A Visual Observation System and Method for the Freeze-Thaw Process of Oocytes and Embryos

Abstract

The application relates to an oocyte and embryo freeze-thaw process visual observation system and a method thereof, the system comprising a low-temperature microscope loaded with a high-speed industrial camera, a high-speed quenching platform and an image processor, and the method comprising the following steps: preparing an oocyte sample; performing a freeze-thaw process and / or a high-speed quenching process loading on the oocyte sample, and collecting video images in real time by the high-speed industrial camera; pre-processing the video images, and then respectively performing intracellular ice formation site judgment, growth type and mode judgment and growth rate calculation, and obtaining corresponding observation results. Compared with the prior art, the application can observe the formation and growth of intracellular and extracellular ice crystals in the oocyte freezing and rewarming process in real time, and process and analyze the ice crystal images in the oocyte and embryo freeze-thaw process.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biomedical image processing, and particularly relates to an oocyte and embryo freeze-thaw process visual observation system and method thereof. BACKGROUND

[0002] Oocyte is an important reproductive cell of female mammals, and oocyte and embryo cryopreservation has become an important means for human assisted reproduction and animal genetic breeding. However, the clinical cryopreservation results show that the oocyte and embryo still have poor embryonic development ability after recovery, which is mainly because the oocyte and embryo will be damaged by intracellular ice crystals during the freeze-thaw process. In order to reduce the damage of intracellular ice to oocyte and embryo, it is necessary to directly observe the ice crystal morphology, ice crystal growth and the influence of ice crystal on the morphology of oocyte and embryo under different cooling rates and different cryoprotectants during the freeze-thaw process.

[0003] At present, the low-temperature microscope is the most commonly used experimental tool for studying intracellular ice behavior, which can observe the changes of cells and their environment during freezing or warming. However, the video camera of the traditional low-temperature microscope usually acquires images at a frame rate of 25 or 30 fps, and the time resolution (i.e. sampling interval) generated is 33 or 40 ms. This technology can only visually show the darkening result of the frozen cells, and cannot capture the growth details of the intracellular ice crystals in the rapidly cooled cells. Karlsson et al. mounted a high-speed camera on the basis of the low-temperature microscope to study the intracellular freezing, successfully captured the darkening images of the intracellular freezing, but the resolution was only 128x128, and the overall imaging effect of the experiment was poor, which could not obtain more information about the growth details of the intracellular ice from the images. Xu et al. quantitatively analyzed the influence of intracellular ice crystals on cell structure according to the light transmission intensity, and found that the cooling rate had a significant effect on the supercooling degree, crystallization time and deformation amount of the cells. However, due to the limitation of the low-temperature microscope used, the maximum cooling rate studied was 90℃ / min, and the initial position, growth rate and morphology of the ice crystals in the cells during the rapid cooling process could not be known. In summary, the existing research methods cannot directly observe the changes of oocyte and embryo during the freeze-thaw process, making it difficult to optimize the freeze-thaw conditions and establish a quality evaluation system and operation specification for freeze-thaw.

[0004] In essence, observing the freeze-thaw process of oocytes and embryos under a cryogenic microscope mainly involves cell pretreatment and loading, high-speed continuous image recording, and image processing and analysis. During cell loading, issues such as cell crushing and defocusing during observation are common. Recording may result in insufficient frame rate or low image resolution, making it impossible to observe the formation and growth of ice crystals inside and outside the oocyte during freeze-thaw. Equipment limitations prevent recording the formation and growth of ice crystals during the ultra-rapid cooling process of oocytes. Furthermore, there is a lack of standardized processing and analysis methods for the acquired freeze-thaw images of oocytes. Summary of the Invention

[0005] The purpose of this invention is to overcome the defects of the prior art by providing a visualization observation system and method for the freezing and thawing process of oocytes and embryos, which can observe the formation and growth of ice crystals inside and outside the cells in real time during the freezing and thawing process of oocytes, and process and analyze the ice crystal images during the freezing and thawing process of oocytes and embryos.

[0006] The objective of this invention can be achieved through the following technical solution: a visualization observation system for the freeze-thaw process of oocytes and embryos, including a low-temperature microscope equipped with a high-speed industrial camera, a high-speed quenching platform and an image processor, wherein the high-speed quenching platform is used to perform high-speed quenching loading on oocyte samples;

[0007] The high-speed industrial camera is used to acquire video images of the freeze-thaw process of oocytes and embryos in real time and transmit them to the image processor;

[0008] The image processor is used to process and analyze video images of the freeze-thaw process of oocytes and embryos to determine the formation sites, growth patterns, and growth rates of intracellular ice.

