves the technical problem that existing technologies are prone to reconstruction distortion in edge contours and high-frequency texture areas.

[0052] Secondly, this invention balances high spatial resolution and three-dimensional imaging capabilities. Traditional light field microscopes suffer from limited lateral resolution due to the trade-off between spatial and angular information sampling. Although Fourier light field microscopes optimize sampling uniformity, they still have resolution bottlenecks. The polarization imaging module of this invention uses a high-resolution CMOS camera and dedicated optical components to acquire high-resolution polarization images. The Fourier light field module, through customized optical parameter design, maintains high spatial resolution while ensuring angular sampling uniformity, thus achieving a balance between large-scale imaging and high-precision three-dimensional reconstruction.

[0053] Third, the dual-path microscopic imaging system of the present invention, which has polarization optical path and Fourier optical field optical path, has flexible adaptability and practical value. It supports switching between 10×, 20×, 50× and 100× multi-magnification objective lenses, and each objective lens is matched with a standard imaging lens. At the same time, the integrated Z-axis piezoelectric nano-displacement stage can realize high-precision displacement control, which can meet the observation needs of microscopic samples of different types and scales.

[0054] Of course, any product implementing this invention does not necessarily need to achieve all of the technical effects described above at the same time.

[0055] Other features and advantages of the invention will become clear from the following detailed description of exemplary embodiments of the invention with reference to the accompanying drawings. Attached Figure Description

[0056] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments of the invention and, together with their description, serve to explain the principles of the invention.

[0057] Figure 1 This is the optical path diagram of the dual-path microscopic imaging system of the present invention, which has a polarization optical path and a Fourier optical field optical path;

[0058] Figure 2 This is a schematic diagram of the modification of the Olympus BX53M microscope device slot in the system of this invention;

[0059] Figure 3 This is a structural diagram of the Fourier lens in the system of this invention;

[0060] Figure 4 This is a physical image of the hexagonal microlens array in the system of this invention;

[0061] Figure 5 This is a structural diagram of the sCMOS camera in the system of this invention;

[0062] Figure 6 It is the Z-axis piezoelectric nano-displacement stage in the system of this invention;

[0063] Figure 7This is a physical image of the dual-path microscopic imaging system of the present invention, which has a polarization optical path and a Fourier optical field optical path.

[0064] Among them: 101, objective lens; 102, first semi-transparent mirror; 103, imaging lens; 104, second semi-transparent mirror; 105, light source; 106, narrowband filter assembly; 107, first camera (CMOS); 201, rotatable polarizer; 300, Fourier light field imaging module; 301, Fourier lens; 302, microlens array; 303, second camera (sCMOS); 401, Z-axis piezoelectric nanostage. Detailed Implementation

[0065] Various exemplary embodiments of the present invention will now be described in detail with reference to the accompanying drawings. It should be noted that, unless otherwise specifically stated, the relative arrangement, numerical expressions, and values ​​of the components and steps set forth in these embodiments do not limit the scope of the invention.

[0066] The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit the invention or its application or use.

[0067] Techniques, methods, and equipment known to those skilled in the art may not be discussed in detail, but where appropriate, such techniques, methods, and equipment should be considered part of the specification.

[0068] In all the examples shown and discussed herein, any specific values ​​should be interpreted as merely exemplary and not as limitations. Therefore, other examples of exemplary embodiments may have different values.

[0069] It should be noted that similar labels and letters in the following figures indicate similar items; therefore, once an item is defined in one figure, it does not need to be discussed further in subsequent figures.

[0070] Existing technologies, such as traditional light field microscopy imaging, suffer from numerous inherent defects. The core problem stems from the trade-off between spatial and angular information sampling in a single exposure. This not only results in relatively low lateral resolution but also leads to an uneven decrease in axial resolution with increasing depth of field, making it difficult to simultaneously meet the demands of large-scale imaging and high-precision 3D reconstruction. Furthermore, its effective depth of field is narrow, only a fraction of that of subsequent Fourier light field microscopy techniques, making it insufficient for the 3D observation of large-volume microscopic samples.

