Adjustable phase-symmetric microscopic imaging method, device, equipment and storage medium

Abstract

The invention relates to the technical field of microscope imaging, in particular to an adjustable phase-contrast microscopic imaging method, device and equipment and a storage medium, and the adjustable phase-contrast microscopic imaging method comprises the following steps: obtaining the thickness of a to-be-imaged sample, and generating a corresponding number of cell depth positions according to the thickness of the to-be-imaged sample; triggering a corresponding phase-symmetry imaging instruction according to the cell depth position, and triggering a refraction adjusting voltage according to the phase-symmetry imaging instruction to adjust the liquid crystal refractive index so as to obtain the adjusted liquid crystal refractive index; according to the liquid crystal refractive index, calculating to obtain a depth-symmetrical image corresponding to each cell depth position, and extracting a light position image from each depth-symmetrical image to obtain a corresponding depth cell image; and synthesizing the depth cell images according to the sequence of the depth positions of the cells to obtain a three-dimensional image of the sample. The method has the effect of forming thick sample imaging or three-dimensional imaging.

Description

Technical Field

[0001] The present application relates to the technical field of microscope imaging, and in particular to an adjustable phase-conforming microscopic imaging method, device, equipment and storage medium. Background Art

[0002] At present, phase-contrast microscopy has been widely used in microscopic imaging methods, mainly for imaging thin and transparent biological cells. It is difficult to see cell images under general bright field illumination, but under a phase-contrast microscope, strong contrast imaging of the cells can be seen.

[0003] In existing phase-contrast microscopes, the light source is illuminated in a ring shape through the illumination phase-contrast ring onto the sample. There is a corresponding phase-contrast ring on the objective lens, which attenuates the illumination light and changes the phase of the illumination. The diffracted wave passing through the sample and the surround wave of the illumination will interfere, and finally form a high-contrast phase-contrast image on the camera. The light beam interference is divided into the surround wave D of the illumination and the diffracted wave S of the sample, and finally forms a particle wave P wave on the camera. When the imaging area is not a cell, but the nutrient solution or cell fluid outside the cell, the light intensity of the D wave and the S wave is I P =I S +I D ; When the imaging area is a cell, the phases of the S wave and the D wave are different. in is the phase difference between the S wave and the D wave. hour, The image contrast is highest at the location of the nutrient medium and cells at this time. A typical monolayer of cultured cells is about 5 microns thick and has a refractive index of about 1.36. The cells are surrounded by a nutrient medium with a refractive index of 1.335, which produces an optical path difference of 0.125 microns, or about one quarter wavelength (green light). In order to create a phase difference of π between the cell and the cell fluid, a -π / 2 phase (1 / 4 wavelength optical path) needs to be generated on the path of the cell fluid. This phase difference can be achieved by processing the objective lens symmetry ring to achieve a phase difference of 1 / 4 wavelength (during processing, the phase difference of the surround wave that blocks the illumination is swapped and thinned to 1 / 4 wavelength). The phase difference between the cell and the cell fluid is π, and the contrast is highest at this time.

[0004] However, existing proportional limiting imaging has limitations. The sample must be very thin and cannot form thick sample imaging or three-dimensional imaging. Summary of the invention

[0005] In order to form thick sample imaging or three-dimensional imaging, the present application provides an adjustable symmetrical microscopic imaging method, device, equipment and storage medium.

[0006] The above-mentioned invention objective of the present application is achieved through the following technical solutions: An adjustable phase-contrast microscopic imaging method, the adjustable phase-contrast microscopic imaging method comprising: Acquire the thickness of the sample to be imaged, and generate a corresponding number of cell depth positions according to the thickness of the sample to be imaged; triggering a corresponding phase-contrast imaging instruction according to the cell depth position, and triggering a refraction adjustment voltage according to the phase-contrast imaging instruction to adjust the refractive index of the liquid crystal to obtain an adjusted refractive index of the liquid crystal; Calculate the depth-contrast image corresponding to each cell depth position according to the liquid crystal refractive index, and extract the light position image from each depth-contrast image to obtain the corresponding depth cell image; The depth cell images are synthesized according to the order of the cell depth positions to obtain a three-dimensional image of the sample.

