Embryo adaptive quantitative phase imaging system and method based on phase shift real-time feedback

Abstract

This invention discloses an embryo adaptive quantitative phase imaging system and method based on real-time phase shift feedback. The system includes an illumination module, an imaging module, and a quantitative phase imaging module arranged sequentially along the optical path. The Nomaski prism in the imaging module is equipped with a mechanical transmission device, enabling high-precision displacement along the shear direction. The quantitative phase imaging module measures the sample phase difference using a spatial light modulator and a four-step phase shift algorithm. Based on the measurement results, the system calculates the required prism displacement and dynamically adjusts the prism position to maintain the total system phase difference at an optimal state of π / 2. This invention solves the problem that traditional differential interferometry systems cannot adapt to dynamic changes in sample phase due to fixed prism positions, achieving real-time optimization of imaging contrast, and is particularly suitable for dynamic observation of embryonic development.

Description

Technical Field

[0001] This invention relates to the field of embryo image acquisition technology, specifically to an embryo adaptive quantitative phase imaging system and method based on real-time phase shift feedback. Background Technology

[0002] Differential interferometry (DIC) contrast microscopy is an optical imaging method based on the principles of constructive and destructive interference of light. Its core lies in converting the spatial derivative of the optical path difference (i.e., the phase gradient) of the sample into a difference in light intensity, thereby forming an image with contrasting brightness. When a light beam passes through the sample, differences in thickness and refractive index in different regions of the sample cause variations in the optical path difference. If the optical path difference changes significantly at a certain point in the sample, such as in the cell edge region, the phase difference between the two beams changes drastically with position, producing a high-contrast transition effect after interference. Conversely, in uniform regions, because the optical path difference gradient approaches zero, the contrast decreases accordingly.

[0003] In traditional differential interferometry microscopy systems, the Nomarsky prism at the rear of the objective lens is typically fixed in position, creating a fixed initial phase difference. However, a significant problem arises during actual observation: the phase distribution varies between different samples, and even within the same sample, phase values ​​in different regions can dynamically change. This is particularly evident when observing the physiological activities of biological samples. Because the prism position is fixed, the system's total phase difference cannot be adjusted according to real-time changes in the sample's phase value, thus limiting the range of interference intensity adjustment. When the sample phase change exceeds the optimized range corresponding to the fixed phase difference, the imaging contrast decreases significantly, making it impossible to clearly capture the sample's detailed information, severely impacting the observation results and data accuracy.

[0004] In existing technologies, to improve imaging contrast, operators typically need to manually adjust the prism position or replace prisms with different parameters. This method is not only cumbersome but also cannot achieve real-time dynamic adjustment, especially for dynamically changing biological samples, making it difficult to meet the needs of real-time observation. Therefore, developing a differential interferometric imaging system that can dynamically adjust the prism position based on changes in sample phase value and achieve real-time optimization of the system's total phase difference has become an urgent technical problem to be solved in this field. Summary of the Invention

[0005] This invention proposes an embryo adaptive quantitative phase imaging system and method based on real-time phase shift feedback, in order to solve the technical problem in the prior art where the position of the Normask prism at the back end of the objective lens of the differential interferometer system is fixed and the total phase difference of the system cannot be dynamically adjusted according to the phase change of the sample.

[0006] To address the aforementioned technical problems, this invention provides an embryo adaptive quantitative phase imaging system based on real-time phase shift feedback, comprising 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 lighting slit modulation structure, and a focusing system. The light emitted by the LED light source passes through the lighting slit modulation structure and the focusing system in sequence before illuminating the embryo sample to be imaged. The imaging module includes an objective lens, a polarizer, a Nomaski prism, an analyzer, and a transmission device. The transmission device is connected to the Nomaski prism and drives the Nomaski prism to move along the shear direction to adjust the system bias phase difference. The quantitative phase imaging module includes a spatial light modulator, a second relay lens, a tube mirror, and an image acquisition device.

