Embryonic quantitative phase imaging system and method based on dual liquid variable focus lens

Abstract

The application discloses an embryo quantitative phase imaging system and method based on a double-liquid variable-focus lens, which is arranged in a time-difference incubator and comprises an illumination module, an imaging module and a quantitative phase imaging module arranged in sequence along an optical path; the illumination module comprises an LED light source, an illumination slit modulation structure and a liquid variable-focus condensing system, the liquid variable-focus condensing system dynamically adjusts an illumination numerical aperture by changing a focal length; the imaging module comprises an objective lens, a polarizer, a Nomarski prism, an analyzer and a liquid imaging lens, the polarizer is arranged on an emergent light path of the objective lens, the Nomarski prism is arranged between the polarizer and the analyzer, and the liquid imaging lens is arranged on an emergent light path of the analyzer; and the quantitative phase imaging module comprises a spatial light modulator and an image collector, and the spatial light modulator is used for phase shift modulation of the optical path.

Description

Technical Field

[0001] This invention relates to the field of embryo detection technology, specifically to a quantitative phase imaging system and method for embryos based on dual liquid zoom lenses. Background Technology

[0002] With the rapid development of assisted reproductive technology, embryo culture, as one of its core components, has placed higher demands on imaging accuracy and real-time monitoring capabilities. Traditional embryo culture mainly relies on periodic artificial observation of embryo morphological characteristics, which has drawbacks such as frequent intervention, subjective operation, and large errors, easily causing fluctuations in the culture environment and affecting the quality of embryo development.

[0003] To overcome the aforementioned problems, time-lapse culture systems have gradually become the mainstream technology in assisted reproductive laboratories. This system integrates an imaging module and a temperature-controlled environment, utilizing a CCD camera and image acquisition and analysis system to achieve 24 / 7 dynamic monitoring of embryonic development during the pronuclear, cleavage, morula, and blastocyst stages. It enables undisturbed recording and analysis of the continuous embryonic development process. Embryologists can then extract morphodynamic parameters at multiple time points, improving the efficiency of selecting high-quality embryos.

[0004] However, existing time-lapse culture equipment still has many shortcomings in imaging technology. First, regarding imaging modes, existing equipment generally uses bright-field imaging or phase-contrast imaging. Bright-field imaging is insensitive to changes in the refractive index of transparent biological samples, resulting in blurred cell boundaries and low overall imaging contrast, making it impossible to clearly observe the fine structures of the embryo. While Hoffman phase-contrast technology can improve contrast, it produces halos and artifacts, adversely affecting image quality. Second, regarding imaging dimensions, conventional systems are mostly two-dimensional imaging, unable to capture the structure of the embryo at different depths, leading to incomplete assessment information and a lack of a complete three-dimensional view of the developmental process. Third, regarding container compatibility, existing differential interferometry imaging technology relies on linearly polarized light illumination, which has significant imaging failure problems with plastic culture dishes, usually requiring the use of glass dishes, thus limiting practical applications. Furthermore, in terms of image analysis, the image analysis of existing systems is still mainly qualitative, with many systems relying on the subjective judgment of doctors or artificial intelligence for image recognition. However, due to the low contrast and missing information in the basic imaging data, the accuracy of automatic analysis is limited, lacking quantifiable evidence.

[0005] In 3D imaging, traditional methods involve physically moving the sample stage or objective lens to change the focal plane of the objective lens relative to the sample, acquiring a series of 2D images along the Z-axis, and then reconstructing 3D topography or volume information using image processing algorithms. However, the speed of this method is severely limited by the inertia, response speed, and positioning accuracy of the mechanical displacement platform. The inherent hysteresis, vibration, and wear problems of mechanical motion make the entire 3D data acquisition process time-consuming, making it unsuitable for dynamic 3D observation of live biological samples. Furthermore, prolonged exposure to light can damage embryonic cells. Summary of the Invention

[0006] This invention proposes a quantitative phase imaging system and method for embryos based on dual liquid zoom lenses. By integrating liquid lenses into both the illumination and imaging optical paths, the system utilizes electrical signals to dynamically and precisely control the angle of the illumination beam and the position of the imaging focal plane. This enables optimal contrast imaging and high-speed three-dimensional scanning of multiple samples and in multiple scenarios without the need for mechanical movement. It is particularly suitable for rapid and low-damage dynamic observation of live embryo samples.

[0007] To solve the above-mentioned technical problems, the present invention provides an embryo quantitative phase imaging system based on dual liquid zoom lenses, which is set in a time difference incubator and includes an illumination module, an imaging module and a quantitative phase imaging module arranged sequentially along the optical path; The lighting module includes an LED light source, a lighting slit modulation structure, and a liquid zoom focusing system. The liquid zoom focusing system dynamically adjusts the lighting numerical aperture by changing the focal length. The imaging module includes an objective lens, a polarizer, a Nomarsky prism, an analyzer, and a liquid imaging lens. The polarizer is disposed in the output light path of the objective lens, the Nomarsky prism is disposed between the polarizer and the analyzer, and the liquid imaging lens is disposed in the output light path of the analyzer. The quantitative phase imaging module includes a spatial light modulator and an image acquisition unit, wherein the spatial light modulator is used to perform phase shift modulation on the optical path.