[0007] By adopting the above technical solution, first, by obtaining the thickness of the sample to be imaged, a corresponding number of cell depth positions can be generated according to the actual size of the sample. This process ensures that all layers of the sample can be accurately covered in the subsequent imaging process, and improves the accuracy and reliability of imaging. Then, the corresponding phase-contrast imaging instructions are triggered according to these cell depth positions, and then the refraction adjustment voltage is triggered to adjust the refractive index of the liquid crystal to obtain the adjusted liquid crystal refractive index. This is one of the core steps of the entire imaging method. By accurately controlling the refractive index of the liquid crystal, the required phase difference can be achieved at different depths, thereby optimizing the imaging quality at each depth position. Subsequently, the depth-contrast image corresponding to each cell depth position is calculated according to the adjusted liquid crystal refractive index. This step involves complex optical calculations, especially the calculation of liquid crystal tilt angle data and data processing based on this. By substituting the liquid crystal tilt angle data into a specific formula, the depth-contrast image corresponding to each cell depth position can be accurately calculated. This method not only improves the resolution of imaging, but also can obtain clear imaging results at different depths. Extracting the light position image from each depth-contrast image further enhances the image quality and makes the information of key parts more prominent. Finally, all deep cell images are synthesized according to the order of cell depth positions to obtain a complete three-dimensional image of the sample. This process requires precise sorting and seamless splicing of each layer of images to ensure that the final generated three-dimensional image has a high degree of authenticity and integrity. In this way, not only can effective imaging of thick samples be achieved, but also three-dimensional reconstruction can be performed, which greatly expands the application scope and technical capabilities of phase-contrast microscopy. In summary, the adjustable phase-contrast microscopy method achieves efficient imaging of samples of different thicknesses by introducing a liquid crystal refractive index adjustment mechanism. Especially in the imaging of thick samples, this method overcomes the limitations of traditional phase-contrast microscopy, not only improves the imaging quality and resolution, but also provides strong support for applications in biological research and other related fields. In addition, this method also has an automatic voltage adjustment function, which simplifies the operation process and improves experimental efficiency.

[0008] In a preferred example, the present application can be further configured as follows: triggering a corresponding phase contrast imaging instruction according to the cell depth position, triggering a refraction adjustment voltage according to the phase contrast imaging instruction to adjust the liquid crystal refractive index, and obtaining an adjusted liquid crystal refractive index, specifically includes: Acquiring preset voltage-refractive index relationship data, and acquiring refractive index data required for imaging according to the cell depth position; The refraction adjustment voltage is calculated based on the voltage-refractive index relationship data and the refractive index data required for imaging.

[0009] By adopting the above technical solution, the corresponding phase-contrast imaging instructions are triggered according to the cell depth position, and then the refraction adjustment voltage is triggered according to these instructions to accurately adjust the refractive index of the liquid crystal. This process not only improves the imaging accuracy, but also expands the application range of phase-contrast microscopy imaging, making it adaptable to samples of different thicknesses. First, the system needs to obtain the thickness information of the sample to be imaged. This step is usually completed by optical measurement or other physical methods to ensure accurate sample thickness data. Next, according to the measured sample thickness, a corresponding number of cell depth positions are generated. These depth positions are the basis for subsequent imaging, and they determine at which level the imaging operation is performed. Subsequently, the system triggers the corresponding phase-contrast imaging instructions according to these cell depth positions. These instructions contain specific imaging parameters, such as the required refractive index. To achieve this, the system pre-stores a set of voltage-refractive index relationship data. These data are obtained through multiple tests during the experiment, and record the specific refractive index changes of the liquid crystal material under different voltages. Therefore, when the refractive index at a specific depth position needs to be determined, the corresponding voltage value can be quickly found according to the pre-stored data. Specifically, the system will first read the relevant information from the preset voltage-refractive index relationship data. This part of the data is usually presented in the form of a table or curve for easy query and use. Next, the required refractive index data is calculated based on the current cell depth position. This calculation process involves multiple factors, including but not limited to the thickness of the sample, the properties of the cell, and the imaging requirements. For example, for thicker samples, a larger refractive index may be required to compensate for the optical path difference; while for thinner samples, a smaller refractive index can be used. Once the required refractive index is determined, the next step is to calculate the actual voltage value that needs to be applied based on the voltage-refractive index relationship data. This calculation process involves complex mathematical models, but the basic principle is relatively intuitive. Assuming that we know that the refractive index of the liquid crystal material changes linearly with the voltage within a certain voltage range, the voltage value corresponding to any given refractive index can be estimated by a simple interpolation method. Of course, the actual situation may be more complicated, because the relationship between the refractive index and voltage of the liquid crystal material may be nonlinear, and there may even be multi-value mapping. In this case, more advanced algorithms are needed for accurate calculations. After the calculation is completed, the system will generate a control signal to drive the power supply to apply a predetermined voltage value to the liquid crystal layer. As the voltage changes, the arrangement of the liquid crystal molecules will also change accordingly, causing dynamic adjustment of the refractive index. This real-time refractive index adjustment capability enables the system to obtain the best imaging effect at different depths. Ultimately, by adjusting the refractive index of the liquid crystal, the system can collect high-quality symmetrical images at various depths, extract images of bright positions from them, and further synthesize three-dimensional images.

[0010] In a preferred example, the present application can be further configured as follows: the depth-contrast image corresponding to each cell depth position is calculated according to the liquid crystal refractive index, and the light position image is extracted from each depth-contrast image to obtain the corresponding depth cell image, specifically including: Calculating according to the refractive index of the liquid crystal to obtain liquid crystal tilt angle data; A depth-contrast image corresponding to each cell depth position is calculated according to the liquid crystal tilt angle data, and a light position image is extracted from each depth-contrast image to obtain a corresponding depth cell image.

