Single-frame phase contrast microscopic imaging method based on multi-polarization-direction light field reconstruction

Abstract

The invention relates to the technical field of optical microscopic detection, in particular to a single-frame phase contrast microscopic imaging method based on multi-polarization-direction light field reconstruction, which comprises the following steps: adding polarization masks in a plurality of polarization directions into a microscopic light path; irradiating the sample to obtain a polarization image with multiple pieces of polarization direction information; carrying out polarization channel separation on the obtained polarization image to obtain sample intensity images corresponding to a plurality of polarization directions; generating image spectrum distribution corresponding to the polarization direction according to the sample intensity image; and performing phase recovery on the obtained image spectrum distribution to obtain a phase contrast microscopic image of the sample. According to the invention, multi-polarization light field information of a sample can be collected into a single polarization image, and a phase contrast microscopic image is obtained through a reconstruction algorithm. According to the polarized light field acquisition scheme, high-contrast and high-stability real-time phase measurement can be realized only by a single polarized image.

Description

Technical Field

[0001] This invention belongs to the field of optical microscopy detection technology, and particularly relates to a single-frame phase-contrast microscopy imaging method based on multi-polarization direction light field reconstruction. Background Technology

[0002] Optical microscopy provides a core tool for observing minute materials, enabling the observation of morphological information of samples at different scales by matching different magnification objectives. In the fields of life sciences and biomedical research, the precise resolution of cellular and subcellular hierarchical structures is a current research focus. However, biological samples generally have weak optical absorption characteristics, resulting in low contrast in traditional bright-field microscopy, directly limiting the clear observation of microscopic structures. While existing technologies such as chemical staining and fluorescent labeling can improve imaging contrast, these exogenous interventions are prone to inducing cellular stress responses and damaging the physiological activity of biological samples, making long-term dynamic tracking difficult.

[0003] Differential phase-contrast (DPC) imaging is a label-free, non-interferometric phase imaging method based on partially coherent illumination modulation, providing a fast, efficient, and high-resolution visualization tool for unstained, transparent samples. DPC technology converts invisible sample phase information into intensity signals that can be directly detected by imaging devices through multiple asymmetric illumination modulations or asymmetric aperture modulations, thus enabling phase-contrast imaging and quantitative phase reconstruction. However, traditional DPC technology relies on multiple illumination acquisitions, resulting in insufficient temporal resolution and making it difficult to adapt to the observation needs of dynamic samples such as living biological samples, flowing cells, and body fluids, thus significantly limiting its applications. Summary of the Invention

[0004] In view of this, the present invention aims to provide a single-frame phase-contrast microscopy imaging method based on multi-polarization direction light field reconstruction. By controlling the polarization state, it achieves single-frame compressed acquisition of light field information to improve imaging speed. It aims to solve the bottleneck problem of traditional phase-contrast imaging technology in dynamic sample observation and promote its application in high-dynamic and high-precision biological observation scenarios.

[0005] To achieve the above objectives, the technical solution created by this invention is implemented as follows: A single-frame phase-contrast microscopy imaging method based on multi-polarization direction light field reconstruction includes: S1: Place the sample in the constructed microscopic optical path and add polarization masks with multiple polarization directions in the microscopic optical path; place the sample in the microscopic optical path and illuminate the sample to obtain a polarization image with information on multiple polarization directions; S2: Perform polarization channel separation on the polarization image obtained in step S1 to obtain sample intensity images corresponding to multiple polarization directions; generate image spectrum distributions for the corresponding polarization directions based on the sample intensity images; S3: Perform phase recovery on the image spectral distribution obtained in step S2 to obtain a phase-contrast microscopic image of the sample.

[0006] Furthermore, in the microscopic optical path constructed in step S1: A beam expander expands the light beam emitted from a light source. The expanded light beam is then focused by a condenser lens and illuminates the sample. The light generated by the illumination of the sample passes through an objective lens. The light beam transmitted from the objective lens is then focused by a tube lens and enters a polarization camera. The polarization camera receives the light beam and forms a polarization image. A polarizing mask is placed at the front focal plane of the condenser lens to allow light of different polarization directions to illuminate the sample simultaneously; or it is placed at the rear focal plane of the objective lens so that the light generated by the sample carries multiple polarization direction information after passing through the polarizing mask and is collected by a polarizing camera.