[0008] Preferably, the liquid imaging lens dynamically adjusts the position of the imaging focal plane by changing the driving voltage, thereby achieving high-speed focal plane scanning along the Z-axis.

[0009] Preferably, the transmission axis direction of the analyzer is perpendicular to the transmission axis direction of the polarizer.

[0010] Preferably, the system includes three operating modes: Mode 1: The liquid zoom focusing system zooms in, while the liquid imaging lens has a fixed focal length, for two-dimensional time-series imaging; Mode 2: The liquid zoom focusing system has a fixed focal length, while the liquid imaging lens zooms to perform three-dimensional tomographic scanning. Mode 3: The liquid zoom focusing system switches to a large aperture angle, and the focal length of the liquid imaging lens is fixed and locked to the focal plane where the target structure is located, in order to obtain a high-resolution two-dimensional image of the target structure.

[0011] This invention also provides a quantitative phase imaging method for embryos based on dual liquid zoom lenses, which, based on the above-described system implementation, includes the following steps: Step S1: By adjusting the focal length of the liquid zoom focusing system, the numerical aperture of the illumination is dynamically adjusted to control the imaging contrast and depth of field; Step S2: After the unpolarized light passes through the embryo sample to be imaged, it is converted into linearly polarized light by passing through the objective lens and polarizer in sequence. It is then decomposed into ordinary light and extraordinary light by the Nomaski prism, and finally interfered by the analyzer to form a differential interference image. Step S3: By controlling the driving voltage of the liquid imaging lens, adjust the imaging focal plane to move along the Z-axis and acquire images of different focal planes; Step S4: Phase-shift modulation is performed using a spatial light modulator to obtain multiple interference images at different phase-shift angles; Step S5: Process multiple interferometric images using a phase reconstruction algorithm to obtain a three-dimensional phase image of the embryo sample to be imaged.

[0012] Preferably, in step S1, the numerical aperture of the illumination is dynamically adjusted by changing the focal length f of the liquid zoom focusing system. This allows for the synchronous adjustment of the system's depth of field (DOF) and imaging contrast (C), where the DOF satisfies the following relationship: ; in, The wavelength of the illumination light, The refractive index of the medium, For camera resolution, This represents the system amplification factor.

[0013] Preferably, when the two pronuclei overlap, the method further includes the following step before step S1: Initial images of the embryo sample to be imaged are acquired, and pronuclear parameters, including the first pronuclear radius, are obtained through analysis. Second pronuclear radius Distance from the center of the ball Calculate the prokaryotic overlap depth According to the prokaryotic overlap depth Determine the required system depth of field (DOF) and adjust the focal length of the liquid zoom focusing system.

[0014] Preferably, the prokaryotic overlap depth The calculation expression is: .

[0015] Preferably, the illumination numerical aperture Focal length of liquid zoom focusing system The following relationship exists between them: ; In the formula, The refractive index of the medium, This indicates the slit width of the illumination slit modulation structure.

[0016] Preferably, in step S5, a deconvolution algorithm based on the transmission intensity equation or an optical field reconstruction algorithm is used to perform phase reconstruction to obtain a three-dimensional refractive index distribution map of the embryo sample to be imaged.

[0017] Compared with the prior art, the beneficial effects of the present invention include at least the following: First, this invention achieves a fully electronically controlled intelligent microscopic imaging system by simultaneously using liquid lenses in both the illumination and imaging optical paths. The liquid lens in the illumination path can dynamically adjust the numerical aperture of the illumination, enabling rapid control of imaging contrast and depth of field; the liquid lens in the imaging path can perform high-speed Z-axis scanning, achieving millisecond-level three-dimensional tomographic imaging. The synergy between the two allows the system to dynamically configure the optimal illumination angle for each scanning focal plane, ensuring the acquisition of images with the best signal-to-noise ratio and contrast throughout the entire three-dimensional volume. This is not merely a simple superposition of two functions, but rather a synergistic effect.

[0018] Secondly, this invention utilizes the millisecond-level electrically controlled zoom speed of a liquid lens to replace mechanical movement, increasing the Z-axis scanning speed by several orders of magnitude, enabling the recording of the three-dimensional morphology of the dynamic development process of living cells. Compared to traditional mechanical scanning methods, liquid lens scanning is lag-free, vibration-free, and wear-free, while significantly reducing light damage to embryonic cells, providing a reliable technical guarantee for long-term dynamic observation of living embryos.

[0019] Third, the illumination module and imaging module of the present invention use unpolarized light to illuminate the sample, and the polarizer is set in the output light path of the objective lens rather than in front of the sample. Therefore, when using plastic culture dishes, the depolarization characteristics of the plastic dishes will not cause the imaging to be without differential interference effect. Adaptive differential interference imaging of glass dishes, plastic dishes and other material containers is realized, which greatly expands the application scenarios of the system.

