A differential interference contrast imaging system, method and time-lapse incubator apparatus

Abstract

This invention discloses a differential interferometric phase-contrast imaging system, method, and time-difference incubator device, belonging to the fields of optical imaging and medical device technology. The system includes an illumination source module, a microscopic imaging module, a polarization interferometry module, and a quantitative phase image reconstruction module. By placing all polarization interferometry components, such as the polarizer and polarization beam splitter, on the image side of the microscopic imaging module, the imaging beam does not need to pass through the plastic culture dish before passing through the polarization elements, thus completely avoiding interference from plastic birefringence on the imaging and achieving high-quality observation of samples in cell biology or materials science fields within plastic culture dishes. Simultaneously, the system uses a polarization camera to simultaneously acquire multiple phase-shifted interferograms in a single exposure, combined with a quantitative phase reconstruction algorithm, achieving high-speed, high-precision quantitative phase imaging, overcoming the shortcomings of traditional mechanical phase-shifting methods, such as slow speed and susceptibility to motion artifacts. Furthermore, the imaging system of this invention can also be integrated into a time-difference incubator.

Description

Technical Field

[0001] This invention relates to the fields of optical imaging and medical device technology, and more specifically to a differential interference phase contrast (DIC) imaging method, system, and time difference incubator device including the system, suitable for plastic culture dishes. Background Technology

[0002] The phase microscopy module in a time-lapse incubator is the most crucial component. Capturing three-dimensional and layered images of biological tissues with a camera is essential for accurately determining their morphology and development. Time-lapse incubators typically employ Hoffman phase imaging technology, which uses oblique incident illumination and spatial modulation filtering to convert the phase gradient of a transparent sample into intensity differences, achieving pseudo-three-dimensional relief visualization of markerless phase objects. However, due to product specifications, the resolution and contrast of the imaging are relatively fixed. Using a fixed-specification Hoffman objective lens for different types and sizes of samples makes it difficult to guarantee image quality, resulting in poor adaptability. Traditional differential interferometry (DIC) is a high-resolution phase imaging technique based on polarized light shearing interference. Linearly polarized light is split into two orthogonally polarized, slightly laterally sheared coherent beams by a Nomaski or Wollaston prism, illuminating adjacent micro-regions of the sample. The two beams generate a phase difference due to the optical path difference of the sample, which then interferes with a second Nomaski or Wollaston prism and an analyzer, converting the optical path difference gradient into intensity or color contrast. The images have a realistic three-dimensional relief effect and extremely high resolution, which can reflect the fine structure and refractive index changes of the sample. However, they are sensitive to birefringent samples and plastic containers and are not suitable for time zone incubators.

[0003] Although traditional DIC microscopes have good contrast and resolution, they still have the following limitations:

[0004] 1. Interference of birefringence on polarization. Traditional DIC microscopes rely heavily on polarized light for imaging. However, plastic culture dishes exhibit birefringence, which severely distorts the direction of polarized light, resulting in significant stray light in the image and a marked decrease in contrast. Furthermore, materials such as crystals or fibers, which are inherently birefringent, can also interfere with polarized light, producing artifacts or signal loss, thus affecting the accuracy of the image. Therefore, traditional DIC microscopes can only use stress-free glass dishes, which are costly, fragile, and inconvenient to use.

[0005] 2. Phase information is difficult to obtain quantitatively. DIC imaging results produce a pseudo-three-dimensional relief effect, which does not directly correspond to the actual height or phase difference. It is only a qualitative analysis and it is difficult to achieve quantitative phase measurement. It is not suitable for accurate analysis of sample thickness or refractive index changes.

[0006] 3. The system structure is complex and adjustment is difficult. The optical path of traditional DIC microscopy systems relies on a precision prism device, and the installation and calibration process is complex. For example, to obtain quantitative information, it is usually necessary to mechanically rotate the analyzer to acquire multiple images, which is slow. Furthermore, due to sample movement, such as embryonic development and Brownian motion, registration errors are introduced. The system is also susceptible to inaccuracies caused by vibration or temperature changes, increasing maintenance costs.

[0007] In addition, although there are attempts in the existing technology to adapt to plastic culture dishes by changing the optical path, such as PlasDIC technology, it usually has problems such as limited use of objective lens numerical aperture and difficulty in achieving high-speed quantitative phase imaging.

[0008] Therefore, there is an urgent need for a DIC method or system that is compatible with plastic culture dishes and can quickly achieve quantitative phase imaging. Summary of the Invention

[0009] In view of the above problems, the present invention is proposed to provide a differential interferometric phase-contrast imaging system, method, and time-difference incubator device that overcomes or at least partially solves the above problems.