[0007] Furthermore, step S2 includes: By using the intensity transformation matrix, the intensity signals of multiple polarization detection directions in the polarization image obtained in step S1 are decoupled according to the multiple polarization directions of the polarization mask to obtain sample intensity images corresponding to multiple polarization directions. The obtained intensity images of multiple samples were subjected to Fourier transform to obtain the image spectral distribution in the corresponding polarization direction.

[0008] Furthermore, the intensity transformation matrix is ​​constructed based on Malus's law, and the elements of the intensity transformation matrix are the square cosine of the angle between each polarization direction in the polarization mask and multiple polarization detection directions in the polarization image.

[0009] Furthermore, the phase recovery process in step S3 includes: Based on the illumination parameters of the polarization direction of the polarization mask and the optical parameters of the objective lens, calculate the phase transfer function corresponding to each polarization direction of the polarization mask. Based on the phase transfer function and the corresponding image spectrum, a TV regularization denoising and reconstruction algorithm is used for joint operation to obtain the high-resolution spectrum of the sample phase. An inverse Fourier transform was performed on the high-resolution spectrum to obtain a phase-contrast microscopic image of the sample.

[0010] Furthermore, in the process of performing TV regularization denoising and reconstruction algorithm, the ADMM algorithm is used for optimization and calculation.

[0011] Furthermore, the phase distribution is obtained using the following formula: ; in, Represents phase distribution, PTF i (u) represents the phase transfer function corresponding to the i-th polarization direction, I i (u) represents the image spectrum corresponding to the i-th polarization direction, ρ represents the regularization intensity, Φ(x) represents the regularization function, and esp represents the error term. F represents the conjugate of the phase transfer function. -1 This represents the inverse Fourier transform.

[0012] Compared with the prior art, the present invention can achieve the following beneficial effects: (1) In the single-frame phase contrast microscopic imaging method based on multi-polarization direction light field reconstruction described in this invention, compared with the traditional scheme, this invention achieves uniform coverage of the light field in the entire circumference through polarization masks with multiple polarization directions, which not only achieves isotropic imaging characteristics, but also significantly improves phase contrast and imaging resolution through complete frequency information acquisition. (2) In the single-frame phase contrast microscopic imaging method based on multi-polarization direction light field reconstruction described in this invention, an innovative architecture combining multi-directional polarization light field and multi-channel synchronous detection is adopted. The polarization camera is used to synchronously acquire polarization incident information in multiple directions, which can complete phase reconstruction under single-frame image acquisition. While maintaining the real-time dynamic imaging speed, the imaging quality is maximized to meet the phase measurement requirements in high dynamic scenes. (3) In the single-frame phase contrast microscopy imaging method based on multi-polarization direction light field reconstruction described in this invention, a regularization constraint reconstruction solution strategy is introduced to effectively suppress the interference of background stray light and system noise on the reconstruction results, improve contrast, and enhance the stability and accuracy of phase measurement. Attached Figure Description

[0013] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments and descriptions of the invention are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings: Figure 1 A schematic flowchart of the single-frame phase-contrast microscopic imaging method based on multi-polarization direction optical field reconstruction as described in the embodiments of the present invention; Figure 2 A schematic diagram of the microscopic optical path described in the embodiments of the present invention; Figure 3 A schematic diagram of the polarization direction of the polarization mask as described in the embodiment of the present invention; Figure 4 The light and dark state diagrams of the three polarization directions described in the embodiments of the present invention; Figure 5 The image spectral distribution of the three polarization directions described in the embodiments of the present invention; Figure 6 The phase-contrast micrographs described in the embodiments of the present invention are provided.