[0007] Preferably, the transmission device is an electric drive device, which is used to achieve high-precision displacement control of the Nomaski prism.

[0008] Preferably, the imaging module further includes a first reflecting mirror, a first relay lens, and a second reflecting mirror, wherein the objective lens, the polarizer, the Nomaski prism, the analyzer, the first reflecting mirror, the first relay lens, and the second reflecting mirror are arranged sequentially along the optical path.

[0009] Preferably, the system bias phase difference introduced when the Normask prism is displaced along the shear direction The following relationship must be satisfied: ; in, The phase difference introduced by the Nomaski prism in its initial position. These are fixed coefficients determined by the parameters of the Nomaski prism. The lateral displacement of the Nomarsky prism; the fixed coefficient satisfy: ; in, The difference between the ordinary and extraordinary refractive indices of the birefringent crystal of the Nomaski prism is given. The wedge angle of the Nomaski prism. The wavelength of the illumination light.

[0010] Preferably, the spatial light modulator is used to phase modulate the reference light and obtain the quantitative phase information of the embryo sample to be imaged through a four-step phase shift algorithm. The spatial light modulator performs four phase shifts on the reference light, with phase shift amounts of 0, π / 2, π, and 3π / 2, respectively.

[0011] This invention also provides an embryo adaptive quantitative phase imaging method based on real-time phase shift feedback, implemented using the aforementioned embryo adaptive quantitative phase imaging system based on real-time phase shift feedback, comprising the following steps: Step S1: Place the embryo sample to be imaged in the imaging system, and measure the sample phase difference introduced by the embryo sample through the quantitative phase imaging module. ; Step S2: Calculate the optimal system offset phase difference based on the optimal imaging contrast condition. This makes the total phase difference of the system satisfy ; Step S3: Based on the optimal system bias phase difference Calculate the required lateral displacement of the Nomaski prism. ; Step S4: Drive the Nomaski prism to move the lateral displacement by the transmission device. To obtain the image with the optimal contrast.

[0012] Preferably, in step S1, the reference light is phase-shifted four times by the spatial light modulator, with phase shifts of 0, π / 2, π, and 3π / 2, respectively, to obtain four interference images with corresponding interference light intensities of π / 2, π / 2, π / 2, and 3π / 2, respectively. , , and The sample phase difference Calculated using the following formula: .

[0013] Preferably, in step S3, the lateral displacement amount Calculated using the following formula: ; in, The phase difference introduced by the Nomaski prism in its initial position. These are fixed coefficients determined by the parameters of the Nomaski prism.

[0014] Preferably, steps S1 to S4 constitute a real-time feedback control loop. When the phase distribution of the embryo sample to be imaged changes, the imaging system repeatedly executes steps S1 to S4 in real time to dynamically adjust the position of the Nomaski prism in order to maintain optimal imaging contrast.

[0015] Preferably, before step S1, an initialization step is included: calibrating the phase difference introduced by the Nomarsky prism in its initial position. And according to the wedge angle of the Nomaski prism The refractive index difference of a birefringent crystal and the wavelength of illumination light Calculate the fixed coefficient .

[0016] The beneficial effects of the present invention include at least the following: This invention, by setting a high-precision transmission device at the Normask prism, can dynamically adjust the prism position according to the real-time changes in the sample phase value, thereby achieving real-time optimization of the total phase difference of the system and keeping the imaging contrast at its best. This solves the problem that traditional differential interferometry systems cannot adapt to phase changes in different samples or different regions of the same sample due to the fixed prism position.

[0017] This invention combines quantitative phase imaging technology, uses a spatial light modulator to modulate the phase of the reference light, and accurately measures the phase difference introduced by the sample through a four-step phase shift algorithm, providing an accurate data basis for calculating the prism displacement and realizing closed-loop control of phase measurement and contrast optimization.

[0018] This invention establishes a precise mathematical relationship between prism displacement and system phase difference. It has the maximum phase sensitivity when the total system phase difference is π / 2, and can quickly calculate the precise displacement required by the prism based on the measured sample phase difference, thus achieving adaptive adjustment.

