A differential interference quantitative phase imaging system, method and cell culture device based on a multi-slit light source

Abstract

This invention discloses a differential interferometric quantitative phase imaging system, method, and cell culture equipment based on a multi-slit light source, belonging to the fields of optical imaging and medical device technology. The system's diffraction illumination source module contains a multi-slit aperture with at least two light-transmitting slits, the center-to-center distance of which satisfies the matching constraint equation. This design enables in-phase interference superposition of the multi-slit illumination light at the sample surface, maintaining high interference contrast while increasing the numerical aperture and light transmittance. The polarization interferometry module uses a Normask prism in conjunction with a polarization camera integrating a micro-polarizer array to acquire four phase-shifted interference images in a single exposure. The absolute quantitative phase distribution of the sample is then calculated by the image reconstruction module. This invention overcomes the physical limitations of traditional single-slit illumination in terms of resolution and contrast, is compatible with clinical plastic culture dishes, and offers advantages such as low phototoxicity, vibration resistance, and rapid quantitative imaging. It can be integrated into cell culture equipment.

Description

Technical Field

[0001] This invention relates to the field of optical imaging and medical device technology, and more specifically to a differential interferometric quantitative phase imaging system, method and cell culture equipment based on a multi-slit light source. Background Technology

[0002] Label-free microscopic imaging modules in cell culture incubators are crucial for acquiring underlying information from cellular biology samples. Because transparent tissues such as embryos and organoids absorb light waves very weakly, specific optical contrast enhancement techniques are necessary to convert the minute changes in refractive index or optical path differences that occur when light waves pass through the sample into light intensity differences that can be recognized by the detector. However, achieving high-resolution, long-term monitoring of transparent cellular biology tissues, especially as progressing towards high-precision quantitative phase analysis, faces a series of extremely demanding and mutually constraining engineering and physical challenges due to the unique closed environment of cell culture incubators. Existing mainstream optical contrast enhancement and microscopic observation techniques suffer from the following insurmountable limitations:

[0003] 1. Qualitative artifacts and extremely high phototoxicity risks of Huffman modulation phase contrast (HMC); Currently, the most commonly used technique in clinical time-lapse embryo incubators and organoid observation is Huffman modulation phase contrast (HMC). While this technique is compatible with widely used porous plastic culture dishes, its spatial amplitude modulation filtering only achieves a "pseudo-3D relief" visual visualization. Due to the limitations of the technology, HMC cannot extract true quantitative phase data such as the absolute thickness, optical path difference, or three-dimensional refractive index of embryos and organoids. Furthermore, its strong spatial amplitude obstruction results in extremely low light energy utilization, producing severe "halo" artifacts not only at the edges of embryonic blastomeres or organoids, hindering boundary determination, but also forcing the system to significantly increase illumination intensity, greatly aggravating the photoradiation dose to fragile cellular biological tissues, and easily inducing phototoxic damage such as developmental delay or arrest.

[0004] 2. Traditional differential interference phase contrast (DIC) is extremely incompatible with clinical plastic culture consumables; Traditional DIC microscopy can provide extremely high-resolution interferometric images, but it is highly dependent on a pure polarized light field. In clinical IVF embryo time-lapse culture and high-throughput organoid drug screening, the large-scale use of disposable plastic multi-well culture plates is unavoidable. The inherent stress birefringence effect of plastic materials severely distorts the polarized light direction, leading to a large amount of stray light and even interference field collapse in traditional DIC images. Therefore, traditional DIC can only use costly, stress-free glass dishes that cannot meet the requirements of high-throughput automated clinical culture, completely eliminating the possibility of them being placed inside clinical cell culture incubators.

[0005] 3. The resolution of existing single-prism differential interferometry (PlasDIC) is limited; To accommodate plastic culture dishes, existing technologies have removed the interference prism at the front of the condenser lens, developing the single-prism PlasDIC technology. However, to maintain the high spatial coherence required for the light source, this technology must use an extremely narrow single slit at the center of the optical axis for illumination. This physical compromise leads to two major technological deadlocks: First, the single slit severely limits the effective numerical aperture of the objective lens, resulting in a lateral resolution far below the theoretical diffraction limit, making it difficult to clearly distinguish the tiny organelles inside the embryo; second, if attempts are made to simply widen the slit or arbitrarily add multiple slits to increase the amount of light and resolution within the existing structure, the interference fringes generated by different spatial luminescent points will undergo severe incoherent superposition and mutual cancellation, resulting in a complete loss of image contrast. This traps existing technologies in a physical bottleneck where high resolution, high brightness, and high contrast cannot be simultaneously achieved.

[0006] 4. The high phototoxicity and environmental sensitivity of traditional quantitative phase imaging (QPI); To obtain the true three-dimensional phase distribution of the internal structures of embryos and organoids, quantitative phase analysis must be introduced. However, most existing high-precision quantitative phase imaging techniques rely on mechanical phase-shifting devices, such as rotating analyzers, which require multiple exposures over time, typically 3 to 4 consecutive images to calculate the phase. This multi-frame acquisition scheme has two major drawbacks in cell biology monitoring: First, multiple exposures significantly increase the total radiation dose to the sample from the illumination light, leading to extremely high phototoxicity over long periods of monitoring, causing developmental delays or even death in extremely fragile cell biology embryos or organoids; second, the long acquisition time lag makes it highly susceptible to motion artifacts introduced by the minute displacements of living cells, and it is extremely sensitive to the minute environmental vibrations generated by fans and compressors inside the incubator.

[0007] In summary, the current field of optical microscopy and life science instrumentation urgently needs a novel label-free quantitative imaging system that can be directly integrated into a cell culture incubator. This system must overcome the mutual constraints of existing technologies while simultaneously meeting the following requirements: "fully compatible with low-cost clinical plastic culture dishes; able to overcome the bottlenecks of lateral resolution and interference contrast inherent in traditional central single-slit illumination at the physical level; and able to achieve rapid, single-shot quantitative phase imaging of embryos and organoids without any mechanical moving parts." This will meet the stringent engineering requirements of next-generation intelligent cell culture incubators for extremely low phototoxicity and high-precision three-dimensional quantitative data. Summary of the Invention

[0008] To address the combined shortcomings of existing label-free microscopy techniques in long-term cell biology monitoring in cell culture chambers, such as limited consumables, low resolution, strong phototoxicity, and extreme lack of shock resistance, this invention aims to provide a differential interferometric quantitative phase imaging system, method, and cell culture equipment based on a multi-slit light source.