[0010] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, embodiments of the present invention provide a differential interferometric phase-contrast imaging system, comprising: arranged sequentially along the optical path: Lighting source module, used to provide lighting light; The microscopic imaging module is used to hold the sample and scan and magnify it for microscopic imaging. A polarization interferometry module is used to perform polarization beam splitting and phase shift modulation on the beam from the microscopic imaging module, and to detect the intensity of the interferometric light carrying sample information; and, A quantitative phase image reconstruction module is used to reconstruct a quantitative phase image of the sample based on the signal detected by the polarization interferometry module. The polarization interference module includes a polarizer, a polarization beam splitter, a polarization converter, and a detector arranged sequentially along the optical path. The polarizer, the polarization beam splitter, and the polarization converter are all located on the image side of the microscopic imaging module. The detector is configured to acquire at least two interferometric images with different phase shifts simultaneously in a single exposure.

[0011] In one embodiment, the illumination source module includes a light source, a Köhler lens, a field stop, a slit stop, and a condenser lens arranged sequentially along the optical path; the slit stop is disposed on the entrance pupil plane or conjugate plane of the condenser lens, the long side direction of its slit is perpendicular to the shearing direction of the polarizing beam splitter, and the slit width b of the slit stop satisfies the following condition: (1) Where B represents the shear distance of the object plane, f represents the focal length of the condenser lens, and λ represents the wavelength of the light source.

[0012] In one embodiment, the polarization beam splitter is a Nomarsky prism, a Wollaston prism, or a Savar plate.

[0013] In one embodiment, the polarization converter is a quarter-wave plate, a liquid crystal variable phase delayer, or a spatial modulator (SLM); when the polarization converter is a quarter-wave plate, its fast axis direction forms a 45° angle with the long axis direction of the polarization beam splitter.

[0014] In one embodiment, the detection device is a polarization camera, whose pixel surface is integrated with a micro-polarization filter array, the micro-polarization filter array containing at least two filter units with different polarization directions, so that the polarization camera can simultaneously acquire at least two interference images corresponding to different polarization directions in a single exposure. The polarization detection directions of the micro-polarization filter array include 0°, 45°, 90° and 135°, enabling the polarization camera to simultaneously acquire four phase-shifted interference images.

[0015] In one embodiment, the detection device includes a rotatable analyzer and an area array detector. By controlling the analyzer to rotate to different angles and cooperating with the area array detector to perform multiple exposures, multiple interferometric images with different phase shifts are acquired in a time-series manner.

[0016] In one embodiment, the quantitative phase image reconstruction module is configured to perform the following steps: Acquire at least four phase-shifted interferometric images synchronously acquired by the detector; Based on the phase-shifting algorithm, the phase gradient distribution of the sample is calculated from the phase-shifted interferometric image and then filtered. The phase gradient distribution after filtering is integrated and regularized to reconstruct the quantitative phase distribution of the sample.

[0017] In one embodiment, the step of calculating the phase gradient distribution based on the phase-shifting algorithm includes: when acquiring four phase-shifting interferometric images I1, I2, I3, and I4, calculating the phase gradient according to the following formula: (2) in, The images show the light intensity at analyzer angles of 0°, 45°, 90°, and 135°. The shear distance of the system; The step of integrating and regularizing the filtered phase gradient distribution includes solving the optimization problem using the ADMM-TV algorithm based on total variational regularization. (3) (4) in, For phase The x-direction derivative; For phase The y-direction derivative; The phase gradient is calculated based on formula (2). Filtered phase gradient, For phase The total variational regularization term, λ is the regularization parameter; i and j represent the number of rows and columns of the image, respectively.

[0018] In a second aspect, embodiments of the present invention provide a differential interferometric phase-contrast imaging method, applied to a differential interferometric phase-contrast imaging system as described in any one of the first aspects, comprising the following steps: Illumination light is generated using an illumination source module; The illumination light is transmitted or reflected through the sample carried in the sample container, and a magnified image of the sample is formed by the microscopic imaging module. Using the polarization interference module located on the image side of the microscopic imaging module, the beam carrying sample information is sequentially polarized, polarized and split, and polarization converted, and at least two interference images with different phase shifts are acquired simultaneously in a single exposure. Based on the interferometric image, a quantitative phase image of the sample is obtained through phase gradient calculation and integral reconstruction algorithm.

[0019] Thirdly, embodiments of the present invention provide a time-difference culture device, including a culture chamber and an environmental control system, and further including a differential interferometric phase-contrast imaging system as described in any one of the first aspects, wherein the differential interferometric phase-contrast imaging system is used to perform sequential quantitative phase imaging of biological samples in a sample container within the culture chamber.