[0019] The steps of this invention constitute a real-time feedback control loop, which can continuously monitor changes in the sample phase distribution and dynamically adjust the prism position. It is particularly suitable for dynamic observation scenarios where the morphology and structure of embryos undergo continuous changes during development, ensuring the stability of imaging quality during long-term observation. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of the structure of the adaptive differential interferometry phase imaging system provided in an embodiment of the present invention; Figure 2 This is a schematic diagram of the optical principle of the Normask prism. Figure 3 The graph shows the relationship between prism displacement and phase change. Figure 4 The graph shows the relationship between prism displacement and system contrast. Figure 5 This is a flowchart of the adaptive differential interferometry phase imaging system. Figure 6 This is a schematic diagram of the process for an embryo adaptive quantitative phase imaging method based on real-time phase shift feedback.

[0021] In the figure: 101-Illumination module; 201-Imaging module; 301-Quantitative phase imaging module; 11-LED light source; 12-Illumination slit modulation structure; 13-Concentrating system; 14-Embryo sample to be imaged; 15-Objective lens; 16-Polarizer; 17-Nomarski prism; 18-Analyzer; 19-First reflecting mirror; 20-First relay lens; 21-Second reflecting mirror; 22-Spatial light modulator; 23-Second relay lens; 24-Tube lens; 25-Image acquisition device. Detailed Implementation

[0022] 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.

[0023] like Figure 1 As shown, this embodiment of the invention provides an embryo adaptive quantitative phase imaging system based on real-time phase shift feedback. The system includes an illumination module 101, an imaging module 201, and a quantitative phase imaging module 301 arranged sequentially along the optical path. The three modules work together to achieve high-contrast adaptive imaging of embryo samples.

[0024] The illumination module 101 uses an LED light source 11 with a wavelength range of 400-700nm, covering the visible light band to meet different imaging requirements. The light emitted by the LED light source 11 is first spatially modulated by the illumination slit modulation structure 12, and then enters the focusing system 13. The focusing system 13 converges the illumination beam, so that the modulated beam illuminates the embryo sample 14 to be imaged at a suitable angle. This illumination method can provide uniform and stable illumination conditions for subsequent differential interferometry imaging.

[0025] The imaging module 201 is the core component of this invention, mainly composed of an objective lens 15, a polarizer 16, a Nomaski prism 17, an analyzer 18, a first reflecting mirror 19, a first relay lens 20, and a second reflecting mirror 21. These optical elements are arranged sequentially along the optical path. A high-precision transmission device is provided at the Nomaski prism 17, preferably an electrically driven device, which enables high-precision displacement control of the prism via electric means, thereby achieving precise adjustment of the system's offset phase difference.

[0026] The quantitative phase imaging module 301 mainly consists of a spatial light modulator 22, a second relay lens 23, a tube lens 24, and an image acquisition device 25 for acquiring images. In this embodiment, the image acquisition device 25 is a CCD camera. The spatial light modulator 22 is used to phase modulate the reference light, enabling precise control of the reference light phase. This embodiment employs a four-step phase-shift algorithm for quantitative phase measurement. The spatial light modulator 22 performs four phase shifts on the reference light, with phase shift amounts of 0, π / 2, π, and 3π / 2, thereby acquiring four interference images with different phase shifts, and then calculating the quantitative phase information of the sample.

[0027] The imaging principle of this invention is based on constructive and destructive interference of light. When the phase difference between two beams of light is an integer multiple of 2π, constructive interference occurs, and the amplitude superposition reaches its strongest point, i.e. When the phase difference is an odd multiple of π, interference cancellation occurs, and the amplitudes cancel each other out. The contrast C of the system imaging is mainly related to the phase difference. The phase difference of the system consists of two parts: one part is the phase difference introduced by the sample, which varies depending on the structure of the sample itself; the other part is the system bias phase difference introduced by the Normask prism, which is directly related to the position of the prism.