[0009] 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 quantitative phase imaging system based on a multi-slit light source, comprising: a diffraction illumination source module arranged sequentially along the optical path, used to provide illumination light, and modulate the illumination light to generate a multi-slit illumination beam; The microscopic imaging module is used to carry the sample and collect and relay the light from the sample; The polarization interferometry module is used to perform polarization beam splitting and phase shift modulation on the light from the microscopic imaging module, and to detect the intensity of the interference light carrying sample information; 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 diffraction illumination source module includes an LED lamp, a light-collecting lens, a multi-slit aperture, and a condenser lens arranged sequentially along the optical path; the multi-slit aperture has at least two light-transmitting slits, and the center-to-center distance between the at least two light-transmitting slits is... satisfy:

[0010] In the formula, m is a positive integer. The center wavelength of the illumination, The focal length of the condenser lens. This refers to the physical shearing amount introduced by the polarization interference module at the sample surface.

[0011] In one embodiment, the microscopic imaging module includes: a stage, an objective lens, a reflecting mirror, a first relay lens, and a second relay lens arranged sequentially along the optical path; The first relay lens and the second relay lens constitute a 4f relay system, which is used to relay the back focal plane conjugate of the objective lens to the external space.

[0012] In one embodiment, the polarization interference module includes: a polarizer, a Nomarski prism, a quarter-wave plate, and a polarization camera arranged along the optical path; The polarizer is used to adjust the light from the microscopic imaging module into linearly polarized light in a preset direction; The Nomaski prism, positioned at the pupil conjugate surface of the microscopic imaging module, is used to shear the linearly polarized light into two beams with spatial shearing. And beams of light with orthogonal polarization directions; The fast axis of the quarter-wave plate forms a 45-degree angle with the principal axis of the Nomaski prism. The polarization camera integrates a micro polarizer array in front of its photosensitive chip, which is used to acquire multiple phase-shifted interference intensity images in a single exposure.

[0013] In one embodiment, the micro-polarizer array is arranged periodically in 2x2 pixel basic units, and the light transmission axis directions of the four micro-polarizers in each basic unit are 0°, 45°, 90° and 135°, respectively.

[0014] In one embodiment, the quantitative phase image reconstruction module is configured to perform the following steps: Four interference intensity images I1, I2, I3, and I4 are demultiplexed from a single frame image acquired by the polarization camera. Calculate the phase gradient distribution of the sample using the following formula. x and y represent the two-dimensional spatial coordinates of the sample plane;

[0015] For the phase gradient distribution Frequency domain integral inversion is performed to obtain the absolute quantitative phase distribution φ(x, y) of the sample.

[0016] In one embodiment, the phase gradient distribution Frequency domain integral inversion is performed to obtain the absolute quantitative phase distribution φ(x, y) of the sample; including: Step S1, for the phase gradient distribution Perform a Fast Fourier Transform to obtain its frequency domain representation. ; Step S2: Construct the Tikhonov regularized inverse filter equation in the frequency domain, and calculate the frequency domain spectrum of the absolute quantitative phase distribution of the sample according to the following formula. :

[0017] in, and These represent the spatial frequency components along the shear direction and perpendicular to the shear direction in the spatial frequency domain coordinates, respectively. This is the regularization damping coefficient. The amount of physical shear introduced by the Nomaski prism at the sample surface; This is the operator corresponding to the partial derivative in the spatial domain in the frequency domain; Step S3, for the frequency domain spectrum Performing the inverse fast Fourier transform yields the absolute quantitative phase distribution in the spatial domain. .

[0018] In one embodiment, the at least two light-transmitting slits are symmetrically distributed about the optical axis of the imaging system.

[0019] In one embodiment, the diffraction illumination source module, the microscopic imaging module, and the polarization interference module are arranged coaxially along the optical path to form a transmission imaging optical path. Alternatively, the illumination light generated by the diffraction illumination source module is redirected by the beam splitter to illuminate the sample, and the light reflected from the sample enters the microscopic imaging module again through the beam splitter, forming a reflective imaging optical path.

[0020] In a second aspect, embodiments of the present invention provide a differential interferometric quantitative phase imaging method based on a multi-slit light source, applied to a differential interferometric quantitative phase imaging system based on a multi-slit light source as described in any one of the first aspects, comprising the following steps: Provide illumination light and utilize the conditions. The multi-slit aperture is modulated to generate a multi-slit illumination beam that illuminates the sample, wherein... The center-to-center distance of the multiple slits is m, where m is a positive integer. The center wavelength of the illumination, The focal length of the condenser lens. The amount of physical shear introduced at the sample surface by the polarization interference module; The light from the sample is collected and sheared into two beams with spatial shear Δx and orthogonal polarization directions using a Nomaski prism; Using a quarter-wave plate and a polarization camera in a polarization interferometer module, four interference intensity images with different phase shifts are acquired simultaneously in a single exposure. The quantitative phase distribution of the sample is calculated based on four interferometric intensity images.

[0021] Thirdly, embodiments of the present invention provide a cell culture device, including a culture chamber and a differential interferometric quantitative phase imaging system based on a multi-slit light source as described in any of the first aspects, disposed within the culture chamber. The differential interferometric quantitative phase imaging system based on a multi-slit light source is used to image biological samples in a culture dish placed on a sample stage within the culture chamber.

[0022] As can be seen from the above technical solution, compared with the prior art, the present invention has the following technical effects: 1. Solve the underlying polarization repulsion problem between "high-resolution interferometric imaging" and "high birefringence plastic consumables"; Existing drawbacks: Traditional high-resolution DICs are extremely dependent on pure polarization fields, and they completely fail when encountering the stress birefringence of clinical plastic culture dishes; while existing single-prism PlasDICs can be compatible with plastic dishes, but at the cost of sacrificing the system's light intake and lateral resolution.

[0023] Technical effect: The present invention aims to break through this barrier and achieve high-resolution interferometric images comparable to or even surpassing those of traditional DIC, without losing the high-frequency diffraction information of the system, while being fully compatible with low-cost, high-throughput clinical disposable plastic multi-well plates.

[0024] 2. Overcome the interference cancellation effect in the single prism optical path and break through the physical bottleneck that high resolution and high contrast cannot be achieved simultaneously; Existing drawbacks: In traditional single-prism imaging systems, simply widening the illumination slit or introducing multiple slits to increase light intake and resolution can lead to different phase differences generated by different spatial light sources. This causes interference fringes to incoherently superimpose and cancel each other out on the detector surface, resulting in reduced image contrast.