[0020] Fourth, this invention combines quantitative phase imaging technology and uses a spatial light modulator to modulate the phase of the optical path. This not only enables the quantitative acquisition of phase information of embryonic cells under short exposure conditions, but also enables three-dimensional high-resolution imaging based on phase reconstruction. It is suitable for long-term, dynamic, and three-dimensional observation of thick samples such as embryonic cells, and is free from artifact interference, with stronger stereoscopic effect and detail representation.

[0021] Fifth, this invention can adaptively adjust according to the developmental stage of the embryo. During the pronuclear stage, when pronuclear overlap occurs, it automatically calculates the required depth of field and adjusts the focal length of the liquid lens by analyzing pronuclear parameters to ensure clear imaging of the pronuclear boundaries. During the multicellular stage, it automatically switches to a large depth-of-field mode to observe cell morphology and number. This intelligent adaptive adjustment capability significantly improves the accuracy of embryologists and artificial intelligence models in identifying embryonic status and developmental potential, thereby increasing the success rate of embryo culture and implantation rate.

[0022] Sixth, the system imaging process of this invention does not require staining of the sample, effectively avoiding the potential toxicity risk of staining agents to live embryos, ensuring the safety and reliability of imaging, and providing an advanced image acquisition method for assisted reproduction and related medical research. Attached Figure Description

[0023] Figure 1 A schematic diagram of the overall optical path structure of the embryo quantitative phase imaging system based on dual liquid zoom lenses provided in an embodiment of the present invention; Figure 2 This is a schematic diagram illustrating the effect of changes in the focal length of a liquid lens on the illumination angle. Figure 3 This is a schematic diagram showing the geometric relationship between the slit width, the aperture angle of the illumination, and the focal length of the condenser lens; Figure 4 A graph showing the relationship between the focal length of the illumination liquid lens and the system contrast and depth of field; Figure 5 A schematic diagram illustrating the principle of synchronous illumination and focal plane scanning achieved through the coordinated operation of two liquid lenses; Figure 6 This is a diagram comparing the imaging performance with that of a traditional imaging system. Figure 7 This is a schematic diagram illustrating the geometric relationship when two pronuclei overlap. Figure 8 A schematic diagram illustrating the extraction of parameters for AI analysis of prokaryotic images; Figure 9 This is a comparison of the imaging effects after insufficient depth of field and after depth of field matching.

[0024] In the figure: 101-Illumination module; 11-LED light source; 12-Illumination slit modulation structure; 13-Liquid zoom focusing system; 14-Embryo sample to be imaged; 201-Imaging module; 15-Objective lens; 16-Polarizer; 17-Nomarski prism; 18-Analyzer; 19-First reflecting mirror; 20-Liquid imaging lens; 21-Second reflecting mirror; 22-First relay lens; 301-Quantitative phase imaging module; 23-Spatial light modulator; 24-Second relay lens; 25-Tube mirror; 26-CCD camera. Detailed Implementation

[0025] 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 protection scope of the present invention.

[0026] Example 1 like Figure 1 As shown, this embodiment of the invention provides an embryo quantitative phase imaging system based on dual liquid zoom lenses, which is set in a time difference incubator and includes an illumination module 101, an imaging module 201 and a quantitative phase imaging module 301 arranged sequentially along the optical path.

[0027] The illumination module 101 uses an LED light source 11 with a wavelength range of 400nm to 700nm. The emitted light passes through the illumination slit modulation structure 12 and then enters the liquid zoom focusing system 13. The liquid zoom focusing system 13 focuses the illumination beam, and by dynamically adjusting the focal length of the liquid zoom lens, it can dynamically adjust the illumination angle of the light source, thereby achieving dynamic adjustment of the imaging contrast. The modulated beam then illuminates the embryo sample 14 to be imaged.

[0028] The imaging module 201 mainly consists of an objective lens 15, a polarizer 16, a Nomaski prism 17, an analyzer 18, a first reflecting mirror 19, a liquid imaging lens 20, a second reflecting mirror 21, and a first relay lens 22. The polarizer 16 is positioned in the output light path of the objective lens 15, the Nomaski prism 17 is positioned between the polarizer 16 and the analyzer 18, and the liquid imaging lens 20 is positioned in the output light path of the analyzer 18. The transmission axis of the analyzer 18 is perpendicular to the transmission axis of the polarizer 16, with an angle typically of 90°. The liquid imaging lens 20, positioned in the output light path of the analyzer, can dynamically adjust the position of the imaging focal plane by changing the driving voltage, achieving high-speed focal plane scanning along the Z-axis.

[0029] The quantitative phase imaging module 301 mainly consists of a spatial light modulator 23, a second relay lens 24, a tube mirror 25, and a CCD camera 26. The spatial light modulator 23 is used for phase-shift modulation of the optical path, and the CCD camera 26 is used for image acquisition. This embodiment does not elaborate on the auxiliary optical structures such as reflectors and relay lenses set in the optical path propagation; the following details the structural principle of the core part of this invention.