[0020] As can be seen from the above technical solution, compared with the prior art, the present invention has the following technical effects: 1. Compatible with plastic culture dishes: By placing core polarization components such as polarizers and polarization beam splitters in the imaging optical path behind the sample, the imaging beam carrying sample information does not need to pass through the plastic culture dish with birefringence before passing through the polarization element. This perfectly solves the problem of image quality degradation caused by plastic birefringence in traditional DIC, allowing the use of low-cost and high-safety plastic culture dishes.

[0021] 2. Rapid quantitative phase imaging: Multiple phase-shifted images are acquired simultaneously in a single exposure using a polarization camera. Alternatively, electrically controlled phase shifting can be achieved through a liquid crystal variable phase delay device (LCVR) or a spatial modulator (SLM), completely eliminating the time delay and image registration error caused by mechanical rotation. This enables high-speed, high-precision quantitative phase measurement of the dynamic processes of biological samples.

[0022] 3. High imaging quality: By setting a slit aperture that meets the "quarter-wavelength condition", the spatial coherence of illumination is effectively controlled, significantly improving the contrast and signal-to-noise ratio of DIC images.

[0023] 4. Wide range of applications: This system and method can be directly integrated into time-variety incubators, enabling long-term, label-free, quantitative three-dimensional observation of biological samples such as embryos and cells. It has significant application value in fields such as assisted reproduction and cell biology. Furthermore, the reflective method is also suitable for observing opaque samples such as material surfaces and wafers. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0025] Figure 1 This is a schematic diagram of the optical path of the differential interference phase-contrast imaging system provided in Example 1; Figure 2 This is a structural diagram of a four-quadrant polarization unit of a single imaging unit of a polarization camera provided in Embodiment 1; Figure 3 This is a diagram showing the results of DIC phase imaging of the plastic microspheres provided in Example 1; Figure 4 This is a schematic diagram of the optical path of the reflective DIC quantitative phase-contrast imaging system provided in Example 2; Figure 5 This is a schematic diagram of the optical path of the imaging system using a conventional camera provided in Embodiment 3; Figure 6 This is a flowchart of the differential interferometric phase-contrast imaging method provided in Example 4; Figure 7 This is a structural diagram of the time difference culture device provided in Example 5. Detailed Implementation

[0026] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] Example 1: like Figure 1 As shown, this embodiment provides a differential interferometric phase-contrast imaging system suitable for differential interferometric phase-contrast (DIC) imaging of plastic culture dishes. The differential interferometric phase-contrast imaging system mainly includes an illumination source module, a microscopic imaging module, a polarization interferometry module, and a quantitative phase image reconstruction module (not shown in the figure, implemented by computer software).

[0028] (1) Lighting source module: Provides the lighting source and aperture. For example... Figure 1 As shown, it consists of an LED lamp 1, a Köhler lens 2, a field aperture 3, a slit aperture 4, and a condenser lens 5. The LED lamp 1 preferably uses a 635nm wavelength LED light source, which has significant advantages in biological sample research. Yellow and red light is less easily absorbed by biological tissues and has strong penetrability. Compared to the phototoxicity of laser light sources, the low phototoxicity of LED light sources ensures long-term imaging. Other wavelengths of LED light sources or lasers can also be used.

[0029] The slit stop 4 is located on the entrance pupil plane or conjugate plane of the condenser lens 5. Its slit is elongated and used to limit coherence in the shear direction, improving the contrast of the shear interference. To produce sufficient contrast, the quarter-wavelength condition must be met, meaning the slit width should not exceed one-quarter of the interference fringe distance in the objective lens's exit pupil. In other words, to ensure imaging contrast, the long side of the slit must be perpendicular to the shear direction of the polarizing beam splitter, and the slit width b must satisfy the quarter-wavelength condition. (1) Where B represents the shear distance of the object plane, f represents the focal length of the condenser lens 5, and λ represents the wavelength of the light source 1.

[0030] (2) Microscopic imaging module: used to hold the sample and perform scanning and microscopic magnification imaging of the sample. For example... Figure 1 As shown, the system includes a stage 6 for supporting the plastic culture dish, a microscope objective 7, a reflecting mirror 8, and a tube microscope 9. The sample is placed in the plastic culture dish and then placed on the stage 6. The focal length and position can be adjusted using a three-dimensional displacement platform.

[0031] (3) Polarization interferometry module: used to polarize and phase-shift the beam from the microscopic imaging module, and to detect the intensity of the interference light carrying sample information. The polarization interferometry module mainly includes a polarizer, a polarization beam splitter, a polarization converter, and a detector arranged sequentially along the optical path.