[0028] like Figure 2 As shown, the Nomaski prism 17 is composed of two birefringent crystals cemented together in a wedge shape, with the optical axes of the two crystals perpendicular to each other. When polarized light enters the birefringent crystal, it is decomposed into two beams of light with mutually perpendicular polarization directions: the ordinary ray (o ray) and the extraordinary ray (e ray). The refractive index of the ordinary ray is... The refractive index of unusual light is Two beams of light travel at different speeds in a crystal, resulting in an optical path difference after following the same geometric path. The total optical path difference experienced by the two beams throughout the prism depends on the difference in the distances they travel in the two crystals.

[0029] The basic function of a Nomaski prism is to split an incident light beam into two orthogonally polarized beams with a fixed shear rate. Moving the prism along its shear direction is equivalent to changing the position of the beam passing through the prism. Due to the characteristics of its wedge-shaped structure, different positions correspond to different crystal thickness ratios, resulting in a linear change in the optical path difference between the ordinary and extraordinary rays. This change in optical path difference directly translates into a change in phase difference.

[0030] Let the wedge angle of the prism be... The refractive indices of the ordinary and unusual rays of a birefringent crystal are respectively... and The displacement of the prism along the shear direction is The optical path difference introduced by the prism displacement is: ; The corresponding phase difference is: ; like Figure 3 As shown in the formula, the phase difference of the system is linearly related to the prism displacement. Therefore, the system's bias phase difference can be dynamically controlled by finely moving the prism position. The phase difference introduced by the prism changes with the prism position; when the prism is laterally displaced... When the prism introduces a system bias phase difference, it can be expressed as: ; in, This represents the phase difference introduced by the prism at its initial position. This is a fixed coefficient, determined by the parameters of the prism, and its expression is: ; in, This is the difference between the ordinary and extraordinary refractive indices of a birefringent crystal, i.e. , The wedge angle of the prism. The wavelength of the illumination light.

[0031] In the adaptive differential interferometry microscopy system of this invention, the electric fields of the two orthogonally polarized beams after passing through the sample can be expressed as follows: and Where A is the amplitude. Angular frequency, and These represent the phases of the two beams. The total phase difference between the two beams... for: ; in, The phase difference introduced to the sample, The system bias phase difference (determined by the prism position) is given. After passing through the analyzer, the two beams interfere with each other, and the interference intensity is: ; like Figure 4 As shown, the system contrast exhibits periodic changes as the prism displacement increases. When the total phase difference of the system... When the phase gradient is π / 2, a small change in the phase gradient will lead to the largest change in intensity, thus the system has the greatest phase sensitivity and the best imaging effect.

[0032] In traditional differential interferometry systems, the prism at the rear end of the objective lens is typically positioned fixedly, creating a fixed initial phase difference. However, in actual observations, the phase distribution varies among different samples, and even within the same sample, the phase values ​​in different regions can dynamically change (e.g., physiological activities of embryonic cells). Because the prism position is fixed, the system's total phase difference cannot be adjusted according to real-time changes in the sample's phase value, thus limiting the range of interference intensity adjustment. When the sample phase change exceeds the optimized range corresponding to the fixed phase difference, the imaging contrast significantly decreases, making it impossible to clearly capture the sample's detailed information, severely impacting observation results and data accuracy.

[0033] This invention addresses the aforementioned problems by dynamically adjusting the position of the Normask prism at the rear of the objective lens to optimize the system's interference intensity in real time, thus maintaining optimal imaging contrast at all times. According to the optimal imaging conditions, when the total phase difference satisfies... At that time, the required lateral movement of the prism can be quickly calculated based on the measured sample phase difference. .

[0034] 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 after it passes through the sample. In this system, a spatial light modulator 22 is used to modulate the phase of the reference light, and phase-shift interferometry (PSI) is used to quantitatively measure the phase information of the sample.