[0030] The imaging principle of this system is based on the constructive and destructive interference properties of light, converting the optical path difference information introduced by the sample into changes in image brightness and contrast. When the illumination beam passes through the embryo sample, an optical path difference is generated due to differences in thickness and refractive index in different regions of the sample. The optical path difference is related to the sample thickness. Related to the refractive index distribution, it can be expressed as: ; in , These represent the refractive indices of different regions in the sample. The wavelength of the illumination light.

[0031] The light beam carrying the sample's optical information enters the polarizer after passing through the objective lens, where it is converted into linearly polarized light according to Malus's law. Assume the transmission axis of the polarizer is aligned with... The included angle of the axis is The incident light intensity is The intensity of the emitted light is... The expression is: ; Linearly polarized light, after passing through a Nomarski prism, is decomposed into ordinary (o) and extraordinary (e) rays due to birefringence. The polarization directions of the o and e rays are perpendicular to each other, and their Jones vectors are respectively [ ]and[ The o-beam and e-beam then pass through an analyzer. The angle between the analyzer's transmission axis and the polarizer's transmission axis is typically 90°. According to Jones matrix theory, the Jones vectors of the two beams after passing through the analyzer need to be transformed accordingly. Let the analyzer's Jones matrix be P, and the Jones vectors of the o-beam and e-beam after passing through the analyzer be P[...]. ] and P[ After polarization analysis, the two beams have a constant phase difference, the same polarization direction, and the same vibration frequency, satisfying the conditions for interference. The intensity I of the light after interference can be calculated by the square of the Jones vector mode, according to the principle of interference: ; Expanding the above formula, we get: ; in and The light intensities and optical path differences of the o-ray and e-ray are respectively. The change in phase information directly affects the numerical change in interference intensity, thus realizing the transformation of the phase information of the sample light into the brightness and contrast changes of the interference image, which achieves the relief three-dimensional effect observed by the camera.

[0032] It is worth emphasizing that this system differs significantly from conventional differential interferometry (DIC) microscopes: this system does not use linearly polarized light to illuminate the sample, but rather unpolarized light, with the polarization analysis device positioned in the optical path behind the objective lens. Therefore, when using plastic culture dishes, the depolarization properties of the plastic material do not cause a loss of differential interferometry imaging, enabling adaptive differential interferometry imaging of glass dishes, plastic dishes, and other containers.

[0033] system imaging contrast It is mainly related to the optical path difference, which consists of two parts: one part is the optical path difference introduced by the sample itself, and the other part is the shear distance of the Nomarsky prism. and the aperture angle of illumination A joint decision.

[0034] When the optical path difference between two beams is an integer multiple of the wavelength, they are in phase and their amplitudes superimpose most strongly. ; When the optical path difference is an odd multiple of half the wavelength, the phases are opposite and the amplitudes cancel each other out. ; The system uses a Nomaski prism of specific specifications with a fixed shear distance. Therefore, the dynamic adjustment of the system's contrast is mainly achieved by changing the illumination aperture angle. This is achieved by the optical path difference introduced by the prism. It can be represented as: ; Imaging contrast The relationship with lighting parameters can be expressed as: ; The traditional method for adjusting the illumination aperture angle is to use a variable aperture condenser lens, which mechanically opens and closes the condenser's aperture stop to change the aperture angle of the illumination beam. However, this mechanical adjustment method has obvious drawbacks: slow response speed, unable to achieve rapid dynamic adjustment; mechanical wear, affecting service life and reliability; limited adjustment accuracy, and difficulty in achieving automated and programmed control.

[0035] This invention integrates a liquid lens into the condenser lens position of the illumination optical path, using electrical signals to dynamically and precisely control the angle of the illumination beam, achieving optimal contrast imaging for multiple samples and multiple scenes without mechanical movement. For example... Figure 2 As shown, Figure 2 The image on the right shows the liquid lens with an initial focal length of 30mm. The focal length of the liquid lens can be rapidly changed by controlling it with an electrical signal, thereby regulating the angle of the illumination light. Figure 2 The image on the left shows the situation when the focal length of the liquid lens is 25mm. It is clearly visible that as the focal length decreases, the angle of the illumination light gradually increases, thereby increasing the numerical aperture (NA) and improving contrast. Using this principle, contrast can be dynamically controlled throughout embryonic development, ensuring optimal imaging at different stages. For example, during the pronuclear stage, high-contrast imaging is needed to observe the number of pronuclei and the state of the nucleoli. At this stage, the focal length of the liquid lens needs to be decreased to increase the numerical aperture. As the embryo develops into the multicellular stage, a greater depth of field is required to observe the number and state of cells, thus better determining the embryonic developmental status. At this stage, the focal length of the liquid lens needs to be increased to decrease the numerical aperture. Traditional time-lapse incubators cannot adjust illumination parameters or rely on mechanical structures for adjustment, resulting in slow imaging speeds, low adjustment precision, and exposing embryonic cells to higher doses of light, negatively impacting embryonic development.

[0036] When the illumination slit is located at the front focal plane of the condenser lens and Kohler illumination is used, the slit width... Illumination aperture angle With the focal length of the condenser lens The following relationships exist: Figure 3 As shown.