[0011] By adopting the above technical solution, the refractive index of the liquid crystal is precisely controlled, and the liquid crystal tilt angle data is accurately calculated. This process ensures the accuracy of the depth-proportional image corresponding to the depth position of each cell, and improves the imaging quality. By extracting the light position image from each depth-proportional image, the image contrast is further enhanced, making the cell details more clearly visible. Ultimately, these high-quality deep cell images can be effectively synthesized to generate a three-dimensional image of the sample, which improves the application range and practicality of the entire microscopic imaging system.

[0012] In a preferred example, the present application can be further configured as follows: the depth-contrast image corresponding to each cell depth position is calculated according to the liquid crystal tilt angle data, and the light position image is extracted from each depth-contrast image to obtain the corresponding depth cell image, specifically including: The liquid crystal tilt angle data is input into the following formula to calculate the depth-contrast image: in, It refers to the phase difference at the end of the objective lens; Where n is a positive integer, It refers to the phase difference of nutrient solution; Among them, I P It refers to the light intensity that forms the particle wave P wave on the camera, I S It refers to the diffraction wave intensity of the sample, I D Refers to the ambient light intensity of the lighting.

[0013] By adopting the above technical solution, the depth-proportional image corresponding to each cell depth position can be accurately calculated based on the liquid crystal tilt angle data, and the light position image can be extracted from each depth-proportional image to obtain the corresponding depth cell image. Specifically, by substituting the liquid crystal tilt angle data into the formula, the phase difference at the objective end can be calculated; the phase difference of the nutrient solution can be calculated by the formula; the relationship between the diffraction wave intensity of the sample and the surround wave intensity of the illumination can be calculated by the formula, and then the intensity of the particle wave P wave formed on the camera can be obtained. These calculation results help to accurately capture cell images at different depths, improve the contrast and clarity of the imaging, and ultimately achieve high-quality three-dimensional imaging.

[0014] In a preferred example, the present application can be further configured as follows: synthesizing the deep cell images according to the order of the cell depth positions to obtain a three-dimensional image of the sample, specifically including: Obtaining the order of the numerical values ​​of the cell position depths, and sorting the depth cell images; The deep cell image is synthesized according to the sorting result to obtain the three-dimensional image of the sample.

[0015] By adopting the above technical solution, it is possible to accurately sort and synthesize cell images at different depths, ensuring that the final three-dimensional image of the sample has high resolution and clarity. Specifically, by arranging the numerical values ​​of the cell position depth in order, it is ensured that the cell images at each depth level are superimposed according to the actual spatial distribution, avoiding imaging distortion caused by image confusion. Furthermore, synthesis processing based on ordered deep cell images not only improves the speed of image reconstruction, but also enhances the image quality, achieving effective three-dimensional imaging of thick samples.

[0016] The second object of the invention is achieved by the following technical solutions: An adjustable phase-contrast microscopic imaging device, the adjustable phase-contrast microscopic imaging device comprising: A depth acquisition module, used to acquire the thickness of the sample to be imaged, and generate a corresponding number of cell depth positions according to the thickness of the sample to be imaged; A refraction adjustment module, used for triggering a corresponding phase-contrast imaging instruction according to the cell depth position, and triggering a refraction adjustment voltage according to the phase-contrast imaging instruction to adjust the refractive index of the liquid crystal to obtain an adjusted refractive index of the liquid crystal; An image extraction module, used to calculate the depth-contrast image corresponding to each cell depth position according to the liquid crystal refractive index, and extract the light position image from each depth-contrast image to obtain the corresponding depth cell image; The image synthesis module is used to synthesize the deep cell images according to the order of the cell depth positions to obtain a three-dimensional image of the sample.

[0017] By adopting the above technical solution, first, by obtaining the thickness of the sample to be imaged, a corresponding number of cell depth positions can be generated according to the actual size of the sample. This process ensures that all layers of the sample can be accurately covered in the subsequent imaging process, and improves the accuracy and reliability of imaging. Then, the corresponding phase-contrast imaging instructions are triggered according to these cell depth positions, and then the refraction adjustment voltage is triggered to adjust the refractive index of the liquid crystal to obtain the adjusted liquid crystal refractive index. This is one of the core steps of the entire imaging method. By accurately controlling the refractive index of the liquid crystal, the required phase difference can be achieved at different depths, thereby optimizing the imaging quality at each depth position. Subsequently, the depth-contrast image corresponding to each cell depth position is calculated according to the adjusted liquid crystal refractive index. This step involves complex optical calculations, especially the calculation of liquid crystal tilt angle data and data processing based on this. By substituting the liquid crystal tilt angle data into a specific formula, the depth-contrast image corresponding to each cell depth position can be accurately calculated. This method not only improves the resolution of imaging, but also can obtain clear imaging results at different depths. Extracting the light position image from each depth-contrast image further enhances the image quality and makes the information of key parts more prominent. Finally, all deep cell images are synthesized according to the order of cell depth positions to obtain a complete three-dimensional image of the sample. This process requires precise sorting and seamless splicing of each layer of images to ensure that the final generated three-dimensional image has a high degree of authenticity and integrity. In this way, not only can effective imaging of thick samples be achieved, but also three-dimensional reconstruction can be performed, which greatly expands the application scope and technical capabilities of phase-contrast microscopy. In summary, the adjustable phase-contrast microscopy method achieves efficient imaging of samples of different thicknesses by introducing a liquid crystal refractive index adjustment mechanism. Especially in the imaging of thick samples, this method overcomes the limitations of traditional phase-contrast microscopy, not only improves the imaging quality and resolution, but also provides strong support for applications in biological research and other related fields. In addition, this method also has an automatic voltage adjustment function, which simplifies the operation process and improves experimental efficiency.