[0014] Explanation of reference numerals in the attached figures: 1. Light source; 2. Beam expander; 3. Condenser; 4. Objective lens; 5. Tube lens; 6. Polarizing camera; 7. Sample; 8. Polarizing mask. Detailed Implementation

[0015] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and do not constitute a limitation thereof.

[0016] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0017] In the description of this invention, it should be understood that the terms "center," "longitudinal," "lateral," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," and "outer," etc., indicating orientations or positional relationships based on the orientations or positional relationships shown in the accompanying drawings, are only for the convenience of describing this invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation on this invention. Furthermore, the terms "first," "second," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, features defined with "first," "second," etc., may explicitly or implicitly include one or more of that feature. In the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0018] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "installation," "connection," and "linking" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection of two components. Those skilled in the art will understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0019] The invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0020] like Figure 1As shown, the single-frame phase-contrast microscopic imaging method based on multi-polarization direction light field reconstruction described in the embodiments of the present invention includes: S1: Place the sample in the constructed microscopic optical path and add polarization masks with multiple polarization directions in the microscopic optical path; place the sample in the microscopic optical path and illuminate the sample to obtain a polarization image with multiple polarization direction information.

[0021] In some embodiments, the microscopic optical path is as follows Figure 2 As shown, the system includes a light source 1, a beam expander 2, a condenser lens 3, an objective lens 4, a tube lens 5, and a polarizing camera 6. The beam expander 2 expands the light beam emitted from the light source 1. The expanded beam is then focused by the condenser lens 3 and illuminates the sample 7. The light generated by the illumination from the sample 7 passes through the objective lens 4. The beam transmitted from the objective lens 4 is then focused by the tube lens 5 and enters the polarizing camera 6. The polarizing camera 6 receives the beam and forms a polarization image. A polarizing mask 8 is positioned at the front focal plane of the condenser lens 3, as shown... Figure 2 As shown in (a), the sample is simultaneously illuminated by light polarized in different directions. The polarizing mask 8 can also be placed at the back focal plane of the objective lens 4, as shown in [example image]. Figure 2 As shown in (b), the light generated by sample 7 carries multiple polarization direction information after passing through polarization mask 8 and is collected by polarization camera 6.

[0022] In this embodiment of the invention, the polarization mask 8 adopts a circular structure and is evenly divided into three sectors along its circumference. The three sectors correspond to polarization directions of 0°, 120°, and 240°, respectively. Figure 3 As shown. The focal length of the condenser lens 3 is 70mm, the objective lens 4 is an Olympus 10× / 0.4NA objective lens, and the polarization camera 6 is a TRI050S-MC with a pixel size of 3.45µm and a resolution of 2448×2048. The polarization camera 6 outputs intensity signals in four polarization detection directions (0°, 45°, 90°, and 135°), meaning that the polarization image output by the polarization camera 6 carries information in four polarization detection directions. The center of the light source 1, the objective lens 4, the tube lens 5, and the center of the polarization camera 6 are initially aligned coaxially, and it is assumed that the imaging center of the polarization camera 6 is on the optical axis. In this embodiment of the invention, a single-frame polarization light field acquisition scheme is adopted to realize that the light field information corresponding to the three polarization directions of the polarization mask 8 can be calculated in a single imaging acquisition process. The brightness and darkness state diagram is shown below. Figure 4 As shown, this allows light field information in three polarization directions to be collected simultaneously. Figure 4 Images (a), (b), and (c) show the brightness and darkness states at polarization directions of 0°, 120°, and 240°, respectively. Polarization images of sample 7 and the background were acquired using polarization camera 6. Based on the polarization image of the background, the polarization image of sample 7 was processed to remove background influence, resulting in a polarization image with multiple polarization direction information.

[0023] S2: Perform polarization channel separation on the polarization image obtained in step S1 to obtain sample intensity images corresponding to multiple polarization directions; generate image spectrum distributions for the corresponding polarization directions based on the sample intensity images.