[0071] While Fourier light field microscopy has improved the sampling uniformity problem of traditional light field microscopy to some extent, it still has not overcome the core technical limitations. First, the fundamental conflict between spatial and angular sampling has not been eliminated. The limited number of sensor pixels must simultaneously carry two types of information, resulting in a significant bottleneck in lateral resolution and making it difficult to accurately reproduce the fine texture of microstructures. Second, this technology can only acquire absolute depth information and lacks the ability to perceive the normal direction of the sample surface, making it unable to determine the concavity and convexity characteristics of microstructures. The reconstruction accuracy in edge contours and high-frequency texture areas is severely insufficient.

[0072] This invention proposes a dual-path microscopic imaging system incorporating polarization and Fourier light field imaging. The system consists of a polarization imaging module and a Fourier light field imaging module. The polarization branch acquires high-resolution polarization images of the sample surface to support subsequent fine-grained normal estimation and topography reconstruction. The light field branch, based on the principle of Fourier light field microscopy, acquires Fourier light field images with spatial-angular information and can estimate the absolute depth information of the sample surface using algorithms. The two light paths are structurally independent but acquire data synchronously. System calibration and spatial registration achieve the unification of the two modalities in both time and space dimensions.

[0073] Reference Figures 1-7 As shown, Figure 1 This is the optical path diagram of the dual-path microscopic imaging system of the present invention, which has a polarization optical path and a Fourier optical field optical path; Figure 2 This is a schematic diagram of the modification of the Olympus BX53M microscope device slot in the system of this invention; Figure 3 This is a structural diagram of the Fourier lens in the system of this invention; Figure 4 This is a physical image of the hexagonal microlens array in the system of this invention; Figure 5 This is a structural diagram of the sCMOS camera in the system of this invention; Figure 6 It is the Z-axis piezoelectric nano-displacement stage in the system of this invention; Figure 7 This is a physical image of the dual-path microscopic imaging system of the present invention, which has a polarization optical path and a Fourier optical field optical path.

[0074] like Figure 1 , Figure 7 As shown, this embodiment proposes a dual-path microscopic imaging system with a polarization optical path and a Fourier optical field optical path, including: a microscope, a polarization imaging module, a second semi-transparent mirror 104, and a Fourier optical field imaging module 300, wherein,

[0075] The microscope includes an objective lens 101, a first semi-transparent mirror 102, an imaging lens 103, and a first camera 107 arranged along the imaging optical path;

[0076] The polarization imaging module includes a rotatable polarizer 201 for acquiring the polarization degree and polarization angle distribution of the sample surface; the rotatable polarizer 201 is located between the imaging lens 103 and the first camera 107.

[0077] The second semi-transparent mirror 104 is located between the imaging lens 103 and the rotatable polarizer 201, and is used to divide the imaging optical path into a polarization branch and an optical field branch that are perpendicular to each other. The polarization imaging module is located on the polarization branch. The first semi-transparent mirror 102 and the second semi-transparent mirror 104 have adjustable degrees of freedom in two dimensions: pitch and deflection. The imaging optical path is transmitted along the horizontal optical axis.

[0078] The Fourier light field imaging module 300, located on the light field branch, is used to acquire Fourier light field images with spatial-angular information. The Fourier light field imaging module 300 includes a Fourier lens 301, a microlens array 302, and a second camera 303 arranged sequentially along the light field branch. The target surface of the second camera 303 is in a conjugate position with the imaging surface of the microscope. The Fourier lens 301 is located on the conjugate Fourier surface of the object plane, and the microlens array 302 is located on the back focal plane of the Fourier lens 301. It is used to collect light beams at least two angles mapped from the exit pupil of the objective lens 101.

[0079] The focal length f of the Fourier lens 301 FL According to Equation 1:

[0080]

[0081] Where M is the system's magnification, NA obj D is the numerical aperture of the objective lens. cam D is the width of the camera's detection area. pupil The diameter of the beam exit pupil;

[0082] The microlens array 302 has a hexagonal arrangement structure composed of microlens units;

[0083] The number of microlens units n on the diagonal of the microlens array 302 对角线 Satisfy the requirements of Equation 2:

[0084]

[0085] Where, d MLA The aperture of the microlens unit;

[0086] The sampling ratio N of the microlens array 302 satisfies the requirements of Equation 3:

[0087]

[0088] Among them, R xy Let λ be the target lateral resolution of the system, and λ be the wavelength of the light source;

[0089] Numerical aperture (NA) of the microlens unit ml Satisfy the requirements of equations 4-5;

[0090]

[0091] Among them, M T S is the overall system magnification factor; P is the pixel size of the second camera, S r Here are the system sampling rate parameters; the aperture d of the microlens unit. MLA Satisfy the requirements of Equation 6:

[0092] d MLA ≥2·f FL ·NA ml Formula 6;

[0093] The focal length f of the microlens unit MLA Satisfy the requirements of Equation 7:

[0094]

[0095] The effective target area of ​​the second camera 303 is 13.3mm × 13.3mm;

[0096] The second camera 303 achieves a quantum efficiency of 95% at a wavelength of 600nm.