[0025] Technical Effects: This invention aims to establish a strict mathematical matching relationship between the spacing of the illumination slits, the illumination wavelength, the focal length of the condenser lens, and the physical shearing of the prism through precise spatial light field control, thereby forcing the "in-phase interference superposition" of light fields from different slits. This significantly increases the large-angle oblique illumination and the total light transmittance of the system while ensuring zero loss in coherent interference contrast.

[0026] 3. Overcoming the classic optical contradiction between large numerical aperture and large depth of field; Existing limitations: When observing in vitro fertilized embryos or thick organoids, the lateral resolution and axial depth of field of traditional microscope objectives are mutually constrained. High resolution inevitably leads to extremely shallow depth of field, making it easy for the upper and lower layers of three-dimensional thick samples to become out of focus and blurred, making it impossible to obtain global high-definition tomographic data.

[0027] Technical Effects: This invention utilizes a specific multi-slit aperture design to achieve multi-slit illumination within the sample observation area. By leveraging its non-divergent properties along the optical axis, it overcomes the depth-of-field limitations of the traditional Rayleigh criterion, enabling the system to simultaneously acquire ultra-high lateral resolution and ultra-long, clear depth of field, ensuring sharp global imaging of thick embryos and organoids.

[0028] 4. Solves the pain points of extremely high phototoxicity and environmental vibration sensitivity in traditional quantitative phase imaging (QPI); Existing drawbacks: Traditional techniques for obtaining quantitative three-dimensional refractive index or dry mass distribution must rely on mechanical polarization devices for time-series multi-image exposure, such as 3 to 4 phase-shifted exposures. This results in a large radiation dose, which can easily lead to phototoxic death of embryos or organoids. Furthermore, it is extremely sensitive to the high-frequency micro-vibrations of the incubator compressor and fan, which can easily produce motion artifacts.

[0029] Technical Effects: This invention aims to eliminate all mechanical phase-shifting components and utilize a hardware collaborative architecture with transient spatial multiplexing to extract high-precision encapsulated phase in a single, extremely short exposure. This reduces the light radiation dose to biological samples to less than a quarter of that achieved with traditional methods. Attached Figure Description

[0030] 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.

[0031] Figure 1 This is a schematic diagram of the optical path of the differential interferometric quantitative phase imaging system based on a multi-slit light source provided in Example 1; Figure 2 This is a schematic diagram of the single-slit illumination optical path provided in Embodiment 1; Figure 3 This is a schematic diagram of the interference enhancement optical path achieved by double-slit diffraction provided in Example 1; Figure 4 This is a schematic diagram of the optical path for wide-angle illumination provided in Embodiment 1 using a multi-slit light source. Figure 5 The image shows the phase acquisition and reconstruction of the mouse embryo provided in Example 1; Figure 6 This is a comparison image of the mouse embryo imaging effect provided in Example 1; Figure 7 The optical path diagram of the reflective quantitative phase differential interferometry system based on a multi-slit light source provided in Example 2 is shown below. Figure 8 This is a diagram of the cell culture chamber provided in Example 4. Detailed Implementation

[0032] 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.

[0033] To address the combined technical shortcomings of existing label-free microscopy techniques in long-term cell biology monitoring in cell culture chambers, such as limited consumables, low resolution, high phototoxicity, and extreme lack of shock resistance, this invention provides a differential interferometry quantitative phase imaging system, method, and cell culture chamber equipment based on a multi-slit light source.

[0034] Example 1: like Figure 1 As shown, this embodiment provides a differential interferometric quantitative phase imaging system based on a multi-slit light source, suitable for differential interferometric phase-contrast (DIC) imaging of plastic culture dishes. This system mainly includes a diffraction illumination source module 101, a microscopic imaging module 102, a polarization interferometry module 103, and a quantitative phase image reconstruction module (not shown in the figure, implemented by computer software), arranged sequentially along the optical path. The diffraction illumination source module, the microscopic imaging module, and the polarization interferometry module are arranged coaxially along the optical path, forming a transmission imaging optical path.

[0035] (1) Diffraction illumination source module 101: used to provide illumination light, modulate the illumination light, and generate a multi-slit illumination beam. For example... Figure 1 As shown, the system includes: an LED lamp 1, a focusing lens 2, a multi-slit aperture 3, and a condenser lens 4 arranged sequentially along the optical path. The LED lamp 1 preferably uses a red light band, such as an LED light source in the 625-635nm band, as red light causes less damage during cell or tissue development. The focusing lens 2, multi-slit aperture 3, and condenser lens 4 collect and shape the LEDs, providing Kohler illumination while simultaneously illuminating the multi-slit source. The multi-slit aperture 3 is located on the entrance pupil plane or conjugate plane of the condenser lens 4. Its slits are elongated to limit coherence in the shear direction, improving the contrast of shear interference. The spacing and width of the slits are designed to enhance imaging contrast.

[0036] The multi-slit aperture has at least two light-transmitting slits, which are symmetrically distributed about the optical axis of the imaging system. Theoretically, the more slits the better, but due to the limitation of the condenser lens's aperture, 2-6 slits are generally preferred. Furthermore, the center-to-center distance between the at least two slits is... satisfy:

[0037] In the formula, m is a positive integer. The center wavelength of the illumination, It is a condenser lens with a focal length of 4. This refers to the physical shearing amount introduced at the sample surface by the polarization interferometry module. The value of is related to the parameters of the prism. The shearing amount is generally half of the resolution, and is usually set to 500nm-2um.

[0038] The long side of the multi-slit stop is perpendicular to the shearing direction of the polarizing beam splitter, and the width b of the multi-slit stop satisfies the following condition:

[0039] in, Represents the shear distance on the object plane. λ represents the focal length of the condenser lens, and λ represents the wavelength of the light source.

[0040] (2) Microscopic imaging module 102, used to carry the sample and perform scanning and microscopic magnification imaging of the sample, such as Figure 1 As shown, the system includes a stage 5, an objective lens 6, a mirror 7, a first relay lens 8, and a second relay lens 9 arranged sequentially along the optical path. The first relay lens 8 and the second relay lens 9 form a 4f relay system, used to conjugate the back focal plane (exit pupil) of the objective lens 6 to the external space. The Normask prism 13 of the polarization interference module is precisely placed on this conjugate plane to ensure uniform shearing interference across the entire field of view.

[0041] Objective lens 6, as one of the core optical components of the imaging system, is responsible for collecting the spatial phase distribution of the sample over a long depth of field. Transmitted light waves.