[0018] The third objective of the present application is achieved through the following technical solutions: A computer device comprises a memory, a processor and a computer program stored in the memory and executable on the processor, wherein the processor implements the steps of the above-mentioned adjustable phase-contrast microscopy imaging method when executing the computer program.

[0019] The fourth objective of the present application is achieved through the following technical solutions: A computer-readable storage medium stores a computer program, which implements the steps of the above-mentioned adjustable phase-contrast microscopy imaging method when executed by a processor.

[0020] In summary, the present application includes at least one of the following beneficial technical effects: 1. By adjusting the refractive index of liquid crystal, phase-contrast imaging of samples of different thicknesses is achieved, which solves the problem that traditional phase-contrast microscopes are limited to thin samples and expands the application range of phase-contrast imaging; 2. By using voltage to control the refractive index of liquid crystal, the image of the bright position can be extracted at different depths, and then a three-dimensional image can be synthesized, which improves the imaging quality and resolution of thick samples; 3. The automated voltage regulation process simplifies the operation flow, improves imaging efficiency, and makes the imaging method more convenient and efficient. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is a flow chart of an adjustable symmetric microscopy imaging method in one embodiment of the present application; Figure 2 is a schematic diagram of the structure of a symmetrical microscope in one embodiment of the present application; Figure 3 is a schematic diagram of the structure of the lighting symmetry ring in one embodiment of the present application; Figure 4 is a schematic diagram of the structure of a symmetric ring inside an objective lens in one embodiment of the present application; Figure 5 is a schematic cross-sectional view of a symmetric ring inside an objective lens in one embodiment of the present application; Figure 6 This is a schematic diagram of the structure of the coating layer in the symmetrical ring in one embodiment of the present application. Figure 7 This is a schematic diagram of the structure of a liquid crystal sheet in a symmetrical ring in an embodiment of the present application. Figure 8 is a schematic diagram of a liquid crystal loading voltage in an embodiment of the present application; Fig. 9 is a flowchart for implementing step S20 of the method in adjustable symmetric microscopy imaging in one embodiment of the present application; Fig.10 is a flowchart for implementing step S30 of the method in adjustable symmetric microscopy imaging in one embodiment of the present application; Fig.11 is a schematic diagram of light beam interference in one embodiment of the present application; Fig.12 is a flowchart for implementing step S40 of the method in adjustable symmetric microscopy imaging in one embodiment of the present application; Fig.13is a principle block diagram of an adjustable symmetrical microscopic imaging system in one embodiment of the present application; Fig.14 It is a schematic diagram of a device in an embodiment of the present application. DETAILED DESCRIPTION

[0022] The present application is further described in detail below in conjunction with the accompanying drawings.

[0023] In one embodiment, if Figure 1 As shown, the present application discloses an adjustable phase-contrast microscopic imaging method, which specifically includes the following steps: S10: Obtain the thickness of the sample to be imaged, and generate a corresponding number of cell depth positions according to the thickness of the sample to be imaged.

[0024] Specifically, refer to Figure 2-5 , is a structural diagram of a symmetric microscope in an embodiment of the present application, including an illumination light source 2, and a symmetric ring 21 inside the illumination light source 2, an objective lens 3 is arranged above the illumination light source 2, a top symmetric ring 4 inside the objective lens 3, an imaging system 5 is arranged on the top of the symmetric ring 4, and a sample 1 is placed between the illumination light source 2 and the objective lens 3. The difference from the general symmetric ring is that the structure of the symmetric ring 4 is divided into 3 layers, the first layer is a coating layer 41, wherein the inner and outer layers of the coating layer 41 are transparent areas 411 with a transmittance of ≥90%, and the middle is a shading area 412, the shading area has a transmittance of 10% to 60% for the P-state particle wave polarized light, and a transmittance of ≤10% for the S-state diffracted wave polarized light, that is, the shading area 412 makes the S-state diffracted wave polarized light almost not transmitted, and the P-state particle wave polarized light partially transmitted.

[0025] Reference Figure 6 , 7 , where 42 is a liquid crystal sheet, where 422 is a liquid crystal layer, with electrically controllable liquid crystal built in, and different phase differences can be achieved by loading different voltages, and 421 is a glue layer, which is provided with water with a refractive index close to that of the liquid crystal. 43 of the symmetric ring 4 is a wave plate layer, which can convert polarized light regulated by the liquid crystal into circularly polarized light. By applying power to the liquid crystal layer 422 of the symmetric ring, the refractive index of the liquid crystal can be changed, so that the phase of the 422 layer can be changed, where the thickness of the coating layer 41 ranges from 0.5mm to 2mm, the thickness of the liquid crystal sheet 42 ranges from 2μm to 50μm, the thickness of the wave plate layer 43 ranges from 0.5mm to 2mm, and the radius range of the shading area 412: the maximum radius is R; the radius range of the shading area 412: inner diameter 0.3R to 0.5R, outer diameter 0.5R to 0.9R.