[0097] It should be noted that the dual-path microscopy imaging system of the present invention, featuring both polarization and Fourier light field paths, is a modification of an existing microscope. Specifically, the microscope used in this invention is based on existing technology. This microscope should have a modular structure, supporting hardware expansion of the illumination and imaging channels, providing a sound physical foundation for the construction of the dual-path microscopy imaging system. The microscope needs to support flexible switching between multiple magnification objectives, and the objectives must be compatible with the imaging lens. The imaging camera (first camera) within the microscope should have high resolution, enabling high-speed image acquisition at 64fps, meeting the frame rate and resolution requirements for microscopic polarization imaging. Furthermore, the microscope is equipped with standard modular slots in both the illumination and imaging channels, supporting the connection of external custom optical components to achieve optical path modulation. As long as the microscope meets the above requirements and can be subsequently modified, this invention will not elaborate on these aspects.

[0098] Specifically, the first semi-transparent mirror 102 on the objective lens 101 reflects the incident light through the objective lens 101 to illuminate the object. The objective lens 101 magnifies the object. The second semi-transparent mirror 104 near the polarization imaging module splits the imaging beam into the polarization imaging module and the Fourier light field imaging module 300. The imaging lens 103, on the one hand, combines with the Fourier lens 301 to form a 4F system, presenting the frequency domain distribution of the original image plane of the objective lens 101 at its back focal plane. Then, the microlens array 302 (MLA) placed here focuses the light of different sub-apertures onto the camera sensor to form a Fourier light field microscopic image containing multiple viewpoint sub-images. On the other hand, it directly passes through the rotatable polarizer 201 to acquire images at different polarization angles.

[0099] It should be noted that the function of the Fourier lens 301 is to project the original image onto the Fourier plane. Together with the imaging lens 103 inside the Olympus BX53M microscope, it forms a 4f system, meaning that parallel light remains parallel after being transmitted through the system.

[0100] The function of the microlens array 302 is to segment the pupil information on the Fourier plane and image it onto the camera target surface. Therefore, the distance between the microlens array 302 and the camera should be strictly equal to the focal length of the microlens, that is, the target surface of the second camera 303 is located on the focal plane of the microlens array 302. In practice, the microlens array 302 can be mounted in an adjustable-length lens sleeve and fixedly connected to the second camera 303. The distance between the microlens array 302 and the second camera 303 can be adjusted by adjusting the length of the sleeve. When the smallest light spot is formed on the image plane, it indicates that the camera target surface is exactly located on the focal plane of the microlens, which is the ideal microlens-camera relative position. At this time, the two can be fixed together by the sleeve to form a stable imaging combination structure.

[0101] This embodiment of the dual-path microscopy imaging system with polarization and Fourier light field paths introduces a beam splitter (BS) 104 into the conventional wide-field microscopy imaging path, splitting the sample reflected beam into two paths, which enter the polarization imaging module and the Fourier light field imaging module 300 respectively, thus achieving synchronous acquisition of polarization and Fourier light field images.

[0102] The polarization imaging module is set in the traditional wide-field microscopic imaging optical path. A rotatable linear polarizer 201 (LP) is inserted between the imaging lens 103 (TL) and the camera. Combined with a high-resolution CMOS camera (first camera 201), images at different polarization angles are acquired to obtain the polarization degree and polarization angle distribution of the sample surface, providing raw polarization data for normal estimation and three-dimensional topography reconstruction.