[0042] (3) Polarization interference module 103 is used to polarize and phase-shift the beam from the microscopic imaging module and detect the intensity of the interference light carrying sample information. The polarization interference module mainly includes a reflector 10, a filter 11, a polarizer 12, a Nomaski prism 13, a quarter-wave plate 14, a tube mirror 15 and a polarization camera 16 arranged sequentially along the optical path.

[0043] 1) Polarizer: Placed in the exit optical path of the objective lens. Its function is to project any polarized light wave carrying the sample phase onto a specific linearly polarized state, such as a state relative to the principal axis of the subsequent prism. The direction provides a polarization reference for subsequent constant amplitude shearing interference.

[0044] 2) Nomaski prism: Positioned laterally and obliquely after the polarizer. It is responsible for splitting the polarized linearly polarized beam into two equal-amplitude beams with a small spatial shear along the lateral shear direction (e.g., the x-axis). Two orthogonally linearly polarized light beams with mutually perpendicular vibration directions. The relative phase difference carried by the two beams. The spatial differential phase of the sample along the x-axis is rigorously characterized.

[0045] 3) Quarter-wave plate: Placed downstream of the Nomaski prism, with its fast axis aligned with the principal axis of the Nomaski prism. Angle configuration. According to the de Sénarmont compensation principle, this waveplate will carry the phase difference. The orthogonal linearly polarized pairs are converted into a single linearly polarized light; and the polarization azimuth angle of the emitted linearly polarized light rotates linearly with the sample phase difference, with the rotation angle being... .

[0046] 4) Tube lens and array polarization camera: The tube lens is responsible for converging imaging. The polarization camera, as the terminal device, has a customized micro-polarizer array integrated on the front surface of its photosensitive chip, which acts as a spatially multiplexed analyzer.

[0047] Among them, the micro polarizer array is Pixels are periodically tiled as basic units, including those along the light transmission axis. The micro-polarization channel. When waveplate-encoded linearly polarized light is projected onto the target surface, according to Malus's Law, the transmitted polarization angle is... Interference light intensity after micro-polarizer satisfy:

[0048] In the formula, For background DC component, For interference contrast. Due to the angle of the polarizer. It is amplified in the interference term. By substituting the angles of the four channels, four interferometric intensity images with strictly orthogonal phase shift relationships can be captured simultaneously in a single exposure:

[0049] (4) Quantitative phase image reconstruction module: used to reconstruct the quantitative phase image of the sample based on the signal detected by the polarization interference module.

[0050] Its processing flow includes: 1) From a single frame image acquired by a polarization camera, four interference intensity images I1, I2, I3, and I4 are demultiplexed. 2) After receiving and demultiplexing the original image, the following operations are performed using the algebraic orthogonal cancellation principle:

[0051] The four-quadrant arctangent function must be used here. To ensure high signal-to-noise ratio and unsigned folding extraction of the complete packaged material. Spatial differential phase within the interval The algorithm directly eliminates the spatially non-uniform DC background. With stripe amplitude Dynamic interference.

[0052] 3) Regarding the phase gradient distribution Frequency domain integral inversion is performed to obtain the absolute quantitative phase distribution φ(x, y) of the sample. Specifically, this includes steps S1~S3: Due to the physical shear of the Normask prism in this system The extracted differential phase is less than or close to the system's diffraction limit. The physical jump variables are extremely small, and the vast majority naturally fall into the range of... Within the principal value interval. Therefore, this invention eliminates the need for a two-dimensional phase unwrapping operation that easily introduces artifacts, and directly performs frequency domain inversion on the continuous differential phase: Step S1 (Spatial Frequency Domain Mapping): Use Fast Fourier Transform to map the continuous differential phase. Transform to frequency domain The partial derivatives in the spatial domain correspond to the operator in the frequency domain. .

[0053] Step S2 (Regularized Inverse Filter Reconstruction): To prevent numerical divergence caused by low-frequency defocus noise and to provide a two-dimensional Laplace smoothing constraint in the uncut y-direction, this invention constructs the Tikhonov regularized inverse filter equation in the frequency domain:

[0054] In the above formula, The frequency domain spectrum is the absolute phase; This is the physical shear multiplier, used to ensure that the dimensions and amplitude of the reconstructed phase are strictly accurate; that is, it is the physical shear introduced by the Nomaski prism at the sample surface. For regularization damping terms; in the denominator This is used to suppress high-frequency noise crosstalk in the non-shear direction.

[0055] The above regularization damping term Used to suppress low-frequency defocusing noise in space and prevent the denominator from diverging when divided by zero. The preferred value range is... In actual calculations, the damping term... The value of is determined collaboratively by the system's dynamic signal-to-noise ratio and the spatial feature size of the target under test, and it follows the following adjustment mechanism: negatively correlated with the system's signal-to-noise ratio: the quantitative phase image reconstruction module evaluates in real time the contrast amplitude of the interference fringes generated by the illumination from the preceding multi-slit light source. When aberrations in plastic culture dishes or microporous grooved dishes cause reduced fringe contrast (decreasing the system's effective signal-to-noise ratio), the system automatically adjusts the frequency. The value of this parameter is adjusted to enhance noise suppression capability; conversely, in high signal-to-noise ratio regions with strong stripe contrast, the value is automatically reduced. Negatively correlated with target feature frequency: to prevent excessive spatial smoothing, The value of is adaptively and iteratively converges to a preset lower bound while satisfying global low-frequency noise floor suppression, in order to maximize the preservation of the absolute phase map. High-frequency details in the text.

[0056] Step S3 (Optical Path Mapping): For Performing an inverse Fourier transform will output a smooth, absolute quantitative phase map. The equivalent optical path thickness distribution h(x,y) of the sample can be transformed through the following relationship:

[0057] in, The refractive index of the sample itself. denoted as the refractive index of the background medium.

[0058] The quantitative phase image reconstruction module runs on a computer and realizes quantitative reconstruction of the sample phase based on four differential interference images with different phase shifts in a single frame detected by a polarization camera.