[0026] Specifically, after obtaining the thickness of the sample, that is, the thickness of the sample to be imaged, it is split according to the thickness. For example, if the sample thickness is 50 μm, a depth position can be selected every 5 μm, for a total of 10 depth positions, as the corresponding cell depth positions.

[0027] S20: triggering a corresponding phase-contrast imaging instruction according to the cell depth position, and triggering a refraction adjustment voltage according to the phase-contrast imaging instruction to adjust the refractive index of the liquid crystal to obtain an adjusted refractive index of the liquid crystal.

[0028] Specifically, Figure 8 As shown, after the corresponding cell depth position is obtained, the corresponding refraction adjustment voltage is generated to adjust the tilt angle of the liquid crystal and trigger the symmetrical imaging instruction at each position, thereby adjusting the liquid crystal refractive index by adjusting the tilt angle of the liquid crystal. After the liquid crystal is powered on according to the refraction adjustment voltage, the adjusted liquid crystal refractive index is obtained according to the voltage value.

[0029] S30: Calculate the depth-contrast image corresponding to each cell depth position according to the liquid crystal refractive index, and extract the light position image from each depth-contrast image to obtain the corresponding depth cell image.

[0030] Specifically, after obtaining the liquid crystal refractive index adjusted by voltage, a depth-proportional image of the cell depth position is calculated by a predetermined algorithm, and the bright position is extracted from the image to obtain a depth-proportional image corresponding to the cell depth position.

[0031] S40: synthesizing the depth cell images according to the order of the cell depth positions to obtain a three-dimensional image of the sample.

[0032] Specifically, according to the order of the Z axis of the sample from bottom to top, each deep cell image is synthesized by a preset algorithm, such as the Feldkamp-Davis-Kress (FDK) algorithm, so as to obtain a three-dimensional image of the sample.

[0033] In this embodiment, first, by obtaining the thickness of the sample to be imaged, a corresponding number of cell depth positions can be generated according to the actual size of the sample. This process ensures that all layers of the sample can be accurately covered in the subsequent imaging process, and improves the accuracy and reliability of imaging. Then, the corresponding phase-contrast imaging instructions are triggered according to these cell depth positions, and then the refraction adjustment voltage is triggered to adjust the refractive index of the liquid crystal to obtain the adjusted liquid crystal refractive index. This is one of the core steps of the entire imaging method. By accurately controlling the refractive index of the liquid crystal, the required phase difference can be achieved at different depths, thereby optimizing the imaging quality at each depth position. Subsequently, the depth-contrast image corresponding to each cell depth position is calculated according to the adjusted liquid crystal refractive index. This step involves complex optical calculations, especially the calculation of liquid crystal tilt angle data and data processing based on this. By substituting the liquid crystal tilt angle data into a specific formula, the depth-contrast image corresponding to each cell depth position can be accurately calculated. This method not only improves the resolution of imaging, but also can obtain clear imaging results at different depths. Extracting the light position image from each depth-contrast image further enhances the image quality and makes the information of key parts more prominent. Finally, according to the order of the cell depth position, all deep cell images are synthesized to obtain a complete three-dimensional image of the sample. This process requires precise sorting and seamless stitching of each layer of images to ensure that the final three-dimensional image has a high degree of authenticity and integrity. In this way, not only can effective imaging of thick samples be achieved, but they can also be reconstructed in three dimensions, greatly expanding the application scope and technical capabilities of phase-contrast microscopy. In summary, the adjustable phase-contrast microscopy method achieves efficient imaging of samples of different thicknesses by introducing a liquid crystal refractive index adjustment mechanism. Especially in the imaging of thick samples, this method overcomes the limitations of traditional phase-contrast microscopy, not only improves the imaging quality and resolution, but also provides strong support for applications in biological research and other related fields. In addition, this method also has an automatic voltage adjustment function, which simplifies the operation process and improves experimental efficiency.

[0034] In one embodiment, if Fig. 9 As shown, in step S20, the corresponding phase contrast imaging instruction is triggered according to the cell depth position, and the refraction adjustment voltage is triggered according to the phase contrast imaging instruction to adjust the liquid crystal refractive index to obtain the adjusted liquid crystal refractive index, which specifically includes: S21: Obtaining preset voltage-refractive index relationship data, and obtaining refractive index data required for imaging according to the cell depth position.

[0035] Specifically, the voltage-refractive index relationship data, i.e., the relationship between the specific amount of applied voltage and the refractive index of the liquid crystal, is obtained by pre-measurement or by using the parameters of the liquid crystal. Further, according to each cell depth position, the preset refractive index data required for imaging is obtained, so that the image presented at the refractive index can better meet the requirements after processing.

[0036] S22: Calculate the refraction adjustment voltage according to the voltage-refractive index relationship data and the refractive index data required for imaging.