[0032] 1) The polarizer provides polarized light to the system. It should be noted that, compared with the traditional DIC, the polarization interference module of this system is placed behind the sample.

[0033] 2) Polarization beam splitter module: This module uses a Nomarski prism to cut the incident linearly polarized light. When the linearly polarized light is incident at 45°, it will be uniformly split into two mutually perpendicular linearly polarized beams.

[0034] 3) The polarization conversion module uses a quarter-wave plate to convert two beams of polarized light with mutually perpendicular vibration directions into one left-handed circularly polarized light and one right-handed circularly polarized light. After passing through polarizers with different polarization directions, a phase shift can be generated. Combined with the polarization camera in the imaging module, a method can be achieved to quickly obtain four phase images.

[0035] 4) Detection device: The camera used in this system is a polarization camera. Each pixel surface integrates a micro-polarization filter with angles of 0°, 45°, 90°, and 135°. This design allows for the capture of light at four polarization angles in a single exposure. Combined with the polarization beam splitting module and polarization conversion module, it can significantly improve the extraction of the phase image. Then, using a four-step phase shift algorithm, a quantitative phase image of the object is obtained, which greatly improves the imaging speed, eliminates errors caused by sample movement and Brownian shift, and achieves high-precision quantitative phase reconstruction.

[0036] All of the above polarization devices are located in the imaging optical path behind the sample. (Refer to...) Figure 1 As shown, the polarization interference module specifically includes a polarizer 10, a first lens 11, a Nomaski prism 12, a quarter-wave plate 13, a second lens 14, a filter 15, and a polarization camera 16. After passing through the tube mirror 9, the light beam from the tube mirror 9 is first converted into linearly polarized light by the polarizer 10, and then split into two orthogonal polarized beams by the first lens 11 and the Nomaski prism 12. The Nomaski prism 12, acting as a polarization beam splitter, has its major axis at a 45° angle to the polarization direction of the polarizer 10, thus decomposing the incident linearly polarized light into two beams of linearly polarized light with mutually perpendicular vibration directions and a small transverse shear δr. The fast axis of the quarter-wave plate 13 forms a 45° angle with the major axis of the Nomaski prism 12, converting the two mutually orthogonal linearly polarized beams split by the Nomaski prism into a left-handed circularly polarized beam and a right-handed circularly polarized beam, which are then imaged by the filter 15 and the polarization camera 16. The Normask prism 12 can also be replaced by the Wollaston prism.

[0037] The polarization camera 16 is key to achieving rapid imaging in this embodiment. It achieves this by adding a polarizer in front of the sensor, such as... Figure 2 As shown, each imaging computing unit of the sensor integrates micro-polarization filters in four directions: 0°, 45°, 90°, and 135°. Therefore, the camera can capture light rays with four different polarization angles in a single exposure. Combined with the left-handed circularly polarized light converted by the polarization beam splitter and the right-handed circularly polarized light, after passing through the micro-polarization filters, interference occurs, resulting in four interference images with different phase shifts. Detection. The intensity of each image can be expressed as:

[0038] in, θ represents the original total incident intensity of the two circularly polarized light beams, i.e., the total light intensity before passing through the analyzer; θ represents the analysis angle. This represents the phase difference between two beams of light, introduced by the Nomaski prism.

[0039] (4) Quantitative phase image reconstruction module: The phase information obtained by DIC interferometry is caused by the optical path difference generated by the gradient change between two adjacent points. Therefore, phase extraction and integration are required, and the sample phase information is restored according to the correspondence between wavelength and phase.

[0040] To obtain a quantitative phase image, the obtained quantitative gradient image needs to be integrated. Direct line integration suffers from strong linear artifacts due to noise accumulation, while Fourier integration requires two-dimensional gradient maps for reconstruction. Here, this embodiment uses the ADMM-TV optimization method to integrate the gradient map, leveraging known prior knowledge for regularization constraints. As a result, the phase of the sample is reconstructed.

[0041] The quantitative phase image reconstruction module runs on a computer and achieves quantitative reconstruction of the sample phase based on four differential interferometric images with different phase shifts detected by a polarization camera. To verify the system's quantitative phase imaging capability, polymethyl methacrylate (PMMA) microspheres with known optical parameters were selected as standard samples. The refractive index of PMMA at the illumination wavelength is approximately 1.49, and it was placed in a deionized water environment with a refractive index of approximately 1.33. Since the refractive index difference between the microsphere and the surrounding medium is fixed and known, the resulting optical path difference, and consequently the phase difference, is definite and calculable, making it an ideal sample for verifying the accuracy of phase measurements.