[0035] Assume the complex amplitude of the reference light is ,in As a reference light amplitude intensity, The spatial phase of the reference light is defined. By writing a specific phase modulation pattern into the spatial light modulator 22, the phase of the reference light can be modulated to... This embodiment employs a four-step phase-shifting algorithm, performing four phase shifts on the reference light with shift amounts of 0, π / 2, π, and 3π / 2, resulting in four interference images with corresponding interference intensities of π / 2, π / 2, and 3π / 2. , , and The phase difference of the sample can be solved using the arctangent function: ; like Figure 5 and Figure 6 As shown, the embryo adaptive quantitative phase imaging method based on real-time phase shift feedback provided by the present invention includes the following steps: Step S1: Place the embryo sample 14 to be imaged in the imaging system, and measure the sample phase difference introduced by the embryo sample 14 using the quantitative phase imaging module 301. Specifically, the reference light is phase-shifted four times by the spatial light modulator 22, with phase shifts of 0, π / 2, π, and 3π / 2, respectively, to obtain four interference images with different phase shifts. The sample phase difference is then calculated using the formula described above.

[0036] Step S2: Calculate the optimal system offset phase difference based on the best imaging contrast condition. Since the total system phase difference... The system exhibits maximum phase sensitivity and optimal imaging performance; therefore, the optimal system offset phase difference should satisfy: .

[0037] Step S3: Calculate the required lateral displacement Δd of the Nomaski prism 17 based on the optimal system offset phase difference. According to the aforementioned relationship between prism displacement and phase difference, the displacement can be calculated using the following formula: ; Step S4: The calculated lateral displacement is obtained by moving the Nomaski prism 17 via a mechanical transmission device. This allows the system to achieve optimal imaging and obtain images with the best contrast.

[0038] In practical applications, steps S1 to S4 constitute a real-time feedback control loop. When the phase distribution of the embryo sample 14 to be imaged changes, the imaging system repeatedly executes steps S1 to S4 in real time, dynamically adjusting the position of the Nomaski prism 17 to maintain optimal imaging contrast. This real-time feedback mechanism is particularly suitable for dynamic observation scenarios where morphology and structure continuously change during embryonic development.

[0039] Before performing step S1, an initialization step is required: calibrating the phase difference introduced by the Normask prism 17 in its initial position. And according to the wedge angle of the prism The refractive index difference of a birefringent crystal and the wavelength of illumination light Calculate the fixed coefficient Once these parameters are calibrated, they can be used directly in subsequent imaging processes without the need for repeated calibration.

[0040] This invention provides an embryo adaptive quantitative phase imaging system and method based on real-time phase shift feedback. By combining quantitative phase measurement technology with a dynamic prism adjustment mechanism, it achieves high-contrast, high-resolution adaptive imaging of embryo samples. The system can automatically adjust imaging parameters according to real-time changes in the sample phase, ensuring optimal imaging results throughout the observation process. This provides an imaging tool for embryo development research and assisted reproductive medicine.

[0041] 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.

[0042] 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. An embryo adaptive quantitative phase imaging system based on real-time phase shift feedback, characterized in that: It includes an illumination module (101), an imaging module (201), and a quantitative phase imaging module (301) arranged sequentially along the optical path. The lighting module (101) includes an LED light source (11), an lighting slit modulation structure (12) and a focusing system (13). The light emitted by the LED light source (11) passes through the lighting slit modulation structure (12) and the focusing system (13) in sequence and then illuminates the embryo sample (14) to be imaged. The imaging module (201) includes an objective lens (15), a polarizer (16), a Nomaski prism (17), an analyzer (18), and a transmission device. The transmission device is connected to the Nomaski prism (17) and drives the Nomaski prism (17) to move along the shear direction to adjust the system bias phase difference. The quantitative phase imaging module (301) includes a spatial light modulator (22), a second relay lens (23), a tube lens (24), and an image acquisition device (25).