[0103] The Fourier light field imaging module 300 adds a Fourier lens 301 (FL) to the conjugate Fourier surface of the object plane to perform an optical Fourier transform on the image plane. Then, a microlens array 302 (MLA) is placed on the back focal plane of the Fourier lens 301. The light emitted from the sample is first collected by the objective lens 101 and approximately collimated by the imaging lens 103. The Fourier lens 301 (FL) then presents the frequency domain distribution of the original image plane of the objective lens 101 on its back focal plane. The microlens array 302 (MLA) placed here then focuses the light of different sub-apertures onto the second camera 303 sensor, forming a Fourier light field microscopic image containing multiple viewpoint sub-images, realizing the joint acquisition of spatial and angular information in a single frame.

[0104] In a Fourier light field microscopy system, the focal length of the Fourier lens 301 determines the size of the image formed on the Fourier plane by the objective lens exit pupil, which is related to the system's magnification M and the objective lens's numerical aperture NA. obj The width of the second camera's detection surface, D cam There is a corresponding relationship. This system uses a magnification of M=100 and NA. obj With an objective lens of 0.8, the effective detection surface of the camera is D. cam =13.3mm, camera pixel size P=6.5μm, take D pupil =D cam Substituting into the formula, we get:

[0105]

[0106] Considering that the effective exit pupil diameter available on the Fourier plane in the actual system is much smaller than the target width of the second camera 303, and taking into account the numerical aperture of the sample illumination and the light transmission limitation, the beam exit pupil diameter is effectively scaled to D. pupil = 4.8mm. At this point, the focal length of the Fourier lens 301 is:

[0107]

[0108] It should be noted that in this embodiment, the actual system and the available lens focal length is mainly 300mm. The beam exit pupil diameter is obtained by reverse calculation using the formula, which is 4.8mm. This diameter can be achieved by adjusting the aperture. That is, in this system, the Fourier lens focal length can be calculated based on the beam exit pupil diameter, or the beam exit pupil diameter can be reversed based on the focal length of lenses available on the market and then adjusted in conjunction with the aperture.

[0109] Based on the above calculations, in this embodiment, the Lubang Optoelectronics AD513-A commercial achromatic lens with a focal length f is ultimately adopted. FL =300mm.

[0110] In this embodiment, the wavelength of the light source is λ = 510 nm.

[0111] After determining the parameters of the Fourier lens 301, the parameters of the microlens array 302 are further designed. The microlens array 302 is arranged on the back focal plane of the Fourier lens 301 and is used to collect multi-angle beams mapped from the exit pupil of the objective lens 101. Therefore, a balance needs to be established between spatial sampling accuracy, angular resolution and system imaging consistency.

[0112] To meet the target lateral resolution R xy ≤1μm, according to the formula:

[0113]

[0114] It can be known that:

[0115]

[0116] In this embodiment, a system sampling ratio N = 3 is selected.

[0117] At the same time, in order to meet the overall system amplification factor M T =50, through the formula:

[0118]

[0119] Where, S r The system sampling rate parameter is in the following form:

[0120]

[0121] It can be seen that,

[0122]

[0123] This leads to the deduction of the microlens aperture d. MLA Must meet:

[0124] d MLA ≥2·f FL ·NA ml =3.06mm;

[0125] Considering the limitations of processing and splicing accuracy, in this embodiment, the microlens aperture is set to d. MLA =3.25mm, and further combined with the formula:

[0126]

[0127] Therefore f MLA Specifically:

[0128]

[0129] In this embodiment, taking into account the effective beam coverage diameter, sampling accuracy, and sub-aperture separation requirements, the microlens focal length f is selected. MLA =120mm, corresponding to an F number of 37, thus determining the parameter configuration scheme of the key optical components in the optical field module of this system.

[0130] Ultimately, the Fourier light field imaging module of this system mainly consists of a Fourier lens 301, a microlens array 302, a high-performance sCMOS camera (second camera 303), and a piezoelectric nanoscale displacement stage. The configuration and functions of each device are as follows:

[0131] (1) Fourier Lens 301: Utilizing the Lubang Optoelectronics AD513-A type achromatic cemented doublet lens, made of N-BK7 / SF2 material, with a focal length of 300mm, a back focal length of 295.4mm, and an aperture of 90%, supporting a wavelength range of 400nm–700nm. This lens is placed behind the original microscopic imaging plane, transforming the intermediate image from the sample to the Fourier plane, thus mapping the exit pupil angle information to space. Its structure is as follows... Figure 3 As shown.