[0059] The core principles of this invention will be further explained in detail below: This system employs a Kohler illumination architecture. Under this architecture, the front focal plane of the condenser lens, i.e., the plane containing the multi-slit aperture, and the focal plane of the objective lens, i.e., the sample observation plane, satisfy a strict Fourier conjugate relationship. For example... Figure 2 The spatial translation of the light source on the front focal plane is physically equivalent to the deflection of the illumination wave vector angle on the sample plane. The focal length of the condenser lens is... The central illumination wavelength is Assume the physical distance from the center of the nth slit on the multi-slit aperture to the optical axis is... The incident angle corresponding to this slit. Paraxial approximation is:

[0060] The transverse spatial wave vector projection of the tilted plane wave on the sample surface for:

[0061] When the above carries wave vector After the illumination light passes through the transparent biological sample, it enters a single interference prism (such as a Nomarski prism) located near the back focal plane of the objective lens. The prism utilizes its birefringence to split the beam into two beams with orthogonal polarization directions and a small physical shearing in the transverse direction. The sub-beam. For the tilted illumination light produced by the nth slit, its own wavefront equation is: x is the spatial coordinate of the shear direction along the x-axis. At a distance of... After shearing, the two beams of light will produce an additional background phase difference at both ends of the shearing distance, which is purely due to the "oblique illumination geometric path".

[0062] At this point, after passing through the analyzer, the intensity distribution of the interference light generated on the detector by the single slit is... Satisfies the two-beam interference equation:

[0063] in, The phase difference caused by the biological sample being tested. The fringe contrast is for single-slit interference; This refers to the total light intensity that the system allows to pass through and reach the detector when illuminating a single slit, i.e., the background light intensity or DC component.

[0064] Because the broadband light source in front of the multi-slit aperture is a spatially incoherent light source, each slit (e.g.) The emitted light beams cannot coherently interfere on the detector surface; their intensities can only be linearly superimposed on a scalar basis. Therefore, the total light intensity received by the area array detector is... Sum the integrals of the light intensity at each slit:

[0065] If the slit spacing is random, or if it is a single slit that is continuously widened, it will cause the offset phase of each emission point to be affected. It becomes a continuously changing or irregular value. During the integration and summation process, each independent cosine term... Severe phase misalignment superposition occurs, causing the cosine oscillation terms (i.e., the AC interference contrast of the image) to cancel each other out, eventually approaching zero. This is the underlying physical reason why existing technologies cannot balance light intake and resolution by simply widening a single slit or arbitrarily adding multiple slits.

[0066] The differential interferometric quantitative phase imaging system based on a multi-slit light source provided in this invention has the following three major advantages: First advantage: The establishment and in-phase superposition of multi-slit matching constraint equations enhance contrast. To maintain optimal interference contrast even with incoherent superposition of multiple slits, this invention imposes an extremely stringent physical boundary condition on the optical path design: all cosine interference terms generated by the slits must be forced to achieve "in-phase superposition." This means that two adjacent slits on the multi-slit aperture ( and The resulting background phase offset difference must be exactly equal to one complete optical cycle, i.e. Integer multiples of:

[0067] Derivation of the above core principles Substituting the expression into the above formula, we get:

[0068] Factor out the common factor and set the physical center distance between adjacent slits as . The equation simplifies to:

[0069] Arrange to go to both sides at the same time Finally, the core optical matching constraint equation of this invention is derived:

[0070] in, It is a positive integer.

[0071] When the slit spacing on the multi-slit aperture When the above matching equation is strictly satisfied, regardless of the number of off-axis slits added, the interference fringes they produce on the detector will achieve 100% precise phase overlap. This design cleverly utilizes the ultra-large off-axis illumination angle brought about by multiple slits, multiplying the lateral Abbe resolution limit and the total light throughput, while significantly reducing phototoxicity; at the same time, it completely eliminates interference cancellation at the physical level, ensuring the high-contrast interference field required for quantitative phase reconstruction, such as... Figure 3 As shown, taking a double seam as an example, when the spacing between the double seams... When changes occur, the interference intensity generated on the detector changes significantly, which in turn causes a significant change in the imaging contrast.

[0072] Second advantage: Wide-angle multi-slit lighting enhances the system's lateral contrast. In traditional single-prism differential interferometry (PlasDIC) imaging systems, to maintain the coherence of the light source and the contrast of the interference fringes, the slits on the spatial multi-slit aperture must be positioned close to the center of the optical axis, and the physical slit width is strictly limited. This configuration results in a decrease in the effective illumination numerical aperture of the condenser lens. Extremely small. According to the Abbe diffraction limit theory, the lateral cutoff frequency and resolution of the system are limited by the sum of the numerical apertures of the objective lens and the condenser lens. Traditional central single-slit illumination seriously wastes the aperture collection capacity of the condenser lens, resulting in low lateral resolution, which is difficult to meet the observation needs of fine organelle structures in cell biology in high-throughput cell culture incubators.

[0073] The quantitative phase differential interferometry system based on a multi-slit light source proposed in this invention is distinctly different from the traditional single-slit system, which is limited by contrast and cannot expand the illumination angle. The system of this invention is configured to push at least two of the transmission slits on the optically matched multi-slit aperture to the far end, off-axis. Because the above constraint equations are satisfied, the symmetrically arranged multi-slits at the far end not only do not cause contrast attenuation, but also achieve in-phase superposition and enhancement of the interference signal. The specific principle is as follows: Figure 4 As shown.

[0074] In single-slit illumination, the maximum aperture angle of the illumination beam is: The effective illumination numerical aperture of the condenser lens Extremely small, because the effective illumination numerical aperture of the condenser lens and Directly related, the specific formula is:

[0075] Here, n represents the refractive index; let the numerical aperture of the objective lens be... When the two light-transmitting slits are located at the edge of the condenser lens, the illumination tilt angle projected onto the sample surface is... The illumination angle is much larger than that of a single slit, resulting in the largest lateral illumination spatial wave vector. :

[0076] By utilizing the large-angle symmetrical oblique illumination provided by multiple slits, the system can transfer higher-frequency, finer diffraction information from the sample into the objective lens's collection aperture. At this point, the system of this invention can transmit the maximum spatial frequency... Significantly expanded to:

[0077] in, As the maximum spatial frequency that the system can receive increases, the imaging resolution of the system will be significantly improved, and the detail resolution will be better. For the largest lateral illumination space wave vector, The numerical aperture of the objective lens. The numerical aperture of the condenser lens. The wavelength is the illumination wavelength.

[0078] The technical solution of this invention completely breaks the technical deadlock of the mutual exclusion between "high interference contrast" and "large-angle illumination" in traditional single-slit interferometric imaging by utilizing matched multiple slits. Without sacrificing any DIC relief contrast, the system multiplies the lateral spatial frequency passband of the optical system, bringing the system's lateral resolution close to the theoretical diffraction limit, thereby achieving ultra-high resolution observation of the fine internal structures of unstained cellular biological samples, such as organelles inside embryonic blastomeres.