[0024] In some embodiments, step S2 includes: S21: Using the intensity transformation matrix, the intensity signals of multiple polarization detection directions in the polarization image obtained in step S1 are decoupled according to the multiple polarization directions of the polarization mask to obtain sample intensity images corresponding to multiple polarization directions.

[0025] The intensity transformation matrix is ​​constructed based on Malus's law, and its elements are the squared cosines of the angles between each polarization direction in the polarization mask and multiple polarization detection directions in the polarization image. The first element T in the intensity transformation matrix T... 1,1 For example, the first element T 1,1 If the polarization direction in the corresponding polarization mask is 0° and the polarization detection direction in the polarization image is 0°, then the angle between the two directions is 0°. According to Malus's law, the intensity transfer coefficient of polarized light after passing through the polarization channel is the square of the cosine of the angle, i.e., T. 1,1 =cos 2 (0) = 1. All other elements in the intensity transformation matrix T are calculated according to the first element T. 1,1 The intensity transformation matrix T is obtained by calculating in the following manner: .

[0026] The sample intensity images corresponding to the three polarization directions are obtained using the following formula: ; Among them, S0, S 120 and S 240 These represent the sample intensity images with polarization directions of 0°, 120°, and 240°, respectively, I0, I 45 I 90 and I 135 These represent the intensity signals with polarization detection directions of 0°, 45°, 90°, and 135°, respectively.

[0027] S22: Perform Fourier transform on the obtained intensity images of multiple samples to obtain the image spectral distribution along the corresponding polarization directions. The image spectral distributions along the three polarization directions are as follows: Figure 5 As shown, that is Figure 5 Images (a), (b), and (c) show the spectral distribution of images with polarization directions of 0°, 120°, and 240°, respectively.

[0028] S3: Perform phase recovery on the image spectral distribution obtained in step S2 to obtain a phase-contrast microscopic image of the sample.

[0029] In some embodiments, the phase recovery process in step S3 includes: S31: Based on the illumination parameters of the polarization direction of the polarization mask and the optical parameters of the objective lens, calculate the phase transfer function corresponding to each polarization direction of the polarization mask. The illumination parameters of the polarization direction of the polarization mask include the illumination direction and the numerical aperture of the polarization mask; the optical parameters of the objective lens include the numerical aperture and focal length.

[0030] S32: Based on the phase transfer function and the corresponding image spectrum, a TV regularized denoising and reconstruction algorithm is used for joint computation to obtain the high-resolution spectrum of the sample phase. During the TV regularized denoising and reconstruction algorithm process, the ADMM algorithm is used for optimization calculation.

[0031] S33: Perform inverse Fourier transform on the high-resolution spectrum to obtain a phase-contrast microscopic image of the sample.

[0032] Steps S32 and S33 can be summarized as follows: ; in, Represents phase distribution, PTF i (u) represents the phase transfer function corresponding to the i-th polarization direction, I i (u) represents the image spectrum corresponding to the i-th polarization direction, ρ represents the regularization intensity, Φ(x) represents the regularization function, and esp represents the error term. F represents the conjugate of the phase transfer function. -1 This represents the inverse Fourier transform. In this embodiment of the invention, i=1 corresponds to a polarization direction of 0°, i=2 corresponds to a polarization direction of 120°, and i=3 corresponds to a polarization direction of 240°.

[0033] Using the single-frame phase-contrast microscopy imaging method based on multi-polarization direction light field reconstruction provided in the embodiments of the present invention, Figure 6 As shown, in the obtained phase micrograph, the cell edges and background regions are clearly demarcated, the background is generally smooth, the cell nucleus and cytoplasm are clearly distinguishable, and some subcellular structures can be observed. Therefore, this invention, through single-frame phase-contrast microscopy based on multi-polarization direction light field reconstruction, achieves phase microscopy imaging under single-frame conditions, effectively improving imaging contrast and suppressing background noise, thereby obtaining higher quality phase imaging results.

[0034] It should be understood that the various forms of processes shown above can be used to reorder, add, or delete steps. For example, the steps described in this invention disclosure can be executed in parallel, sequentially, or in different orders, as long as the desired result of the technical solution disclosed in this invention can be achieved, and this is not limited herein.