[0042] Its processing flow includes: Step 1, Data Acquisition: Using PMMA spheres with a refractive index of 1.49, in a deionized water environment, a plastic petri dish carrying the sample was placed on the stage, and four phase-shifting interferometry images were simultaneously acquired using a detection device. That is, different polarization unit directions on the polarization camera can collect images such as... Figure 3 The four differential interferometric images with different phase shifts shown in section (a) correspond to 0 0.5 , There's still 1.5 left. Four differential interferometric images can significantly shorten the time for acquiring phase-shifted images and reduce errors caused by image registration.

[0043] Step 2, Phase gradient calculation and filtering: Based on the phase shift algorithm, the phase gradient distribution of the sample is calculated from the phase shift interferometric image and then filtered.

[0044] The phase gradient calculation employs a four-step phase-shift algorithm. (2) in, The light intensity diagrams for analyzer angles of 0°, 45°, 90°, and 135° are respectively related to the light intensity at these angles. Figure 3 Part (a) 0 0.5 , There's still 1.5 left. Four differential interference images. The shear distance of the system; the calculated result is the phase gradient distribution, such as... Figure 3 As shown in section (b). Median filtering can be used to handle noise and discontinuities in the results.

[0045] Gradient graph filtering: In this embodiment, median filtering is used for processing, and the result is as follows. Figure 3 As shown in section (c), median filtering is a non-linear image smoothing technique that effectively suppresses noise and preserves edge information by replacing the value of each pixel with the median of all pixel values ​​within its neighborhood window. First, an odd-sized sliding window (e.g., 3×3) is defined. Centered on the current pixel, all pixel values ​​within its neighborhood are extracted. The pixel values ​​are then sorted by size, and the median value of the sorted sequence is taken as the latest value of the current pixel. Discontinuous points, due to their significant differences from surrounding values, are placed at the beginning or end of the sort and can therefore be filtered out.

[0046] Its mathematical expression is:

[0047] in The original phase gradient map is calculated according to formula (2). This is the phase gradient map of the filtered output. It is a sliding window.

[0048] Step 3, Phase Reconstruction: The phase gradient distribution map after filtering is integrated and regularized to reconstruct the quantitative phase distribution of the sample.

[0049] Filtered unidirectional phase gradient The phase is reconstructed by solving the total variational regularization problem. : (3) (4) For phase The x-direction derivative; For phase The y-direction derivative; where i represents the row and j represents the column. Phase diagram The value after difference along the x-direction at pixel position i, column j. The calculated phase gradient Filtered phase gradient, For phase The total variational regularization term is given, where λ is the regularization parameter. Phase reconstruction employs a total variational (TV) regularization optimization model and is solved iteratively using the alternating direction multiplier method (ADMM). The k-th iteration includes the following update formula:

[0050]

[0051]

[0052] Among them, the deviation of the constraint conditions is used to measure and accumulate the deviation, so that the auxiliary variable and the phase gradient tend to be consistent during the iteration process; It is the auxiliary variable in the x-direction during the k-th iteration. It is the auxiliary variable in the y-direction during the k-th iteration. It is the x-direction dual variable in the k-th iteration. It is the dual variable in the y-direction of the k-th iteration. The phase obtained in the (k+1)th iteration The gradient. d An auxiliary variable introduced to represent phase. The gradient term separates the regularization term from the data fidelity term, facilitating iterative solutions using the alternating direction multiplier method. b is the dual variable used to measure and accumulate the deviation of the constraints, making the auxiliary variable during iteration... d It tends to be consistent with the phase gradient. This is a penalty parameter used to control the weight of the constraint term in the Lagrangian function; it is not a physical parameter of the sample itself. The soft thresholding operator for a vector is defined as follows:

[0053] in, The input vector represents the gradient correlation at the current pixel. This is a threshold parameter used to control the contraction intensity. The process continues until convergence, yielding the final quantitative phase map. ,like Figure 3 As shown in section (d), the microsphere phase imaging effect of PMMA material is well verified.

[0054] The differential interferometric phase-contrast imaging system provided in this invention adds a slit in front of the condenser lens to control the spatial coherence of the illumination source. It removes the polarizing prism at the illumination end of a traditional DIC system, setting only a polarizing beam-splitting prism at the imaging end to achieve shearing interference of the imaging beam, ensuring it is unaffected by birefringent material samples. A quarter-wave plate and a polarizing camera are added after the polarizing beam-splitting prism. The fast axis of the quarter-wave plate makes a 45° angle with the long axis of the DIC prism, converting the two linearly polarized beams sheared by the DIC prism into circularly polarized light. After passing through the analyzer, the intensity change of the interference is represented by the phase delay, and the phase delay changes with the angle of the analyzer. The polarizing camera, through an integrated micro-polarizer array, captures the polarization intensity in four directions (0°, 45°, 90°, and 135°) at each pixel, thus enabling simultaneous imaging of four phases. Combined with a phase reconstruction algorithm, quantitative phase imaging of the sample is achieved, significantly improving the speed of quantitative phase imaging.