[0037] Specifically, after obtaining the refractive index data required for imaging corresponding to each cell depth position, the refractive index is input into the voltage-refractive index relationship data to obtain the corresponding refraction adjustment voltage.

[0038] In one embodiment, if Fig.10 As shown, in step S30, the depth-contrast image corresponding to each cell depth position is calculated according to the liquid crystal refractive index, and the light position image is extracted from each depth-contrast image to obtain the corresponding depth cell image, which specifically includes: S31: Calculate according to the refractive index of the liquid crystal to obtain liquid crystal tilt angle data.

[0039] Specifically, the initial refractive index ne of the liquid crystal is obtained, and according to the refractive index n of the liquid crystal 折射率 , and through n 折射率 =ne*cosβ, the liquid crystal tilt angle β is calculated.

[0040] S32: Calculate the depth-contrast image corresponding to each cell depth position according to the liquid crystal tilt angle data, and extract the light position image from each depth-contrast image to obtain the corresponding depth cell image.

[0041] Specifically, the liquid crystal tilt angle data is input into the following formula to calculate the depth-contrast image: in, It refers to the phase difference at the end of the objective lens, n 样品 is the refractive index at the sample, n 营养液 It refers to the refractive index at the nutrient solution; Where n is a positive integer, It refers to the phase difference of nutrient solution; Among them, I P It refers to the light intensity that forms the particle wave P wave on the camera, I S It refers to the diffraction wave intensity of the sample, I D Refers to the ambient light intensity of the lighting.

[0042] Specifically, combined Fig.11 , the light beam interferes and is divided into the surrounding wave D of the illumination and the diffraction wave S of the sample, and finally forms a particle wave P wave on the camera. By obtaining the refractive index before and after the refraction adjustment voltage, that is, the refractive index n at the sample 样品 , the refractive index n of the nutrient solution 营养液 , and the corresponding cell depth position d, the objective end phase difference is calculated Then, through the phase difference of the objective lens end Calculate the nutrient solution phase difference Further, according to the objective lens end phase difference Difference in nutrient solution The phase difference between the S wave and the D wave, that is, the phase difference between the surround wave and the diffraction wave, is calculated, and the light intensity of the particle wave is calculated to obtain the cell image at that depth.

[0043] In one embodiment, if Fig.12 As shown, in step S40, the deep cell images are synthesized according to the order of the cell depth positions to obtain a three-dimensional image of the sample, which specifically includes: S41: Obtain the numerical order of the cell position depths and sort the depth cell images.

[0044] Specifically, the cell images are sorted in the order of the numerical values ​​of the cell position depths, from bottom to top after the samples are placed.

[0045] S42: synthesizing the deep cell image according to the sorting result to obtain a three-dimensional image of the sample.

[0046] Specifically, after the cell depth images are sorted, a preset algorithm is used to synthesize the images to obtain a three-dimensional image of the sample.

[0047] It should be understood that the size of the serial numbers of the steps in the above embodiments does not mean the order of execution. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0048] In one embodiment, an adjustable phase-contrast microscopic imaging device is provided, and the adjustable phase-contrast microscopic imaging device corresponds one-to-one with the adjustable phase-contrast microscopic imaging method in the above embodiment. Fig.13 As shown, the adjustable phase-conforming microscopic imaging device includes a depth acquisition module, a refraction adjustment module, an image extraction module and an image synthesis module. The detailed description of each functional module is as follows: A depth acquisition module is used to acquire the thickness of the sample to be imaged and generate a corresponding number of cell depth positions according to the thickness of the sample to be imaged; A refraction adjustment module, used for triggering a corresponding phase-contrast imaging instruction according to the cell depth position, triggering a refraction adjustment voltage according to the phase-contrast imaging instruction to adjust the liquid crystal refractive index, and obtaining an adjusted liquid crystal refractive index; An image extraction module is used to calculate the depth-contrast image corresponding to each cell depth position according to the refractive index of the liquid crystal, and extract the light position image from each depth-contrast image to obtain the corresponding depth cell image; The image synthesis module is used to synthesize the deep cell images according to the order of the cell depth positions to obtain a three-dimensional image of the sample.

[0049] Optionally, the refraction adjustment module includes: The refractive index acquisition submodule is used to obtain preset voltage-refractive index relationship data and obtain the refractive index data required for imaging according to the cell depth position; The voltage acquisition submodule is used to calculate the refraction adjustment voltage according to the voltage-refractive index relationship data and the refractive index data required for imaging.

[0050] Optionally, the image extraction module includes: The tilt angle calculation submodule is used to calculate according to the refractive index of the liquid crystal to obtain the liquid crystal tilt angle data; The image extraction submodule is used to calculate the depth-contrast image corresponding to each cell depth position according to the liquid crystal tilt angle data, and extract the light position image from each depth-contrast image to obtain the corresponding depth cell image.