[0037] ; The focal length change of the liquid lens in the lighting module alters the aperture angle of the illumination, directly affecting the numerical aperture of the illumination. : ; in The refractive index of the medium, The slit width of the illumination slit modulation structure, This is the focal length of the liquid lens.

[0038] Numerical aperture of the objective lens It will directly affect the resolution of the image. Depth of field Based on the Abbe diffraction limit and according to the Rayleigh criterion, the system resolution... It can be represented as: ; System depth of field It can be represented as: ; in ρ is the refractive index of the medium, which is 1 in air; For the camera's resolution; This represents the system's magnification factor.

[0039] When the system's illumination numerical aperture Smaller than the numerical aperture of the objective lens At that time, the imaging depth of the system is mainly affected by the numerical aperture of the illumination. Therefore: ; Will Substituting the expression, we can obtain the depth of field. With the focal length of the liquid lens Relationship: ; like Figure 4 As shown, the numerical aperture of the system illumination changes with the focal length of the liquid illumination lens. The depth of field of the system will also change accordingly. and imaging contrast The contrast and depth of field change synchronously. When the focal length decreases, the illumination numerical aperture increases, improving contrast but decreasing depth of field; when the focal length increases, the illumination numerical aperture decreases, decreasing contrast but increasing depth of field. This characteristic allows the system to dynamically balance contrast and depth of field according to different imaging requirements.

[0040] Traditional 3D imaging methods acquire a series of 2D images along the Z-axis by physically moving the sample stage or objective lens to change the focal plane of the objective lens relative to the sample. Then, image processing algorithms reconstruct the 3D topography or volume information. However, the speed of this method is severely limited by the inertia, response speed, and positioning accuracy of the mechanical displacement platform. The inherent hysteresis, vibration, and wear problems of mechanical motion make the entire 3D data acquisition process time-consuming, making it unsuitable for dynamic 3D observation of live biological samples.

[0041] This invention dynamically alters the backplane distance or equivalent optical path length of the entire imaging system by controlling the driving voltage of the liquid imaging lens, thereby enabling the system's imaging focal plane to move rapidly and precisely along the Z-axis within the sample. By applying a pre-set sequence of rapidly changing voltages to the liquid lens, the focal plane of the imaging system can be commanded to perform high-speed reciprocating scans within the sample. Replacing slow mechanical movement with the millisecond-level electrically controlled zoom speed of the liquid lens, the Z-axis scanning speed can be increased by several orders of magnitude, enabling the recording of the three-dimensional morphology of dynamic live cell development processes while significantly reducing photodamage to embryonic cells.

[0042] like Figure 5 As shown, this invention utilizes liquid lenses simultaneously in both the illumination path (as a condenser lens) and the imaging path, achieving a fully electronically controlled intelligent microscopic imaging system. While the imaging path liquid lens alone can perform Z-axis scanning, the illumination conditions for each focal plane are fixed. This invention allows for synchronized and coordinated scanning of the illumination angle (controlled by the illumination path liquid lens) and the imaging focal plane (controlled by the imaging path liquid lens). The optimal illumination angle differs at different depths of the sample: shallower layers may require smaller angles for high contrast, while deeper layers require larger angles to penetrate the scattering medium. Through the linkage of the two liquid lenses, the system can dynamically configure the optimal illumination angle for each scanning focal plane, ensuring the acquisition of images with the best signal-to-noise ratio and contrast throughout the entire three-dimensional volume.

[0043] Quantitative phase imaging is an optical imaging technique that obtains the microstructure and physical properties of a sample by measuring the phase change of a light wave as it passes through the sample. This system utilizes a spatial light modulator to phase-modulate a reference light. Assume the complex amplitude of the reference light is... : ; in As a reference light amplitude intensity, The imaginary unit, For the spatial phase of the reference light, , Let be the coordinates of the light in space. By writing a specific phase modulation pattern into the spatial light modulator 23, the phase of the reference light can be modulated to . The modulated reference optical complex amplitude is: ; The modulated reference light intensity is: ; This system employs phase-shift interferometry (PSI) to extract the phase information of the sample. By varying the phase modulation amount of the spatial light modulator 23, multiple interference images with different phase shifts are obtained. These images are then processed using phase-shift algorithms, such as three-step or four-step phase-shift algorithms, to achieve quantitative measurement of the sample's phase information. By measuring the phase data at different layers of the sample, complete original information can be provided for 3D reconstruction. Taking the four-step phase-shift algorithm as an example, the reference light undergoes four phase shifts, with the shift amounts being... , , , Four interference images were obtained, with corresponding interference light intensities of: , , , The phase difference of the sample can be solved using the arctangent function. : ; By utilizing interference light intensity data under four different phase shifts and through a series of calculations, the phase information of the sample can be extracted, enabling quantitative measurement and imaging of the sample's microstructure and physical properties. The imaging effect is comparable to that of traditional imaging systems. Figure 6 As shown, the left side of the figure shows the imaging results of a conventional imaging system, and the right side shows the imaging results of an embodiment of the present invention.