[0055] Example 2: This embodiment also provides a differential interferometric phase-contrast imaging system, which belongs to the category of reflective DIC imaging systems, such as... Figure 4 As shown. It is particularly suitable for observing opaque samples, such as material surfaces and the surface morphology of wafers. The system includes... (1) Lighting source module: provides a lighting source; such as Figure 4 As shown, it consists of a laser light source 21, a first beam expander lens 22, a second beam expander lens 23, a first tube mirror 24, and a beam splitter 25.

[0056] (2) Microscopic imaging module: used to hold the sample and perform scanning and microscopic magnification imaging of the sample. For example... Figure 4As shown, it includes: a microscope objective 26, a stage 27, and a second tube mirror 29; (3) Polarization Interference Module: Used to polarize and phase-shift the beam from the microscopic imaging module, and to detect the intensity of the interference light carrying sample information. The polarization interference module mainly includes a polarizer 28, a polarization beam splitter, a polarization conversion module, and a detection device arranged sequentially along the optical path.

[0057] 1) The polarizer 28 provides polarized light to the system. It should be noted that, compared with the traditional DIC, the polarization interference module of this system is placed on the opposite side of the sample.

[0058] 2) Polarization beam splitter module: The Savart 30 plate is used to cut the incident linearly polarized light. When the linearly polarized light is incident at 45°, it will be uniformly split into two mutually perpendicular linearly polarized beams.

[0059] 3) Polarization conversion module: A quarter-wave plate 31 is used to convert two beams of polarized light with mutually perpendicular vibration directions into one left-handed circularly polarized light and one right-handed circularly polarized light. After passing through polarizers with different polarization directions, a phase shift can be generated. Combined with the polarization camera 32 of the imaging module, a method to quickly obtain four phase images can be realized.

[0060] 4) Detection device: The polarization camera 32 used in this system integrates micro-polarization filters on the surface of each pixel with angles of 0°, 45°, 90° and 135°. This design allows light to be captured at four polarization angles in a single exposure. Combined with the polarization beam splitting module and polarization conversion module, the extraction of phase map can be significantly improved. Then, a four-step phase shift algorithm is used to obtain a quantitative phase image of the object.

[0061] (4) Quantitative phase image reconstruction module: Same as in Example 1.

[0062] All of the above polarization devices are located on opposite sides of the optical path of the beam splitter 25, respectively, along with the sample. Figure 4 The system includes a laser source 21 or other LED source, a first beam expander 22, a second beam expander 23, a first microscope lens 24, a beam splitter 25, a microscope objective 26, a stage 27, a polarizer 28, a second microscope lens 29, a Savart plate 30, a quarter-wave plate 31, and a polarizing camera 32. The laser source offers better coherence, which can make the DIC effect more pronounced, but care must be taken to avoid damaging biological samples.

[0063] It should be noted that there are multiple ways to implement polarization beam splitters and polarization converters. For example, the combination of the Sava plate 30 and the quarter-wave plate 31 used in this embodiment can be replaced by a liquid crystal variable phase delay device (LCVR) or a spatial light modulator (SLM), the latter of which can simultaneously achieve polarization beam splitting and electrically controlled phase shifting, thereby simplifying the optical path structure. Furthermore, the detection device can also be a combination of a rotatable analyzer 17 and a conventional camera 18 as described in Embodiment 3, achieving phase shifting through time-series acquisition.

[0064] The optical path works as follows: After being expanded, the laser beam is reflected by the beam splitter 25 and illuminates the sample surface on the stage 27 through the microscope objective 26. The light reflected from the sample surface passes through the microscope objective 26 again, and is transmitted through the beam splitter 25, sequentially passing through the polarizer 28, the second tube mirror 29, the Sava plate 30 (which acts as a polarization beam splitter), and the quarter-wave plate 31, finally forming an interference image on the polarization camera 32. The phase is then reconstructed by the computer. The specific reconstruction process is described in Example 1 and will not be repeated here.

[0065] Example 3: This embodiment is a variation of Embodiment 1, intended to illustrate other implementations of the polarization detector. For example... Figure 5 As shown, in this variant, a polarization camera 16 is replaced by a combination of a conventional camera 18 and a rotatable analyzer 17. The analyzer 17 is rotated by a controlled motor, stopping sequentially at four angles: 0°, 45°, 90°, and 135°. The conventional camera 18 then exposes and acquires four interference images for each angle. The subsequent phase calculation and reconstruction process is the same as in Example 1. This scheme can also achieve quantitative phase imaging, but it is a "time-sequential phase shift," and the imaging speed is limited by the mechanical rotation and the time required for multiple exposures. It cannot achieve "single-exposure synchronous phase shift" like the polarization camera used in Example 1, and registration errors may occur due to sample movement when imaging dynamic samples. This comparison further highlights the superior imaging speed of the polarization camera scheme in Example 1.