[0132] (2) Microlens array 302: It adopts a custom-made hexagonal arrangement structure, which contains 19 microlens units. The circumcircle diameter of each sub-lens is 3.75mm, the incircle diameter is 3.25mm, the focal length is 120mm, and the F number is 37.

[0133] physical objects Figure 4 As shown, its array period and aperture are designed through system parameter matching to ensure the uniformity of angular encoding and the effective viewing angle range.

[0134] Regarding the number of microlenses, if the lenses need to cover the entire camera detection surface, then...

[0135]

[0136] Therefore, the number of microlenses in the six directions of the hexagon is 5 (e.g., Figure 4 (As shown), the total number should be 19.

[0137] (3) sCMOS camera (second camera 303): The Dhyana 400BSI V3 high-performance back-illuminated sCMOS camera from Xintu Optoelectronics is selected, which has a resolution of 2048×2048 pixels, a pixel size of 6.5μm×6.5μm, an effective target area of ​​13.3mm×13.3mm, and supports 100fps real-time imaging. Its structure is as follows: Figure 5 As shown, this camera has a quantum efficiency of over 65%, reaching a peak efficiency of 95% at a wavelength of 600nm. It possesses excellent imaging dynamic range and low noise performance, making it suitable for acquiring Fourier light field microscopic images.

[0138] In some optional embodiments provided by the present invention, the system further includes a Z-axis piezoelectric nanodisplacement stage 401 for realizing nanoscale displacement of the microscopic stage in the Z-axis direction; the displacement range of the Z-axis piezoelectric nanodisplacement stage 401 is 100 μm.

[0139] Specifically, in this embodiment, the Z-axis piezoelectric nano-displacement stage 401 is selected from the P12A.Z100S one-dimensional Z-axis piezoelectric nano-positioning stage of Coretek, such as... Figure 6 As shown. It is equipped with a high-resolution sensor to detect the position in real time and feed it back to the piezoelectric controller. The controller adjusts the voltage to correct the displacement through a PID algorithm, which can achieve an electronically controlled displacement range of 100μm along the Z-axis of the microscopic stage, a single displacement resolution of 3nm, and a repeatability accuracy of 0.04% of the stroke.

[0140] In some optional embodiments provided by the present invention, the spectral ratio of the first semi-transparent mirror 102 and the second semi-transparent mirror 104 is 50:50.

[0141] Specifically, a semi-transparent mirror divides the light path into two paths, with transmitted light and emitted light each accounting for 50%.

[0142] In some optional embodiments provided by the present invention, the system further includes a light source 105, which emits light that illuminates the reflective surface of the first semi-transparent mirror 102, and then deflects the light to illuminate the sample, forming an illumination path;

[0143] A narrow-band filter assembly 106 is also provided in the lighting path, which is located between the light source 105 and the first semi-transparent reflector 102.

[0144] Specifically, this system introduces a narrowband filter assembly 106 (U-AN360) with a center wavelength of 510nm into the illumination path, and installs a rotatable linear polarizer 201 (U-AN360P) with high-precision graduations in the imaging path (imaging optical path), such as... Figure 2 As shown.

[0145] In some optional embodiments provided by the present invention, the objective lens 101 includes a 10× objective lens, a 20× objective lens, a 50× objective lens, and a 100× objective lens.

[0146] Specifically, the system supports flexible switching of multiple magnification objectives. The 10×, 20×, 50× and 100× objectives are equipped with numerical apertures of 0.3, 0.45, 0.5 and 0.8 respectively, all of which are matched with a standard imaging lens with a focal length of 180mm.

[0147] In some optional embodiments provided by the present invention, the second camera 303 is a back-illuminated camera.

[0148] The Dhyana 400BSI V3 high-performance back-illuminated sCMOS camera selected in this embodiment has good imaging dynamic range and low noise performance, and is suitable for acquiring Fourier light field microscopic images.

[0149] In some optional embodiments provided by the present invention, the microscope is an Olympus BX53M microscope.

[0150] Specifically, this system is built on the commercial microscopy platform Olympus BX53M microscope. This platform has a modular structure, supports hardware expansion of the illumination and imaging channels, and provides a good physical foundation for building a dual-optical-path system.

[0151] The core microscope components used in the system are shown in Table 1.