[0009] Furthermore, the quartz crucible equipped with the cryomicroscope has a C-shaped ring at the bottom to prevent oocytes and embryos from being crushed.

[0010] Furthermore, the carrier of the high-speed quenching platform is a sapphire glass sheet, which is fixed on the sample feeder.

[0011] A method for visually observing the oocyte system and the frozen-thaw process of embryos includes the following steps:

[0012] S1. Prepare oocyte samples;

[0013] S2. For oocyte samples, a freeze-thaw process and / or a high-speed quenching process are applied, and video images are acquired in real time by a high-speed industrial camera;

[0014] S3. The video images are preprocessed, and then the formation sites of intracellular ice, growth types and patterns, and growth rates are determined, and the corresponding observation results are obtained.

[0015] Furthermore, step S2 specifically includes the following steps:

[0016] S21. After transferring the oocyte sample to a quartz crucible using an oocyte transfer needle, cover it with a coverslip.

[0017] S22. Perform a freeze-thaw process with a controllable cooling rate and / or a high-speed quenching process, while a high-speed industrial camera captures the corresponding video images in real time.

[0018] Further, the specific process of step S21 is as follows: First, the tip of the oocyte transfer needle is arranged in a liquid column-air column-liquid column pattern. The siphon effect is used to slow down the flow of liquid and control the oocyte at the tip of the oocyte transfer needle. Then, the oocyte is gently blown into the droplet of liquid in the C-shaped ring at the bottom of the quartz crucible with the smallest volume. After the oocyte sinks to the bottom and stabilizes, a coverslip is placed on top and the bottom surface of the quartz crucible is wiped with a lint-free cloth to prevent residual liquid.

[0019] Furthermore, the high-speed quenching process in step S22 specifically includes:

[0020] Replace the quartz crucible with a sapphire glass plate;

[0021] A layer of solid adhesive is evenly coated inside the steel ring of the sample feeder, and the sapphire glass plate is embedded in the sample feeder to prevent the sapphire glass plate from falling out of the sample feeder during rapid movement.

[0022] The sample feeder was then placed on a constant temperature table to dry overnight. After the solid adhesive had completely solidified, the residual adhesive on the sapphire strip and the sample feeder steel ring was cleaned with anhydrous ethanol.

[0023] Install the prepared sample feeder onto the cold stage, and then connect the cold stage to the Dewar flask containing liquid nitrogen.

[0024] Before cooling the silver block, the sampler containing the oocyte sample was placed on the silver block for focusing, and then the sampler was moved to the temperature column.

[0025] The temperature of the silver block in the center of the cold table was lowered to -180℃, and the temperature column was kept at 20℃.

[0026] By pushing the fast push handle along the X-axis towards the cooling stage, the movement of the fast push handle will pull the sample feeder to move the sample from the heating column to the silver block, thus completing the high-speed quenching loading of the sample.

[0027] Furthermore, the preprocessing of the video images in step S3 is as follows:

[0028] In the Kinovea software, the recorded high-speed video is exported frame by frame. The number of frames after the high-speed industrial camera is triggered is divided by the camera's temporal resolution to determine the elapsed time after triggering. Based on the cooling rate used in each group of experiments, the temperature information corresponding to each image is obtained.

[0029] The complete intracellular ice growth process is extracted from the video captured by the high-speed camera and the images are exported frame by frame. The image of the previous frame before the formation of intracellular ice is selected as a mask.

[0030] Once extracellular ice crystals have formed, the oocyte no longer shifts during the entire process of intracellular ice formation. The Labelme annotation tool is used to segment the oocyte and background image.

[0031] The generated JSON file is converted into an image, and the obtained mask is used to batch process the key image sequences of intracellular ice.

[0032] Furthermore, the process of determining the formation site of intracellular ice in step S3 is specifically as follows:

[0033] To classify the radial distribution of intracellular ice formation sites, the oocyte's projection area was divided into an internal region and a peripheral region. The internal region accounted for 90% of the total projection area of ​​the circle, while the opposite peripheral region accounted for 10% of the total projection area of ​​the circle.

[0034] Since the diameter of oocytes is affected by individual differences, a mask of the corresponding scale is first created when processing data. The mask is then enlarged or reduced proportionally according to different cell sizes, so as to quickly identify the region of intracellular ice formation sites.

[0035] By exporting intracellular ice images from high-speed video recording frame by frame, the formation sites of intracellular ice can be determined based on the division of the oocyte projection region.