[0044] In summary, the system of this invention uses a first liquid lens in the illumination optical path to perform rapid and programmed scanning of the aperture angle or angular value of the incident beam, thereby acquiring a series of images of the sample under different illumination angles. Simultaneously, a second liquid lens in the imaging optical path performs high-speed focal plane scanning, ensuring that each set of images at different illumination angles covers the three-dimensional spatial structure of the sample.

[0045] The system acquires a massive five-dimensional image dataset containing three-dimensional spatial (X, Y, Z), temporal (T), and illumination angle information. By processing this multi-dimensional data using a dedicated quantitative phase reconstruction algorithm (such as a deconvolution algorithm based on the transmission intensity equation or a light field reconstruction algorithm), the phase delay distribution generated after the light wave passes through the sample can be accurately calculated, and the three-dimensional refractive index distribution map or optical thickness map of the sample can be reconstructed. A significant difference between the illumination module 101 and imaging module 201 of this invention and conventional differential interferometry (DIC) microscopes is that linearly polarized light is no longer used to illuminate the sample. Therefore, when using plastic dishes, the depolarization characteristics of the plastic dishes do not result in a lack of differential interferometry imaging, thus achieving adaptive differential interferometry imaging for glass dishes, plastic dishes, and other containers.

[0046] Dynamic zoom using a liquid lens enables dynamic changes in the illumination aperture, allowing for dynamic adjustment of the illumination NA value and thus dynamic changes in the imaging contrast C. Combined with the liquid lens in the imaging optical path, this achieves ultrafast, dynamically adjustable 3D imaging. The system's contrast can change ultrafastly at different embryonic stages, on the order of milliseconds, ensuring optimal 2D imaging at every stage of embryonic development. Simultaneously, ultrafast tomographic scanning using the liquid lens at the imaging end achieves high-contrast 3D imaging, utilizing the optimal 2D images from each layer for 3D reconstruction. Compared to traditional methods, the ultrafast response of the liquid lens effectively reduces light damage to the embryo. (Traditional 3D imaging methods are time-consuming, cause significant embryonic damage, and cannot adaptively adjust to the embryonic developmental stage, failing to guarantee optimal imaging throughout the entire developmental process.) Imaging sequence: The camera sends a signal to control the LED light source to turn on, and at the same time the liquid lens starts to zoom and position itself to the optimal imaging state. At this time, the illumination liquid lens stops zooming, and the liquid lens at the imaging end starts to zoom, realizing three-dimensional tomographic scanning under the optimal two-dimensional imaging state.

[0047] The system of this invention includes three working modes, which can be flexibly switched according to different observation needs: Mode 1 (2D Time-Series Imaging Mode): The liquid zoom focusing system zooms to use different aperture angles for imaging, while the liquid imaging lens has a fixed focal length, enabling long-term 2D time-series imaging. This mode can obtain optimal 2D imaging data throughout the entire embryonic development process. The system adaptively adjusts illumination parameters according to the embryonic developmental stage, providing optimal imaging results at different developmental stages.

[0048] Mode 2 (3D Tomography Mode): After determining the optimal 2D imaging effect of the system, the liquid zoom focusing system maintains a fixed focal length to keep the illumination angle constant, while the liquid imaging lens performs rapid Z-axis scanning by zooming. This mode can acquire the complete 3D structure of cells within 0.5 seconds for 3D tomography.

[0049] Mode 3 (High-Resolution Detail Analysis Mode): The liquid zoom focusing system switches to a large aperture angle state to obtain high resolution. The focal length of the liquid imaging lens is fixed on a key structure of the cell to obtain a two-dimensional image with the highest resolution, enabling fine observation of the key structure.

[0050] The system's imaging control process is as follows: the camera sends a signal to turn on the LED light source, while the illumination liquid lens begins to zoom and position itself to the optimal imaging state; after the illumination parameters are determined, the liquid lens at the imaging end begins to zoom, achieving three-dimensional tomographic scanning under the optimal two-dimensional imaging state. The entire process is efficiently coordinated through hardware synchronous control.

[0051] The system in this embodiment of the invention possesses intelligent adaptive imaging capabilities, enabling automatic parameter adjustment based on the embryonic developmental state and stage. Taking the observation of the pronuclear stage of the embryo as an example, accurate identification of the number and morphology of pronuclei is particularly important. Figure 7 As shown, when two pronuclei overlap, since the pronuclei have a spherical structure, based on the radius of the first pronucleus... Second pronuclear radius and the distance from the center of the ball The maximum depth of overlap between the two pronuclei can be calculated. : ; When the pronuclei overlap, in order to ensure that the system can achieve optimal imaging of the morphology and structure of the two pronuclei, the imaging depth of field must be guaranteed. Greater than the maximum depth of overlap between two pronuclei In practical use, ensure Just greater than That's enough, but it shouldn't exceed that by too much, because An excessive increase in [something] will reduce the system's imaging resolution and imaging contrast.