[0152] Table 1. Olympus BX53 System Accessories List

[0153] Microscope accessories name light source BX3M-LEDR reflective LED light source, U-LLGAD mercury lamp light source Reflected light path and bracket BX3M-URAS-S, BX3M-ILH Objective lens group LMPLFLN 10×, 20×, 50×, 100× Imaging camera DP28 polarization components U-AN360P (Rotating Polarizer), U-AN360 (Filter Assembly)

[0154] This system supports flexible switching between multiple magnification objectives. The equipped 10×, 20×, 50×, and 100× objectives correspond to numerical apertures of 0.3, 0.45, 0.5, and 0.8, respectively, all matching the standard imaging lens with a focal length of 180mm. The imaging camera uses an Olympus DP28 CMOS sensor with a pixel size of 3.45μm × 3.45μm, boasting a high resolution of 8.9 megapixels and enabling high-speed image acquisition at 64fps, meeting the frame rate and resolution requirements for microscopic polarization imaging. Furthermore, the platform is equipped with standard modular slots in both the illumination and imaging channels, supporting the connection of external custom optical components for optical path modulation. The system incorporates a narrowband filter assembly U-AN360 with a center wavelength of 510nm in the illumination path and a rotatable linear polarizer U-AN360P with high-precision graduations in the imaging path. Figure 2 As shown. The above hardware configuration, combined with the DP28 camera, constitutes a high-resolution polarization imaging module for acquiring multi-angle polarization images.

[0155] The core of this invention focuses on the architectural design and core component parameter matching of an integrated dual-beam-path microscopic imaging system. Specifically, it features: First, an innovative dual-beam-path synchronous acquisition architecture that precisely splits the sample reflected beam into polarization imaging and Fourier light field imaging paths using a semi-transparent mirror, enabling simultaneous acquisition of polarization and Fourier light field images. Second, a customized parameter system for the Fourier light field imaging module. Based on core indicators such as system magnification, objective lens numerical aperture, and camera probe width, a Fourier lens with a focal length of 300mm is precisely calculated and selected, while a hexagonal microlens array is matched to achieve uniform angular sampling of the objective lens exit pupil frequency domain distribution. Fourth, an integrated system with multi-component collaboration. Based on the Olympus BX53M commercial microscopic platform, it integrates specialized devices such as narrowband filters, high-precision rotatable linear polarizers, high-performance back-illuminated sCMOS cameras, and Z-axis piezoelectric nanostages to construct an integrated microscopic imaging system with both polarization information acquisition capabilities and Fourier light field three-dimensional depth sensing capabilities, forming a complete technological closed loop.

[0156] As can be seen from the above embodiments, the dual-path microscopic imaging system with polarization optical path and Fourier optical field optical path provided by the present invention achieves at least the following beneficial effects:

[0157] 1. Existing traditional light field microscopes can only acquire spatial-angular information and cannot perceive the normal direction of the sample surface. However, this invention uses a dual-light path design, where the polarization branch provides polarization characteristics of the sample surface to achieve normal estimation and judgment of the concavity and convexity characteristics of the microstructure, and the Fourier light field branch provides absolute depth information. The fusion of the two modal data greatly improves the reconstruction accuracy of the microstructure and effectively solves the technical problem that existing technologies are prone to reconstruction distortion in edge contours and high-frequency texture areas.

[0158] 2. This invention balances high spatial resolution and three-dimensional imaging capability. Traditional light field microscopes suffer from limited lateral resolution due to the trade-off between spatial and angular information sampling. Although Fourier light field microscopes optimize sampling uniformity, they still have resolution bottlenecks. The polarization imaging module of this invention uses a high-resolution CMOS camera and dedicated optical components to acquire high-resolution polarization images. The Fourier light field module, through customized optical parameter design, maintains high spatial resolution while ensuring angular sampling uniformity, thus achieving a balance between large-scale imaging and high-precision three-dimensional reconstruction.

[0159] 3. The dual-path polarization-Fourier light field microscopic imaging system of the present invention has flexible adaptability and practical value. It supports switching between 10×, 20×, 50× and 100× multi-magnification objective lenses, and each objective lens is matched with a standard imaging lens. At the same time, the integrated Z-axis piezoelectric nano-displacement stage can achieve high-precision displacement control, which can meet the observation needs of microscopic samples of different types and scales.