[0036] 1) Marginal region type intracellular ice sites, where ice crystal formation sites appear at the boundary of the oocyte;

[0037] 2) Intracellular ice sites of internal region type: The sites where intracellular ice crystals form are close to the cell edge but not in contact with it. The formation site is determined to be in the internal region of the projection by comparing the region boundary with a mask.

[0038] 3) Intracellular ice sites where the edge and interior regions coexist: Ice crystals appear together at the oocyte boundary and in the interior region.

[0039] Furthermore, the process of determining the growth type and pattern in step S3 is specifically as follows:

[0040] By analyzing the exported images of intracellular ice behavior in oocytes, the differences in intracellular ice behavior during the cooling process were observed. Based on the experimental images, two different types of intracellular ice were identified during the oocyte cooling process: "dark" cells and "trembling" cells.

[0041] The “dark-type” cells form intracellular ice 2–7 ms after contact with extracellular ice crystals. The ice crystals grow rapidly, and the cells eventually appear dark black. The ice crystal growth pattern of the “dark-type” cells is “diffusion”. The formation sites of “diffusion” ice crystals are distributed in both the edge and interior regions, and as the temperature decreases, they spread throughout the entire cell in a point-like diffusion manner.

[0042] The "trembling" cells do not immediately produce ice crystals after contact with extracellular ice crystals. Intracellular ice sites are formed 600-900 ms after the extracellular ice crystals are formed. Subsequently, the ice crystals grow rapidly, and the ice crystals at the edge of the cell are lighter in color, while the color of the ice crystals in the central area is the same as that of the dark-colored ice crystals in the "trembling" cells. The ice crystal growth pattern of the "trembling" cells is "aggregation". The formation sites of "aggregation" ice crystals are only distributed in the edge area of ​​the cell. In the early stage of growth, the ice crystals aggregate towards the center of the cell with the entire cell outline as the ice front moving interface. The speed of ice front movement is relatively fast, and then the entire cell is covered.

[0043] Furthermore, the growth rate calculation process in step S3 is specifically as follows:

[0044] Each pixel of the image to be processed is processed individually, a threshold is selected, and its gray value is compared with the set threshold to obtain a binarized image.

[0045] The threshold segmentation function is:

[0046] T = T[x,y,p(x,y),f(x,y)]

[0047] Where x and y are the horizontal and vertical coordinates of the pixel, respectively, p(x,y) is the local feature of the pixel, and f(x,y) is the pixel gray value. The image after thresholding is defined as:

[0048]

[0049] In this model, pixels marked with a grayscale value of 1 correspond to key pixels, i.e., intracellular ice regions, while pixels marked with a grayscale value of 0 correspond to the background. By counting the number of pixels occupied by ice crystals and calculating the intracellular ice formation area, the intracellular ice growth rate is obtained as follows:

[0050]

[0051] Where V is the growth rate of intracellular ice crystals, and A i A represents the instantaneous area of ​​intracellular ice in the current frame image.b t represents the intracellular ice area of ​​the previous frame, and t is the time interval between two adjacent frames.

[0052] Compared with the prior art, the present invention has the following advantages:

[0053] I. This invention utilizes a cryogenic microscope equipped with a high-speed industrial camera, a high-speed quenching platform, and an image processor. The high-speed quenching platform performs high-speed quenching loading on oocyte samples; the high-speed industrial camera acquires real-time video images of the oocyte and embryo freeze-thaw process and transmits them to the image processor; the image processor processes and analyzes these video images to determine the formation sites, growth patterns, and growth rates of intracellular ice. This allows for real-time observation of the formation and growth of intracellular and extracellular ice crystals during oocyte freezing and thawing, and enables the processing and analysis of ice crystal images during the freeze-thaw process of oocytes and embryos.

[0054] Second, this invention introduces a high-speed industrial camera suitable for observing the temperature rise and fall process of oocytes into a low-temperature microscopy system, which can monitor the growth process of ice crystals during the freezing and thawing of oocytes in real time. During the freeze-thaw process of oocytes, not only is high-speed image acquisition achieved, but the experimental data obtained also has reference value for optimizing the optimal low-temperature preservation conditions of oocytes.

[0055] Third, in this invention, a C-shaped ring is placed at the bottom of the quartz crucible equipped with a low-temperature microscope. After the oocyte is loaded into the center of the crucible, a coverslip is placed on top. Because of the C-shaped ring, the coverslip will not press directly on the oocyte, thus ensuring the integrity of the cell and preventing the oocyte and embryo from being crushed.