[0052] like Figure 8 As shown, after acquiring embryo images in the initial state, the system can obtain relevant parameters of the pronucleus through AI analysis, including the diameter of the pronucleus. , and the distance between the centers of the pronuclei The maximum depth of overlap between two pronuclei can be calculated using the above formula. When the system performs formal time-lapse imaging, the imaging depth of field needs to be dynamically adjusted. To ensure coverage of the maximum overlapping depth This ensures that the boundaries of the pronucleus can be clearly imaged, thus helping AI and doctors to better identify the developmental status of the embryo.

[0053] Taking actual embryo images as an example, AI analysis yielded... , Distance between the centers of the balls The maximum depth of overlap between the two pronuclei can be calculated using the above formula. At this point, the system's depth of field is required. Need to be greater than Only then can the morphology and boundaries of the two pronuclei be clearly observed. However, under standard microscope configuration, using a 20x objective lens with NA 0.45, the system's numerical aperture of illumination is limited. The system's imaging depth of field is limited because it is matched to the objective lens but cannot be dynamically adjusted. Fixed as At this point, optimal imaging of the overlapping area of ​​the pronucleus is not possible, the boundary of the pronucleus will be blurred, and the identification of the embryo's state will be affected.

[0054] This system identifies Need to reach Then, the focal length of the liquid lens will be adjusted directly according to the theoretical curve. In this embodiment, the system rapidly zooms at millisecond speeds to adjust the focal length of the illumination liquid lens. Precisely adjusted to 22mm, at which point the system... Just for It can clearly image the pronucleus. Theoretically, further increasing... It can also improve the system However, according to Figure 4 The contrast curve shown, when At that time, the system contrast Will follow The value decreases rapidly with each increase, so it is not advisable to increase it further. A balance needs to be struck between depth of field and contrast.

[0055] like Figure 9 As shown, the left figure represents the system focal length. , The imaging effect at that time, the right image shows the focal length of the system's liquid lens after AI analysis. Precise zoom to 22mm, The imaging effect at that time. It can be clearly seen that the image on the left cannot analyze the number and boundary of the pronuclei well, while the image on the right can clearly see the boundary of the pronuclei, and can better assess the number and state of the pronuclei.

[0056] The present invention provides a quantitative phase imaging method for embryos based on dual liquid zoom lenses, comprising the following steps: Step S1: By adjusting the focal length of the liquid zoom focusing system, the numerical aperture of the illumination is dynamically adjusted to control the imaging contrast and depth of field. Appropriate illumination parameters are configured according to the observation requirements of the embryo's current developmental stage.

[0057] Step S2: After the unpolarized light passes through the embryo sample to be imaged, it is converted into linearly polarized light by passing through the objective lens and polarizer in sequence. It is then decomposed into two beams of ordinary light and extraordinary light with perpendicular polarization directions by the Nomaski prism. After passing through the analyzer to make the polarization directions of the two beams consistent, they interfere to form a differential interference image.

[0058] Step S3: By controlling the driving voltage of the liquid imaging lens, the imaging focal plane is adjusted to move along the Z-axis, and images of different focal planes are acquired to achieve three-dimensional tomographic scanning.

[0059] Step S4: Phase-shift modulation is performed using a spatial light modulator to obtain multiple interference images at different phase-shift angles. For example, a four-step phase-shift method can be used to obtain the phase shift amount. , , , Four interference images.

[0060] Step S5: Process multiple interferometric images using a phase reconstruction algorithm to obtain a three-dimensional phase image of the embryo sample to be imaged. Phase reconstruction can be performed using a deconvolution algorithm based on the transmission intensity equation or an optical field reconstruction algorithm to obtain a three-dimensional refractive index distribution map of the embryo sample to be imaged.

[0061] During pronuclear stage embryo imaging, the process prior to step S1 may include: acquiring an initial image of the embryo sample to be imaged, and obtaining pronuclear parameters, including the first pronuclear radius, through AI analysis. Second pronuclear radius Distance from the center of the ball Calculate the prokaryotic overlap depth According to the prokaryotic overlap depth Determine the required system depth of field And adjust the focal length of the liquid zoom focusing system. Adjust to an appropriate value to ensure that the depth of field covers the overlapping area of ​​the original core.

[0062] The technical features of the above embodiments can be combined arbitrarily. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described; only preferred embodiments of the present invention are illustrated. The descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the present invention. As long as the combination of these technical features does not contradict each other, it should be considered within the scope of this specification.

[0063] It should be noted that those skilled in the art can make various modifications and improvements without departing from the inventive concept, and these all fall within the scope of protection of this invention. Therefore, the scope of protection of this invention should be determined by the appended claims.