2. The embryo adaptive quantitative phase imaging system based on real-time phase shift feedback according to claim 1, characterized in that: The transmission device is an electric drive device, which is used to achieve high-precision displacement control of the Normask prism (17).

3. The embryo adaptive quantitative phase imaging system based on real-time phase shift feedback according to claim 1, characterized in that: The imaging module (201) further includes a first reflector (19), a first relay lens (20), and a second reflector (21). The objective lens (15), the polarizer (16), the Nomaski prism (17), the analyzer (18), the first reflector (19), the first relay lens (20), and the second reflector (21) are arranged sequentially along the optical path.

4. The embryo adaptive quantitative phase imaging system based on real-time phase shift feedback according to claim 1, characterized in that: The system bias phase difference introduced when the Normask prism (17) is displaced along the shear direction. The following relationship must be satisfied: ； in, The phase difference introduced by the Nomaski prism (17) in its initial position, For fixed coefficients determined by the parameters of the Nomaski prism (17), The lateral displacement of the Nomaski prism (17); the fixed coefficient satisfy: ； in, The difference between the ordinary and extraordinary refractive indices of the birefringent crystal of the Nomaski prism (17) is given. The wedge angle of the Nomaski prism (17) is... The wavelength of the illumination light.

5. The embryo adaptive quantitative phase imaging system based on real-time phase shift feedback according to claim 1, characterized in that: The spatial light modulator (22) is used to perform phase modulation on the reference light. The quantitative phase information of the embryo sample (14) to be imaged is obtained through a four-step phase shift algorithm. The spatial light modulator (22) performs four phase shifts on the reference light, with phase shift amounts of 0, π / 2, π and 3π / 2, respectively.

6. An embryo adaptive quantitative phase imaging method based on real-time phase shift feedback, characterized in that: The embryo adaptive quantitative phase imaging system based on real-time phase shift feedback, as described in any one of claims 1 to 5, is implemented by comprising the following steps: Step S1: Place the embryo sample (14) to be imaged in the imaging system, and measure the sample phase difference introduced by the embryo sample (14) through the quantitative phase imaging module (301). ; Step S2: Calculate the optimal system offset phase difference based on the optimal imaging contrast condition. This makes the total phase difference of the system satisfy ; Step S3: Based on the optimal system bias phase difference Calculate the required lateral displacement of the Nomaski prism (17). ; Step S4: Drive the Nomaski prism (17) to move the lateral displacement by the transmission device. To obtain the image with the optimal contrast.

7. The embryo adaptive quantitative phase imaging method based on real-time phase shift feedback according to claim 6, characterized in that: In step S1, the reference light is phase-shifted four times by the spatial light modulator (22) with phase shifts of 0, π / 2, π, and 3π / 2, respectively, to obtain four interference images with corresponding interference light intensities of π / 2, π / 2, π / 2, and 3π / 2, respectively. , , and The sample phase difference Calculated using the following formula: 。 8. The embryo adaptive quantitative phase imaging method based on real-time phase shift feedback according to claim 6, characterized in that: In step S3, the lateral displacement Calculated using the following formula: ； in, The phase difference introduced by the Nomaski prism (17) in its initial position, The fixed coefficient is determined by the parameters of the Nomaski prism (17).

9. The embryo adaptive quantitative phase imaging method based on real-time phase shift feedback according to claim 6, characterized in that: Steps S1 to S4 constitute a real-time feedback control loop. When the phase distribution of the embryo sample (14) to be imaged changes, the imaging system repeats steps S1 to S4 in real time to dynamically adjust the position of the Nomaski prism (17) to maintain optimal imaging contrast.

10. The embryo adaptive quantitative phase imaging method based on real-time phase shift feedback according to claim 6, characterized in that: The step S1 is preceded by an initialization step: calibrating the phase difference introduced by the Normask prism (17) in its initial position. and according to the wedge angle of the Nomaski prism (17). The refractive index difference of a birefringent crystal and the wavelength of illumination light Calculate the fixed coefficient .

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