[0056] IV. Before transferring oocyte samples using the oocyte transfer needle, the tip of the transfer needle is arranged in a liquid-air-liquid column pattern. The siphon effect slows down the liquid flow, keeping the oocyte at the tip of the transfer needle. The oocyte is gently blown into the droplet with the smallest possible volume. After the cell settles to the bottom and stabilizes, a coverslip is placed on top, and the bottom of the crucible is wiped with a lint-free cloth to prevent residual liquid. The reduced liquid volume decreases the drift of the oocyte during the cooling process, thereby effectively solving the problem of microscope defocusing.

[0057] V. This invention modifies the high-speed quenching platform by setting the carrier as a sapphire glass plate and optimizing the high-speed quenching loading process. Before cooling the silver block, the sampler carrying the oocyte sample is placed on the silver block for focusing. Then, the sampler is moved to the temperature column, and the temperature of the silver block at the center of the cooling stage is reduced to -180°C while the temperature column is kept at 20°C. Then, by pushing the quick push handle along the X-axis towards the cooling stage, the movement of the quick push handle pulls the sampler to move the sample from the temperature column to the silver block, thereby completing the high-speed quenching loading of the sample and enabling the visualization observation of ultra-high cooling rates.

[0058] VI. This invention exports and preprocesses video images captured by high-speed industrial cameras. It uses the Labelme annotation tool to segment oocytes and embryonic backgrounds, then converts the generated JSON files into images. The obtained masks are used for batch processing of key intracellular ice image sequences, and each pixel in the image to be processed is processed individually. The preprocessed images are better suited for subsequent analysis of intracellular ice formation sites, growth types and patterns, and growth rates, ensuring the reliability of the observation results.

[0059] VII. This invention proposes an analytical method for the formation sites of intracellular ice in oocytes and embryos. Research has revealed three types of intracellular ice formation sites: edge region type, internal region type, and a combination of edge and internal regions. This invention also proposes a method for determining the growth type and pattern of intracellular ice in oocytes. Research has shown that during intracellular ice growth, oocytes can be classified into "dark cells" and "trembling cells," with intracellular ice growth patterns of "diffusion" and "aggregation," respectively. Furthermore, this invention proposes a method for analyzing the intracellular ice growth rate. Using processed binary oocyte images, grayscale marking is used to identify changes in intracellular ice area, and mathematical relationships are used to obtain the growth rate of intracellular ice at different cooling rates. This provides an effective and reliable visualization observation scheme, enabling comprehensive and accurate observation of the formation and growth of intracellular and extracellular ice crystals during oocyte freezing and thawing. Attached Figure Description

[0060] Figure 1 This is a schematic diagram of the method flow of the present invention;

[0061] Figure 2 A schematic diagram of a C-ring;

[0062] Figure 3 This is a schematic diagram of the egg transfer process;

[0063] Figure 4 This is a schematic diagram of a high-speed quenching stage;

[0064] Figure 5 This is a schematic diagram illustrating the division of the oocyte projection region in the embodiment;

[0065] Figure 6a This is a schematic diagram showing the edge region as the initial site of ice crystal formation in the oocyte in the embodiment;

[0066] Figure 6b This is a schematic diagram showing the internal region as the initial site of ice crystal formation in the oocyte in the embodiment;

[0067] Figure 6c This is a schematic diagram illustrating that the initial formation sites of ice crystals in the oocyte in this embodiment are the coexistence of internal and peripheral regions;

[0068] Figures 7a to 7i This is a schematic diagram of intracellular ice growth types in the embodiments;

[0069] Figure 8a This is a schematic diagram illustrating the diffusion-type growth pattern of intracellular ice in the embodiment.

[0070] Figure 8b This is a schematic diagram illustrating the aggregated growth pattern of intracellular ice in the embodiment.

[0071] Figure 9 This is a schematic diagram of the automatic intracellular ice tracking process based on grayscale level changes in the embodiment.

[0072] Figure 10 This is a schematic diagram of the intracellular ice growth rate curve in the embodiment;

[0073] The markings in the diagram are as follows: 1. Quick-release lever, 2. Temperature column, 3. Sapphire glass plate, 4. X-axis movement joystick, 5. Quick-release handle, 6. Silver block. Detailed Implementation

[0074] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0075] Example

[0076] A visualization observation system for the freeze-thaw process of oocytes and embryos includes a cryogenic microscope equipped with a high-speed industrial camera, a high-speed quenching platform and an image processor, wherein the high-speed quenching platform is used to perform high-speed quenching loading on oocyte samples.

[0077] High-speed industrial cameras are used to acquire video images of oocytes and embryos during the freeze-thaw process in real time and transmit them to an image processor;

[0078] The image processor is used to process and analyze video images of the freeze-thaw process of oocytes and embryos to determine the formation sites, growth patterns, and growth rates of intracellular ice.