Claims

1. A quantitative phase imaging system for embryos based on dual liquid zoom lenses, set up in a time-difference incubator, characterized in that: It includes an illumination module, an imaging module, and a quantitative phase imaging module arranged sequentially along the optical path; The lighting module includes an LED light source, an illumination slit modulation structure, and a liquid zoom focusing system. The liquid zoom focusing system dynamically adjusts the numerical aperture of the illumination by changing the focal length. The illumination light emitted by the lighting module is unpolarized light. The liquid zoom focusing system uses a liquid zoom lens. The illumination slit modulation structure is located on the front focal plane of the liquid zoom lens, and the lighting module uses Kohler illumination. The liquid zoom lens is positioned in the output light path of the illumination slit modulation structure. The numerical aperture of the illumination... The following relationship exists between the focal length f of the liquid zoom focusing system and the focal length f: ； In the formula, The refractive index of the medium, This indicates the slit width of the illumination slit modulation structure; The imaging module includes an objective lens, a polarizer, a Nomaski prism, an analyzer, a first reflecting mirror, a liquid imaging lens, a second reflecting mirror, and a first relay lens. The polarizer is disposed in the output light path of the objective lens, the Nomaski prism is disposed between the polarizer and the analyzer, the liquid imaging lens is disposed in the output light path of the analyzer via the first reflecting mirror, and the second reflecting mirror and the first relay lens are disposed sequentially in the output light path of the liquid imaging lens. The quantitative phase imaging module includes a spatial light modulator, a second relay lens, a tube mirror, and an image acquisition device. The spatial light modulator is disposed on the output light path of the first relay lens and is used to perform phase shift modulation on the light path. The second relay lens, the tube mirror, and the image acquisition device are sequentially disposed on the output light path of the spatial light modulator. The image acquisition device is a CCD camera. The illumination numerical aperture Smaller than the numerical aperture of the objective lens The shear distance of the Nomaski prism is a fixed value, and the imaging contrast of the system is adjusted by changing the illumination aperture angle through the liquid zoom focusing system. The liquid zoom focusing system and the liquid imaging lens are synchronously controlled and linked through hardware, and are configured as follows: the image acquisition unit sends a signal to control the LED light source to turn on, and at the same time, the liquid zoom focusing system starts to zoom, determines the required system depth of field (DOF) according to the developmental state of the embryo sample to be imaged, and adjusts the focal length of the liquid zoom focusing system to a value corresponding to the required depth of field, thereby positioning the optimal imaging state; the focal length of the liquid zoom focusing system remains fixed, and the liquid imaging lens starts to zoom, moving the imaging focal plane along the Z-axis and acquiring images of different focal planes; while the focal length of the liquid zoom focusing system remains fixed, the spatial light modulator applies different phase shifts to the optical path, and the image acquisition unit acquires multiple interference images with different phase shifts to reconstruct the quantitative phase image of the embryo sample to be imaged.

2. The embryo quantitative phase imaging system based on dual liquid zoom lenses according to claim 1, characterized in that: The liquid imaging lens dynamically adjusts the position of the imaging focal plane by changing the driving voltage, thereby achieving high-speed focal plane scanning along the Z-axis.

3. The embryo quantitative phase imaging system based on dual liquid zoom lenses according to claim 1, characterized in that: The transmission axis of the analyzer is perpendicular to the transmission axis of the polarizer.

4. A quantitative phase imaging method for embryos based on dual liquid zoom lenses, implemented using the system described in any one of claims 1 to 3, characterized in that, Includes the following steps: Step S1: By adjusting the focal length of the liquid zoom focusing system, the numerical aperture of the illumination is dynamically adjusted to control the imaging contrast and depth of field; Step S2: After the unpolarized light passes through the embryo sample to be imaged, it is converted into linearly polarized light by passing through the objective lens and polarizer in sequence. It is then decomposed into ordinary light and extraordinary light by the Nomaski prism, and finally interfered by the analyzer to form a differential interference image. Step S3: By controlling the driving voltage of the liquid imaging lens, adjust the imaging focal plane to move along the Z-axis and acquire images of different focal planes; Step S4: Phase-shift modulation is performed using a spatial light modulator to obtain multiple interference images at different phase-shift angles; Step S5: Process multiple interferometric images using a phase reconstruction algorithm to obtain a three-dimensional phase image of the embryo sample to be imaged; When the two pronuclei overlap, the process before step S1 includes: Initial images of the embryo sample to be imaged are acquired, and pronuclear parameters, including the first pronuclear radius, are obtained through analysis. Second pronuclear radius Distance from the center of the ball Calculate the prokaryotic overlap depth According to the prokaryotic overlap depth Determine the required system depth of field (DOF) and adjust the focal length of the liquid zoom focusing system; The prokaryotic overlap depth The calculation expression is: ； The illumination numerical aperture Focal length of liquid zoom focusing system The following relationship exists between them: ； In the formula, The refractive index of the medium, This indicates the slit width of the illumination slit modulation structure.

5. The method for quantitative phase imaging of embryos based on dual liquid zoom lenses according to claim 4, characterized in that: In step S1, the numerical aperture of the illumination is dynamically adjusted by changing the focal length f of the liquid zoom focusing system. This allows for the synchronous adjustment of the system's depth of field (DOF) and imaging contrast (C), where the DOF satisfies the following relationship: ； in, The wavelength of the illumination light, The refractive index of the medium, For camera resolution, This represents the system magnification factor.

6. The method for quantitative phase imaging of embryos based on dual liquid zoom lenses according to claim 4, characterized in that: In step S5, a deconvolution algorithm or an optical field reconstruction algorithm based on the transmission intensity equation is used to perform phase reconstruction and obtain a three-dimensional refractive index distribution map of the embryo sample to be imaged.

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