[0051] Optionally, the image extraction submodule includes: The data calculation unit is used to input the liquid crystal tilt angle data into the following formula to calculate the depth proportional image: in, It refers to the phase difference at the end of the objective lens, n 样品 is the refractive index at the sample, n 营养液 It refers to the refractive index at the nutrient solution; Where n is a positive integer, It refers to the phase difference of nutrient solution; Among them, I P It refers to the light intensity that forms the particle wave P wave on the camera, I S It refers to the diffraction wave intensity of the sample, I D Refers to the ambient light intensity of the lighting.

[0052] Optionally, the image synthesis module includes: The image sorting submodule is used to obtain the numerical order of the cell position depth and sort the deep cell images; the image synthesis submodule is used to synthesize the deep cell images according to the sorting results to obtain a three-dimensional image of the sample.

[0053] For the specific definition of the adjustable phase-contrast microscopic imaging device, please refer to the definition of the adjustable phase-contrast microscopic imaging method above, which will not be repeated here. Each module in the above-mentioned adjustable phase-contrast microscopic imaging device can be implemented in whole or in part by software, hardware and a combination thereof. The above-mentioned modules can be embedded in or independent of the processor in the computer device in the form of hardware, or can be stored in the memory of the computer device in the form of software, so that the processor can call and execute the operations corresponding to the above modules.

[0054] In one embodiment, a computer device is provided. The computer device may be a server, and its internal structure diagram may be as follows: Fig.14 As shown. The computer device includes a processor, a memory, a network interface and a database connected through a system bus. Among them, the processor of the computer device is used to provide computing and control capabilities. The memory of the computer device includes a non-volatile storage medium and an internal memory. The non-volatile storage medium stores an operating system, a computer program and a database. The internal memory provides an environment for the operation of the operating system and the computer program in the non-volatile storage medium. The network interface of the computer device is used to communicate with an external terminal through a network connection. When the computer program is executed by the processor, an adjustable commensurate microscopic imaging method is implemented.

[0055] In one embodiment, a computer device is provided, including a memory, a processor, and a computer program stored in the memory and executable on the processor, wherein the processor implements the following steps when executing the computer program: Obtaining the thickness of the sample to be imaged, and generating a corresponding number of cell depth positions according to the thickness of the sample to be imaged; triggering a corresponding phase-contrast imaging instruction according to the cell depth position, and triggering a refraction adjustment voltage according to the phase-contrast imaging instruction to adjust the refractive index of the liquid crystal to obtain an adjusted refractive index of the liquid crystal; The depth-contrast image corresponding to each cell depth position is calculated according to the refractive index of the liquid crystal, and the light position image is extracted from each depth-contrast image to obtain the corresponding depth cell image; According to the order of the cell depth positions, the depth cell images are synthesized to obtain a three-dimensional image of the sample.

[0056] In one embodiment, a computer readable storage medium is provided, on which a computer program is stored, and when the computer program is executed by a processor, the following steps are implemented: Obtaining the thickness of the sample to be imaged, and generating a corresponding number of cell depth positions according to the thickness of the sample to be imaged; triggering a corresponding phase-contrast imaging instruction according to the cell depth position, and triggering a refraction adjustment voltage according to the phase-contrast imaging instruction to adjust the refractive index of the liquid crystal to obtain an adjusted refractive index of the liquid crystal; The depth-contrast image corresponding to each cell depth position is calculated according to the refractive index of the liquid crystal, and the light position image is extracted from each depth-contrast image to obtain the corresponding depth cell image; According to the order of the cell depth positions, the depth cell images are synthesized to obtain a three-dimensional image of the sample.

[0057] Those skilled in the art can understand that all or part of the processes in the above-mentioned embodiment methods can be completed by instructing the relevant hardware through a computer program, and the computer program can be stored in a non-volatile computer-readable storage medium. When the computer program is executed, it can include the processes of the embodiments of the above-mentioned methods. Among them, any reference to memory, storage, database or other media used in the embodiments provided in this application can include non-volatile and / or volatile memory. Non-volatile memory can include read-only memory (ROM), programmable ROM (PROM), electrically programmable ROM (EPROM), electrically erasable programmable ROM (EEPROM) or flash memory. Volatile memory can include random access memory (RAM) or external cache memory. As an illustration and not limitation, RAM is available in many forms, such as static RAM (SRAM), dynamic RAM (DRAM), synchronous DRAM (SDRAM), double data rate SDRAM (DDRSDRAM), enhanced SDRAM (ESDRAM), synchronous link (Synchlink) DRAM (SLDRAM), memory bus (Rambus) direct RAM (RDRAM), direct memory bus dynamic RAM (DRDRAM), and memory bus dynamic RAM (RDRAM).

[0058] Those skilled in the art can clearly understand that for the convenience and simplicity of description, only the division of the above-mentioned functional units and modules is used as an example. In actual applications, the above-mentioned functions can be distributed and completed by different functional units and modules as needed, that is, the internal structure of the device can be divided into different functional units or modules to complete all or part of the functions described above.

[0059] The embodiments described above are only used to illustrate the technical solutions of the present application, rather than to limit them. Although the present application has been described in detail with reference to the aforementioned embodiments, a person skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. Such modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present application, and should all be included in the protection scope of the present application.