[0079] The third advantage: generating multi-slit illumination beams to achieve a dual improvement in image contrast and resolution; To overcome the contradiction between high resolution and high contrast, in a preferred embodiment of the present invention, the optically matched multi-slit aperture is configured with two edge-transmitting slits that are strictly symmetrically distributed about the optical axis. After passing through a condenser lens, the symmetrical multi-slits excite and form a multi-slit light source in the spatial region of the sample surface, illuminating the image. By precisely modulating the spatial frequency of the incident light field, significant optimization of system performance is achieved. Specifically, let the beam along... Propagated along the optical axis, the symmetrical multi-slit projection at the sample surface results in two inclined plane waves with complex amplitude electric fields. and The expression is:

[0080]

[0081] in, and These are the projections of the wave vector in the transverse and axial directions, respectively. Let be the electric field amplitude. Two plane waves interfere and superimpose in phase within the sample region, forming the total spatial light intensity field. The square of the complex amplitude mode:

[0082] Expanding and simplifying using Euler's formula, the intensity distribution equation of the standing wave interference field can be obtained as follows:

[0083] This equation reveals two core physical properties that directly contribute to improvements in contrast and resolution: Contrast Enhancement: The modulation term in the equation This indicates that the sample region was illuminated with a highly contrasting periodic structured fringe. This strongly modulated interference pattern effectively excites high-frequency details in the sample, significantly enhancing the image's feature contrast even under complex background noise. Resolution improvement: The transverse spatial frequency of the interference field is increased from... The decision was made. By optimizing the slit spacing, a large transverse wave vector component can be obtained, thereby converting higher-frequency spatial information of the sample into the cutoff frequency of the optical system. This means that the system can achieve resolution performance exceeding that of conventional illumination while maintaining the advantages of large numerical aperture (NA).

[0084] In addition, it also has the advantage of long depth of field full-field 3D topography restoration through hardware and software synergy: In traditional large-aperture microscopes, thick samples deviating from the extremely shallow focal plane can lead to increased contrast in polarization interference fringes. Because the out-of-focus diffusion decays rapidly.

[0085] This invention utilizes a long depth-of-field beam generated by pre-amplifier spatial modulation to provide through-hole illumination of the sample. Within a depth-of-field range of tens of micrometers, the interference AC contrast of each depth section is [not specified]. The signal-to-noise ratio is consistently maintained at an extremely high level. Therefore, this algorithm maintains uniform phase transfer sensitivity, and combined with vibration-free single-shot acquisition by a polarization camera, it achieves high-fidelity, quantitative physical morphology reconstruction of thick cell biology samples over a wide depth range.

[0086] In this embodiment, imaging tests were performed using mouse embryo samples, such as... Figure 5 The image shows the complete results of quantitative phase reconstruction of an unstained transparent mouse embryo sample using the quantitative phase image reconstruction module provided in Embodiment 1 of the present invention. The entire reconstruction process unfolds layer by layer according to the following hardware and software collaborative steps: First, the terminal area array polarization camera 16 directly captures a frame of the original spatial multiplexing interference intensity image that integrates a micro polarizer array through a single transient ultra-short exposure. Subsequently, the quantitative phase image reconstruction module executes a demultiplexing algorithm to extract from the single frame image four spatially orthogonal phase shifts with four different phase shifts (respectively...). Independent interferometric intensity sub-images ,like Figure 5 As shown in the left sub-figure; Next, the quantitative phase image reconstruction module uses the algebraic orthogonal cancellation principle to calculate the spatial differential phase distribution. Directly eliminates the non-uniform DC background in the entire space. With stripe amplitude Dynamic noise interference; Finally, by constructing a Tikhonov-regularized inverse filter equation in the frequency domain and performing a two-dimensional integral inversion, the output is as follows: Figure 5 Quantitative phase distribution map of mouse embryos marked "Final Phase" .from Figure 5 The final reconstruction result of the "FinalPhase" clearly shows that the reconstructed quantitative phase image eliminates spatial low-frequency defocus noise and phase wrapping artifacts. The boundaries of each blastomere, the zona pellucida structure, and the quantitative optical path difference (or equivalent thickness distribution) of the fine structures inside the cells of the mouse embryo were all imaged with high contrast and high resolution, which fully demonstrates the excellent performance of the single-exposure quantitative morphology restoration of this invention.

[0087] In the embodiments, we also compared the system imaging effects under single-slit and multi-slit light source illumination using mouse embryonic cells, such as... Figure 6 As shown, to further quantify and verify the significant advantages of the present invention's matched multi-slit light source illumination in overcoming the bottleneck of traditional single-slit illumination technology at the physical level, this embodiment conducts a direct and quantitative comparative analysis of the imaging effects of traditional single-slit oblique illumination and the present invention's matched multi-slit large-angle oblique illumination scheme under the same imaging optical path configuration and the same mouse embryo sample. Among them, Figure 6 Part (a) shows the mouse embryo differential interferometry reconstructed image obtained using conventional narrow single-slit illumination close to the optical axis center. Because conventional single-slit illumination severely wastes the aperture-gathering capacity of the condenser lens, the effective illumination numerical aperture of the condenser lens is reduced. The system's transverse spatial cutoff frequency is extremely small, therefore... Figure 6 The fine structures within the embryo in middle (a) are noticeably blurred, and the overall dynamic contrast is low. In contrast, Figure 6 Part (b) demonstrates how the core optical matching constraint equations are fully satisfied using the present invention. Reconstructed images of mouse embryos acquired under symmetrical multi-slit illumination. Because the multi-slits satisfy the constraint equations, they achieve "in-phase interference superposition enhancement" of interference signals on the detector target surface. The large-angle symmetrical oblique illumination provided by the multi-slits completely transfers the higher-frequency fine diffraction information of the sample into the collection aperture of the objective lens.

[0088] Thus, as Figure 6 As shown in section (b), not only is the lateral resolution of the system improved, making the edges of previously blurry, delicate organelles inside the embryo globally sharp, but the physical dynamic contrast and sharp depth of field of the image are also enhanced in a dual manner. To provide a quantitative explanation, this embodiment respectively... Figure 6 Part (a) and Figure 6 In the same mouse embryonic cell region in part (b), a parallel quantitative spatial line was extracted across its cellular structure, as shown by the yellow dashed line in the two sub-images. The dynamic intensity (grayscale) distribution curves of the pixels along the line are embedded in the lower right corner of each sub-image. By quantitatively comparing these two sets of intensity distribution curves along the line, it can be significantly found that the traditional single-slit scheme... Figure 6 The intensity curve in section (a) fluctuates extremely flatly, with a weak modulation index (grayscale gradient change rate), indicating a severe physical loss in its ability to capture high-frequency details; while the multi-slit matching scheme of this invention... Figure 6 The intensity distribution curve corresponding to part (b) shows significantly higher and denser grayscale fluctuation amplitudes and extremely steep edge response characteristics. This analysis result, from the perspective of optical and image quantitative analysis, proves that the present invention can broaden the lateral spatial frequency passband of the optical system and achieve imaging effects that simultaneously achieve high resolution, high brightness, and high contrast without sacrificing any differential interference relief contrast.