[0066] also, Figure 5 The quarter-wave plate 13 can also be replaced by a liquid crystal variable phase delay device (LCVR) to achieve digital phase shift and avoid phase shift errors caused by mechanical rotation.

[0067] Example 4: Based on the same inventive concept, this invention also provides a differential interferometric phase-contrast imaging method, applied to the differential interferometric phase-contrast imaging system as described in any one of embodiments one, two, and three above, with reference to... Figure 6 As shown, the method includes the following steps: S1. Use the lighting source module to generate illumination light; S2. The illumination light is transmitted or reflected through the sample carried in the sample container, and a magnified image of the sample is formed by the microscopic imaging module. S3. Using the polarization interference module located on the image side of the microscopic imaging module, the beam carrying sample information is sequentially polarized, polarized and split, and polarized and converted, and at least two interference images with different phase shifts are acquired simultaneously in a single exposure. S4. Based on the interference image, a quantitative phase image of the sample is obtained through phase gradient calculation and integral reconstruction algorithm.

[0068] Example 5: Using any one of the imaging systems in Embodiments 1, 2, and 3 of this invention as the core observation module, refer to... Figure 7 As shown, the system is integrated into a culture chamber with precise temperature, humidity, and gas concentration control. Software-controlled image sequence acquisition at specific times and locations with automatic focusing constitutes a complete time-lapse culture device, including: Time difference incubator outer shell 100: Provides a sealed cavity, which serves to insulate against heat, light and provide physical protection, ensuring that the internal environment is not disturbed by the outside world.

[0069] Optical imaging module 200: This module integrates the differential interference phase-contrast imaging system of the present invention. Its illumination source module and polarization interference module are compactly designed and fixed in the optical path of the device; the objective lens 7 of the microscopic imaging module is aligned with the interior of the culture chamber. Under control commands, this module can automatically perform focusing, exposure, and image acquisition.

[0070] Sample stage 300: Located within the culture chamber, it supports and secures one or more plastic culture dishes. This sample stage typically features temperature control to ensure the samples are at their optimal temperature. Furthermore, it can be connected to a displacement mechanism to enable sequential observation of multiple culture dishes or different locations within the same culture dish.

[0071] Environmental control system 400: includes heating device, humidification device and mixed gas, such as CO2, N2 and O2 supply and control module, used to accurately maintain temperature, humidity and gas concentration in the culture chamber to meet the long-term culture needs of biological samples such as embryos and cells.

[0072] Linear slide rail or displacement mechanism 500: used to drive the optical imaging module 200 or the sample stage 300 to perform one-dimensional or multi-dimensional movements, thereby enabling sequential imaging of different culture dishes or multiple preset positions within the same culture dish without turning on the incubator.

[0073] During operation, the environmental control system 400 first establishes and maintains a stable biochemical environment within the culture chamber. Subsequently, the control software drives the displacement mechanism 500 to move the target sample to the imaging position according to preset time intervals, triggering the optical imaging module 200 to execute a differential interferometric phase-contrast imaging procedure as described in Example 4, acquiring a quantitative phase image in a single exposure. This process is repeated cyclically, forming a time-series quantitative phase image dataset.

[0074] In summary, this time-difference culture device successfully combines a rapid, quantitative differential interferometry phase-contrast imaging system compatible with plastic culture dishes with precise environmental control. It can observe cells located in plastic culture dishes, overcoming the limitation of traditional DIC microscopes that are incompatible with plastic culture dishes. It can be used as an imaging observation system in time-difference incubators. Through the acquired quantitative phase image sequence, it can be used to automatically analyze the dynamic changes of parameters such as morphology, volume, and dry weight of embryos or cells, which has significant advantages and significance in assisted reproductive applications.

[0075] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on its differences from other embodiments. Similar or identical parts between embodiments can be referred to interchangeably. For the apparatus disclosed in the embodiments, since they correspond to the methods disclosed in the embodiments, the description is relatively simple; relevant parts can be referred to the method section.