[0160] While specific embodiments of the invention have been described in detail by way of examples, those skilled in the art should understand that the examples are for illustrative purposes only and not intended to limit the scope of the invention. Those skilled in the art should understand that modifications can be made to the above embodiments without departing from the scope and spirit of the invention. The scope of the invention is defined by the appended claims.

Claims

1. A dual-path microscopic imaging system with a polarization optical path and a Fourier optical field optical path, characterized in that, include: The system includes a microscope, a polarization imaging module, a second semi-transparent mirror, and a Fourier light field imaging module. The microscope includes an objective lens, a first semi-transparent mirror, an imaging lens, and a first camera arranged along the imaging optical path; The polarization imaging module includes a rotatable polarizer for acquiring the polarization degree and polarization angle distribution of the sample surface. The rotatable polarizer is located between the imaging lens and the first camera. The second semi-transparent mirror is located between the imaging lens and the rotatable polarizer, and is used to divide the imaging optical path into a polarization branch and an optical field branch that are perpendicular to each other. The polarization imaging module is located on the polarization branch. The first semi-transparent mirror and the second semi-transparent mirror have adjustable degrees of freedom in two dimensions: pitch and deflection. The imaging optical path is transmitted along the horizontal optical axis. The Fourier light field imaging module, located on the light field branch, is used to acquire a Fourier light field image with spatial-angular information. The Fourier light field imaging module includes a Fourier lens, a microlens array, and a second camera arranged sequentially along the light field branch. The target surface of the second camera is in a conjugate position with the imaging surface of the microscope. The Fourier lens is located on the conjugate Fourier surface of the object plane, and the microlens array is located on the back focal plane of the Fourier lens, used to collect light beams at least two angles mapped from the exit pupil of the objective lens. The focal length f of the Fourier lens FL According to Equation 1: Where M is the amplification factor of the system, and NA obj D is the numerical aperture of the objective lens. cam D is the width of the detection surface of the second camera; pupil The diameter of the beam exit pupil; The microlens array has a hexagonal arrangement structure composed of microlens units; The number of microlens units on the diagonal of the microlens array is n 对角线 Satisfy the requirements of Equation 2: Where, d MLA The aperture of the microlens unit; The sampling ratio N of the microlens array satisfies the requirement of Equation 3: Among them, R xy Let λ be the target lateral resolution of the system, and λ be the wavelength of the light source; The numerical aperture NA of the microlens unit ml Satisfy the requirements of equations 4-5; Among them, M T S is the overall system magnification factor; P is the pixel size of the second camera, S r This refers to the system sampling rate parameter; The aperture d of the microlens unit MLA Satisfy the requirements of Equation 6: d MLA ≥2·f FL ·NA ml Formula 6; The focal length f of the microlens unit MLA Satisfy the requirements of Equation 7: The effective target area of ​​the second camera is 13.3mm × 13.3mm; The second camera achieves a quantum efficiency of 95% at a wavelength of 600nm.

2. The dual-optical-path microscopic imaging system according to claim 1, characterized in that, The dual-optical-path microscopic imaging system also includes a Z-axis piezoelectric nano-displacement stage, which is used to realize nanoscale displacement of the microscopic stage in the Z-axis direction. The Z-axis piezoelectric nanostage has a displacement range of 100 μm.

3. The dual-optical-path microscopic imaging system according to claim 1, characterized in that, The spectral splitting ratio of both the first and second semi-transparent mirrors is 50:

50.

4. The dual-optical-path microscopic imaging system according to claim 1, characterized in that, The dual-light-path microscopic imaging system also includes a light source. The light emitted by the light source illuminates the reflective surface of the first semi-transparent mirror, and the light is deflected to illuminate the sample, forming an illumination path. A narrow-band filter assembly is also provided in the lighting path, and the narrow-band filter assembly is located between the light source and the first semi-transparent reflector.

5. The dual-optical-path microscopic imaging system according to claim 1, characterized in that, The objectives include 10×, 20×, 50×, and 100× objectives.

6. The dual-optical-path microscopic imaging system according to claim 1, characterized in that, The second camera is a back-illuminated camera.

7. The dual-optical-path microscopic imaging system according to claim 1, characterized in that, The microscope in question is an Olympus BX53M microscope.

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