[0035] The specific embodiments described above do not constitute a limitation on the scope of protection of this invention. Those skilled in the art should understand that various modifications, combinations, sub-combinations, and substitutions can be made according to design requirements and other factors. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this invention should be included within the scope of protection of this invention.

Claims

1. A single-frame phase-contrast microscopic imaging method based on multi-polarization direction light field reconstruction, characterized in that, include: S1: Place the sample in the constructed microscopic optical path and add polarization masks with multiple polarization directions in the microscopic optical path; place the sample in the microscopic optical path and illuminate the sample to obtain a polarization image with information on multiple polarization directions; S2: Perform polarization channel separation on the polarization image obtained in step S1 to obtain sample intensity images corresponding to multiple polarization directions; generate image spectrum distributions for the corresponding polarization directions based on the sample intensity images; S3: Perform phase recovery on the image spectral distribution obtained in step S2 to obtain a phase-contrast microscopic image of the sample.

2. The single-frame phase-contrast microscopic imaging method based on multi-polarization direction light field reconstruction according to claim 1, characterized in that, In the microscopic optical path constructed in step S1: A beam expander expands the light beam emitted from a light source. The expanded light beam is then focused by a condenser lens and illuminates the sample. The light generated by the illumination of the sample passes through an objective lens. The light beam transmitted from the objective lens is then focused by a tube lens and enters a polarization camera. The polarization camera receives the light beam and forms a polarization image. A polarizing mask is placed at the front focal plane of the condenser lens to allow light of different polarization directions to illuminate the sample simultaneously; or it is placed at the rear focal plane of the objective lens so that the light generated by the sample carries multiple polarization direction information after passing through the polarizing mask and is collected by a polarizing camera.

3. The single-frame phase-contrast microscopic imaging method based on multi-polarization direction light field reconstruction according to claim 1, characterized in that, Step S2 includes: By using the intensity transformation matrix, the intensity signals of multiple polarization detection directions in the polarization image obtained in step S1 are decoupled according to the multiple polarization directions of the polarization mask to obtain sample intensity images corresponding to multiple polarization directions. The obtained intensity images of multiple samples were subjected to Fourier transform to obtain the image spectral distribution in the corresponding polarization direction.

4. The single-frame phase-contrast microscopic imaging method based on multi-polarization direction light field reconstruction according to claim 3, characterized in that, The intensity transformation matrix is ​​constructed based on Malus's law. The elements of the intensity transformation matrix are the square cosines of the angles between each polarization direction in the polarization mask and multiple polarization detection directions in the polarization image.

5. The single-frame phase-contrast microscopic imaging method based on multi-polarization direction light field reconstruction according to claim 2, characterized in that, The phase recovery process in step S3 includes: Based on the illumination parameters of the polarization direction of the polarization mask and the optical parameters of the objective lens, calculate the phase transfer function corresponding to each polarization direction of the polarization mask. Based on the phase transfer function and the corresponding image spectrum, a TV regularization denoising and reconstruction algorithm is used for joint operation to obtain the high-resolution spectrum of the sample phase. An inverse Fourier transform was performed on the high-resolution spectrum to obtain a phase-contrast microscopic image of the sample.

6. The single-frame phase-contrast microscopic imaging method based on multi-polarization direction light field reconstruction according to claim 5, characterized in that, In the process of performing TV regularization denoising and reconstruction algorithm, ADMM algorithm is used for optimization and calculation.

7. The single-frame phase-contrast microscopic imaging method based on multi-polarization direction light field reconstruction according to claim 5, characterized in that, The phase distribution is obtained using the following formula: ； in, Represents phase distribution, PTF i (u) represents the phase transfer function corresponding to the i-th polarization direction, I i (u) represents the image spectrum corresponding to the i-th polarization direction, ρ represents the regularization intensity, Φ(x) represents the regularization function, and esp represents the error term. F represents the conjugate of the phase transfer function. -1 This represents the inverse Fourier transform.

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