[0076] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A differential interferometric phase-contrast imaging system, characterized in that, Including those arranged sequentially along the optical path: Lighting source module, used to provide lighting light; The microscopic imaging module is used to hold the sample and scan and magnify it for microscopic imaging. A polarization interferometry module is used to perform polarization beam splitting and phase shift modulation on the beam from the microscopic imaging module, and to detect the intensity of the interferometric light carrying sample information; and, A quantitative phase image reconstruction module is used to reconstruct a quantitative phase image of the sample based on the signal detected by the polarization interferometry module. The polarization interference module includes a polarizer, a polarization beam splitter, a polarization converter, and a detector arranged sequentially along the optical path. The polarizer, the polarization beam splitter, and the polarization converter are all located on the image side of the microscopic imaging module. The detector is configured to acquire at least two interferometric images with different phase shifts in a single exposure. The illumination source module includes a light source, a Köhler lens, a field stop, a slit stop, and a condenser lens arranged sequentially along the optical path; the slit stop is disposed on the entrance pupil plane or conjugate plane of the condenser lens, the long side direction of its slit is perpendicular to the shearing direction of the polarizing beam splitter, and the slit width b of the slit stop satisfies the following condition: 　　　　　　　　　　　　　　（1） Where B represents the shear distance of the object plane, f represents the focal length of the condenser lens, and λ represents the wavelength of the light source.

2. The differential interferometric phase-contrast imaging system according to claim 1, characterized in that, The polarization beam splitter consists of a Normask prism, a Wollaston prism, and a Savar plate.

3. The differential interferometric phase-contrast imaging system according to claim 2, characterized in that, The polarization converter is a quarter-wave plate, a liquid crystal variable phase delayer, or a spatial modulator (SLM); when the polarization converter is a quarter-wave plate, its fast axis direction forms a 45° angle with the long axis direction of the polarization beam splitter.

4. The differential interferometric phase-contrast imaging system according to claim 1, characterized in that, The detection device is a polarization camera, whose pixel surface is integrated with a micro-polarization filter array. The micro-polarization filter array contains at least two filter units with different polarization directions, so that the polarization camera can simultaneously acquire at least two interference images corresponding to different polarization directions in a single exposure. The polarization detection directions of the micro-polarization filter array include 0°, 45°, 90° and 135°, enabling the polarization camera to simultaneously acquire four phase-shifted interference images.

5. The differential interferometric phase-contrast imaging system according to claim 1, characterized in that, The detection device includes a rotatable analyzer and an area array detector. By controlling the analyzer to rotate to different angles and cooperating with the area array detector to perform multiple exposures, multiple interferometric images with different phase shifts are acquired in a time sequence.

6. The differential interferometric phase-contrast imaging system according to claim 1, characterized in that, The quantitative phase image reconstruction module is configured to perform the following steps: Acquire at least four phase-shifted interferometric images synchronously acquired by the detector; Based on the phase-shifting algorithm, the phase gradient distribution of the sample is calculated from the phase-shifted interferometric image and then filtered. The phase gradient distribution after filtering is integrated and regularized to reconstruct the quantitative phase distribution of the sample.

7. The differential interferometric phase-contrast imaging system according to claim 6, characterized in that, The step of calculating the phase gradient distribution based on the phase-shifting algorithm includes: when four phase-shifting interferometric images I1, I2, I3, and I4 are acquired, the phase gradient is calculated according to the following formula: 　　　　　　　　　（2） in, The images show the light intensity at analyzer angles of 0°, 45°, 90°, and 135°. The shear distance of the system; The step of integrating and regularizing the filtered phase gradient distribution includes solving the optimization problem using the ADMM-TV algorithm based on total variational regularization. 　　　　　　　（3） 　　　　　（4） in, For phase The x-direction derivative; For phase The y-direction derivative; The phase gradient is calculated based on formula (2). Filtered phase gradient, For phase The total variational regularization term, λ is the regularization parameter; i and j represent the number of rows and columns of the image, respectively.

8. A differential interferometric phase-contrast imaging method, characterized in that, The method applied to the differential interferometric phase-contrast imaging system as described in any one of claims 1-7 includes the following steps: Illumination light is generated using an illumination source module; The illumination light is transmitted or reflected through the sample carried in the sample container, and a magnified image of the sample is formed by the microscopic imaging module. Using the polarization interference module located on the image side of the microscopic imaging module, the beam carrying sample information is sequentially polarized, polarized and split, and polarization converted, and at least two interference images with different phase shifts are acquired simultaneously in a single exposure. Based on the interferometric image, a quantitative phase image of the sample is obtained through phase gradient calculation and integral reconstruction algorithm.

9. A time-lapse culture device, comprising a culture chamber and an environmental control system, characterized in that, It also includes a differential interferometric phase-contrast imaging system as described in any one of claims 1-7, the differential interferometric phase-contrast imaging system being used for sequential quantitative phase imaging of biological samples in sample containers within the culture chamber.

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