[0089] Example 2: This embodiment also provides a differential interferometric quantitative phase imaging system based on a multi-slit light source, which belongs to the reflective imaging system. In this system, the illumination light generated by the diffraction illumination source module is redirected by a beam splitter to illuminate the sample. The light reflected from the sample is then redirected by a beam splitter to enter the microscopic imaging module, thus forming a reflective imaging optical path.

[0090] like Figure 7 As shown, the system includes (1) Diffraction illumination source module: used to provide illumination light, modulate the illumination light, and generate a multi-slit illumination beam; such as Figure 7 As shown, it consists of an LED light 1, a light-collecting lens 21, a multi-slit aperture 3, a first condensing lens 22, and a second condensing lens 24.

[0091] (2) Microscopic imaging module: used to hold the sample and perform scanning and microscopic magnification imaging of the sample. For example... Figure 7 As shown, it includes: stage 5, objective lens 6, beam splitter 23, first relay lens 8 and second relay lens 9; (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 filter 11, a polarizer 12, a Nomaski prism 13, a quarter-wave plate 14, a tube mirror 15, and a polarization camera 16 arranged sequentially along the optical path.

[0092] (4) Quantitative phase image reconstruction module: Same as in Example 1.

[0093] The optical path works as follows: the illumination light generated by the diffraction illumination source module is modulated by a multi-slit aperture to generate an off-axis multi-slit illumination beam with a specific spatial geometric spacing. This multi-slit illumination beam is deflected after being projected onto the beam splitter (beam splitter 23) and then illuminates the sample surface on the stage 5 through the objective lens 6. In this architecture, the objective lens 6 serves as the condenser lens for the entire illumination system, and the focal length of the condenser lens 4 in the aforementioned matching constraint equation is... The equivalent focal length is replaced by that of objective lens 6. After the illumination beam is reflected from the sample surface, its reflected wavefront carries the absolute quantitative phase difference distribution of the two-way reflection caused by the microscopic morphology or local reflectivity gradient of the sample surface. It then passes downward through objective lens 6 and is transmitted through the aforementioned second condenser lens 24 and beam splitter 23 into the microscopic imaging module. The reflected light is relayed to the external space via a 4f relay system consisting of the first relay lens 8 and the second relay lens 9, which conjugates the back focal plane of objective lens 6. The Nomaski prism 13, located at the conjugate pupil plane, splits the linearly polarized reflected beam into two linearly polarized sub-beams with small spatial shear and orthogonal polarization directions along the transverse shear direction. Subsequently, the orthogonal beams pass through the quarter-wave plate 14, which linearly encodes the spatial differential phase gradient into a polarization azimuth rotation of a single linearly polarized light. Finally, the tube lens 15 focuses the light beam onto the photosensitive chip of the polarization camera 16, which integrates a micro polarizer array. It simultaneously captures four reflection interference intensity images with strict orthogonal phase shift relationships in a single exposure, and then uses the quantitative phase image reconstruction module to reconstruct the absolute quantitative phase distribution of the sample.

[0094] Compared to the transmissive architecture of Embodiment 1, the core technological advantage of this embodiment lies in its coaxial reflective optical path architecture, which uses the objective lens as a condenser lens. This significantly improves system integration and greatly reduces the size of the entire module. It eliminates the relative drift of the split optical path axis caused by high-frequency micro-vibrations in the environment from a physical level, resulting in excellent overall optical stability. This makes it extremely suitable for compact integration into the space-constrained cell culture chamber. Furthermore, since the reflected light does not need to penetrate the bottom plate of the culture dish, it not only fundamentally eliminates the stress birefringence aberration interference inherent in porous plastic consumables, but also combines the equal amplitude shearing modulation of the relay conjugate pupil plane to continuously maintain a uniform high dynamic contrast interference field across the entire field of view during long-term, large-field-of-view imaging. This enables high-contrast, long-term, large-field-of-view quantitative three-dimensional dynamic monitoring of live cell biological samples in the culture chamber.

[0095] Example 3: Based on the same inventive concept, this invention also provides a differential interferometric quantitative phase imaging method based on a multi-slit light source, applied to the differential interferometric quantitative phase imaging system based on a multi-slit light source as described in any of the above embodiments one and two. The method includes the following steps: S1. Provide illumination light and utilize the conditions that are met. The multi-slit aperture is modulated to generate a multi-slit illumination beam that illuminates the sample, wherein... The center-to-center distance of the multiple slits is m, where m is a positive integer. The center wavelength of the illumination, The focal length of the condenser lens. The amount of physical shear introduced at the sample surface by the polarization interference module; S2. Collect the light from the sample and use the Nomaski prism to cut it into two beams with spatial shearing Δx and orthogonal polarization directions; S3. Using the quarter-wave plate and polarization camera of the polarization interferometer module, four interference intensity images with different phase shifts are acquired simultaneously in a single exposure. S4. Based on the four interference intensity images, calculate the quantitative phase distribution of the sample.

[0096] Example 4: Using either the imaging system of Embodiments 1 or 2 of the present invention as the core observation module, refer to... Figure 8 As shown, this embodiment of the invention also provides a cell culture device. This device integrates the imaging system described in the foregoing embodiments as a core observation module into a cell culture chamber with precise temperature, humidity, and gas concentration control functions. Specifically, the device mainly includes a cell culture chamber shell 110, an optical module 111, and a sample stage 112 for carrying the culture medium.

[0097] Cell culture incubator outer shell 110: 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.

[0098] Optical module 111: Integrates the differential interferometric quantitative phase imaging system of this invention. Its diffraction illumination source module and polarization interferometry module are compactly designed and fixed in the optical path of the device; the objective lens of the microscopic imaging module is aimed at the inside of the culture chamber. Under control commands, this module automatically focuses and acquires image sequences at multiple points in the multi-well culture plate on the sample stage 112 at preset time intervals, realizing long-term, automated, and quantitative monitoring of the embryonic or organoid development process.