Claims

1. An adjustable phase-contrast microscopy method, characterized in that: The adjustable phase-contrast microscopy method comprises: Acquire the thickness of the sample to be imaged, and generate a corresponding number of cell depth positions according to the thickness of the sample to be imaged; triggering a corresponding phase-contrast imaging instruction according to the cell depth position, and triggering a refraction adjustment voltage according to the phase-contrast imaging instruction to adjust the refractive index of the liquid crystal to obtain an adjusted refractive index of the liquid crystal; Calculate the depth-contrast image corresponding to each cell depth position according to the liquid crystal refractive index, and extract the light position image from each depth-contrast image to obtain the corresponding depth cell image; The depth cell images are synthesized according to the order of the cell depth positions to obtain a three-dimensional image of the sample.

2. The adjustable phase-contrast microscopy method according to claim 1, characterized in that: The triggering of the corresponding phase contrast imaging instruction according to the cell depth position, and triggering of the refraction adjustment voltage according to the phase contrast imaging instruction to adjust the refractive index of the liquid crystal to obtain the adjusted refractive index of the liquid crystal specifically includes: Acquiring preset voltage-refractive index relationship data, and acquiring refractive index data required for imaging according to the cell depth position; The refraction adjustment voltage is calculated based on the voltage-refractive index relationship data and the refractive index data required for imaging.

3. The adjustable phase-contrast microscopy method according to claim 1, characterized in that: The step of calculating the depth-contrast image corresponding to each cell depth position according to the liquid crystal refractive index, and extracting the light position image from each depth-contrast image to obtain the corresponding depth cell image specifically includes: Calculating according to the refractive index of the liquid crystal to obtain liquid crystal tilt angle data; A depth-contrast image corresponding to each cell depth position is calculated according to the liquid crystal tilt angle data, and a light position image is extracted from each depth-contrast image to obtain a corresponding depth cell image.

4. The adjustable phase-contrast microscopy method according to claim 3, characterized in that: The method of calculating the depth-contrast image corresponding to each cell depth position according to the liquid crystal tilt angle data, and extracting the light position image from each depth-contrast image to obtain the corresponding depth cell image specifically includes: The liquid crystal tilt angle data is input into the following formula to calculate the depth-contrast image: in, It refers to the phase difference at the end of the objective lens, n 样品 is the refractive index at the sample, n 营养液 It refers to the refractive index at the nutrient solution; Where n is a positive integer, It refers to the phase difference of nutrient solution; Among them, I P It refers to the light intensity that forms the particle wave P wave on the camera, I S It refers to the diffraction wave intensity of the sample, I D Refers to the ambient light intensity of the lighting.

5. The adjustable phase-contrast microscopy method according to claim 1, characterized in that: The synthesizing the deep cell images according to the order of the cell depth positions to obtain the sample three-dimensional image specifically includes: obtaining the order of the numerical values ​​of the cell position depths and sorting the deep cell images; The deep cell image is synthesized according to the sorting result to obtain the three-dimensional image of the sample.

6. An adjustable phase-conforming microscopic imaging device, characterized in that: The adjustable symmetric microscopic imaging device comprises: a depth acquisition module, used to acquire the thickness of the sample to be imaged, and generate a corresponding number of cell depth positions according to the thickness of the sample to be imaged; A refraction adjustment module, used for triggering a corresponding phase-contrast imaging instruction according to the cell depth position, and triggering a refraction adjustment voltage according to the phase-contrast imaging instruction to adjust the refractive index of the liquid crystal to obtain an adjusted refractive index of the liquid crystal; An image extraction module, used to calculate the depth-contrast image corresponding to each cell depth position according to the liquid crystal refractive index, and extract the light position image from each depth-contrast image to obtain the corresponding depth cell image; The image synthesis module is used to synthesize the deep cell images according to the order of the cell depth positions to obtain a three-dimensional image of the sample.

7. The adjustable phase-conforming microscopic imaging device according to claim 6, characterized in that: The refraction adjustment module comprises: A refractive index acquisition submodule is used to acquire preset voltage-refractive index relationship data, and acquire the refractive index data required for imaging according to the cell depth position; The voltage acquisition submodule is used to calculate the refraction adjustment voltage according to the voltage-refractive index relationship data and the refractive index data required for imaging.

8. The adjustable phase-conforming microscopic imaging device according to claim 6, characterized in that: The image extraction module comprises: A tilt angle calculation submodule, used to calculate according to the liquid crystal refractive index to obtain liquid crystal tilt angle data; The image extraction submodule is used to calculate the depth-contrast image corresponding to each cell depth position according to the liquid crystal tilt angle data, and extract the light position image from each depth-contrast image to obtain the corresponding depth cell image.

9. A computer device comprising a memory, a processor, and a computer program stored in the memory and executable on the processor, characterized in that: When the processor executes the computer program, the steps of the adjustable phase-contrast microscopy imaging method according to any one of claims 1 to 5 are implemented.

10. A computer-readable storage medium storing a computer program, characterized in that: When the computer program is executed by a processor, the steps of the adjustable phase-contrast microscopy imaging method according to any one of claims 1 to 5 are implemented.

Images