[0099] Sample stage 112: Located within the culture chamber, it is used to support and hold one or more plastic culture dishes. This sample stage typically has a temperature control function to ensure that the samples are at the optimal temperature.

[0100] During system operation, the device is equipped with control software to drive the optical module 111 and related motion mechanisms to perform timed, multi-site, and autofocused image sequence acquisition, thereby forming a complete automated dynamic cultivation and observation system.

[0101] Addressing the shortcomings of existing technologies, this device enables high-quality direct observation of biological cells placed in conventional plastic culture dishes, effectively overcoming the technical barrier of traditional differential interferometry (DIC) microscopes being incompatible with plastic culture dishes due to the birefringence effect of the plastic material. Based on the quantitative phase image sequence acquired by optical module 111, the system can be further used to automatically extract and analyze the dynamic changes in morphological characteristics, volume, cell dry weight, and other parameters of embryos or cells during development. This system has significant technical advantages and application value in embryo quality assessment and non-destructive monitoring of live cells in the field of assisted reproduction.

[0102] 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.

[0103] 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 quantitative phase imaging system based on a multi-slit light source, characterized in that, Including those arranged sequentially along the optical path: A diffraction illumination source module is used to provide illumination light and modulate the illumination light to generate a multi-slit illumination beam; The microscopic imaging module is used to carry the sample and collect and relay the light from the sample; The polarization interferometry module is used to perform polarization beam splitting and phase shift modulation on the light from the microscopic imaging module, and to detect the intensity of the interference light carrying sample information; 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 diffraction illumination source module includes an LED lamp, a light-collecting lens, a multi-slit aperture, and a condenser lens arranged sequentially along the optical path; the multi-slit aperture has at least two light-transmitting slits, and the center-to-center distance between the at least two light-transmitting slits is... satisfy: In the formula, m is a positive integer. The center wavelength of the illumination, The focal length of the condenser lens. This refers to the physical shearing amount introduced by the polarization interference module at the sample surface.

2. The imaging system according to claim 1, characterized in that, The microscopic imaging module includes: a stage, an objective lens, a reflecting mirror, a first relay lens, and a second relay lens arranged sequentially along the optical path; The first relay lens and the second relay lens constitute a 4f relay system, which is used to relay the back focal plane conjugate of the objective lens to the external space.

3. The imaging system according to claim 1, characterized in that, The polarization interference module includes: a polarizer, a Nomarski prism, a quarter-wave plate, and a polarization camera arranged along the optical path; The polarizer is used to adjust the light from the microscopic imaging module into linearly polarized light in a preset direction; The Nomaski prism, positioned at the pupil conjugate surface of the microscopic imaging module, is used to shear the linearly polarized light into two beams with spatial shearing. And beams of light with orthogonal polarization directions; The fast axis of the quarter-wave plate forms a 45-degree angle with the principal axis of the Nomaski prism. The polarization camera integrates a micro polarizer array in front of its photosensitive chip, which is used to acquire multiple phase-shifted interference intensity images in a single exposure.

4. The imaging system according to claim 3, characterized in that, The micro-polarizer array is arranged periodically in 2x2 pixel basic units, and the transmission axis directions of the four micro-polarizers in each basic unit are 0°, 45°, 90° and 135° respectively.

5. The imaging system according to claim 3, characterized in that, The quantitative phase image reconstruction module is configured to perform the following steps: Four interference intensity images I1, I2, I3, and I4 are demultiplexed from a single frame image acquired by the polarization camera. Calculate the phase gradient distribution of the sample using the following formula. x and y represent the two-dimensional spatial coordinates of the sample plane; For the phase gradient distribution Frequency domain integral inversion is performed to obtain the absolute quantitative phase distribution φ(x, y) of the sample.

6. The imaging system according to claim 5, characterized in that, For the phase gradient distribution Frequency domain integral inversion is performed to obtain the absolute quantitative phase distribution φ(x, y) of the sample; include: Step S1, for the phase gradient distribution Perform a Fast Fourier Transform to obtain its frequency domain representation. ; Step S2: Construct the Tikhonov regularized inverse filter equation in the frequency domain, and calculate the frequency domain spectrum of the absolute quantitative phase distribution of the sample according to the following formula. : in, and These represent the spatial frequency components along the shear direction and perpendicular to the shear direction in the spatial frequency domain coordinates, respectively. This is the regularization damping coefficient. The amount of physical shear introduced by the Nomaski prism at the sample surface; This is the operator corresponding to the partial derivative in the spatial domain in the frequency domain; Step S3, for the frequency domain spectrum Performing the inverse fast Fourier transform yields the absolute quantitative phase distribution in the spatial domain. .

7. The imaging system according to claim 1, characterized in that, The at least two light-transmitting slits are symmetrically distributed about the optical axis of the imaging system.

8. The imaging system according to any one of claims 1-7, characterized in that, The diffraction illumination source module, the microscopic imaging module, and the polarization interference module are arranged coaxially along the optical path to form a transmission imaging optical path. Alternatively, the illumination light generated by the diffraction illumination source module is redirected by the beam splitter to illuminate the sample, and the light reflected from the sample enters the microscopic imaging module again through the beam splitter, forming a reflective imaging optical path.

9. A quantitative phase imaging method based on multi-slit light source using differential interferometry, characterized in that, The differential interferometric quantitative phase imaging system based on a multi-slit light source, as described in any one of claims 1-8, comprises the following steps: Provide illumination light and utilize the conditions. The multi-slit aperture is modulated to generate a multi-slit illumination beam that illuminates the sample, wherein... The center-to-center distance of the multiple slits is m, where m is a positive integer. The center wavelength of the illumination, The focal length of the condenser lens. The amount of physical shear introduced at the sample surface by the polarization interference module; The light from the sample is collected and sheared into two beams with spatial shear Δx and orthogonal polarization directions using a Nomaski prism; Using a quarter-wave plate and a polarization camera in a polarization interferometer module, four interference intensity images with different phase shifts are acquired simultaneously in a single exposure. The quantitative phase distribution of the sample is calculated based on four interferometric intensity images.

10. A cell culture device, characterized in that, The invention includes a culture chamber and a differential interferometric quantitative phase imaging system based on a multi-slit light source as described in any one of claims 1-8, disposed within the culture chamber. The differential interferometric quantitative phase imaging system based on a multi-slit light source is used to image biological samples in a culture dish placed on a sample stage within the culture chamber.

Images