[0079] Based on the above system, a method for visually observing the oocyte system and the embryo freeze-thaw process is implemented, such as... Figure 1 As shown, it includes the following steps:

[0080] S1. Prepare oocyte samples;

[0081] S2. For oocyte samples, a freeze-thaw process and / or a high-speed quenching process are applied, and video images are acquired in real time by a high-speed industrial camera;

[0082] S3. The video images are preprocessed, and then the formation sites of intracellular ice, growth types and patterns, and growth rates are determined, and the corresponding observation results are obtained.

[0083] This embodiment applies the above technical solution, and its main contents include:

[0084] I. The process of obtaining and processing oocytes is as follows:

[0085] a) Select female ICR closed colony mice aged 6-8 weeks, and stimulate the mouse ovaries with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) to induce a large number of follicles to mature and ovulate, in order to obtain a sufficient number of mature oocytes.

[0086] b) Within 15–16 hours after hCG injection, mice were rapidly euthanized by cervical dislocation. The abdominal skin was disinfected with 75% ethanol, and the skin was cut open to expose the peritoneum. The peritoneum was then cut open with another pair of sterile ophthalmic scissors, and the internal organs were turned upwards to expose the uterus, fallopian tubes, and ovaries. The upper part of the uterus was grasped with forceps and lifted, and excess fat pads were removed. An incision was made between the ovary and the fallopian tube, with the incision as close to the ovary as possible. The forceps were then moved to the fallopian tube, which was grasped, and the connection between the fallopian tube and the uterus was severed, thus completing the separation of the fallopian tube. The fallopian tube was quickly transferred to preheated M2 culture medium for rinsing. The culture dish containing the fallopian tube was placed on the stage of a stereomicroscope. The dilated area of ​​the fallopian tube was located under the microscope, and the ampulla of the fallopian tube was punctured with a 1 mL syringe. The cumulus-oocyte complex (COCs) was pulled from the fallopian tube into the oocyte collection drop.

[0087] c) Gently aspirate COCs into preheated hyaluronidase using a transfer needle. Gently pipette the COCs for 1–2 minutes until the granulosa cells are loosened, then immediately transfer them to M2 culture medium. Avoid prolonged exposure to the digestive enzymes, which could damage the oocytes. If the corona radiata is still attached to the zona pellucida of the oocyte, continue to pipette repeatedly in the M2 droplet until the granulosa cells are completely detached. Wash the naked oocytes sequentially in M2 culture medium droplets 3–5 times to thoroughly remove hyaluronidase. Select oocytes with intact morphology, homogeneous cytoplasm, and smooth polar body surfaces, and place them into pre-equilibrated M2 culture medium droplets. The number of oocytes in each droplet should be controlled to be 10–20.

[0088] II. Preparation and loading process of oocyte samples:

[0089] a) Gently heat the middle section of the Pasteur pipette by placing it on the outer edge of an alcohol lamp flame until softened. Then, quickly stretch it with your right hand. After the pipette cools, cut a section of appropriate diameter using a ceramic knife to use as an egg transfer needle. The sample container for the low-temperature stage is a shallow quartz crucible with an inner diameter of 15.2 mm, a depth of 2.56 mm, and a bottom thickness of 40 μm. Coating the surface of the silver stage with an appropriate amount of high thermal conductivity silicone oil makes the contact surface adhere better and also reduces the displacement of the quartz crucible, thereby avoiding defocusing problems.

[0090] b) Due to the large size and high water content of oocytes, they are prone to ice crystal formation during freeze-thaw cycles. To obtain complete images of the oocytes for subsequent processing and analysis, a C-shaped ring (e.g., with an outer diameter of 14 mm, an inner diameter of 13 mm, and a thickness of 50 μm) is placed at the bottom of the quartz crucible. Figure 2 (As shown). After the oocyte is loaded into the center of the crucible, a coverslip is placed on top. Because of the C-shaped ring, the coverslip does not press directly on the oocyte, thus ensuring the integrity of the cell and preventing the oocyte and embryo from being crushed.

[0091] c) Using a micropipette, pipette 1.2 μL of the solution and drop it into the center of the quartz crucible, as shown. Figure 3 As shown, before transferring oocytes with a transfer needle, the tip of the transfer needle is arranged in a liquid-air-liquid column pattern. The siphon effect slows down the flow of liquid, keeping the oocyte at the tip of the transfer needle. The oocyte is gently blown into the droplet with the smallest possible volume. After the cell settles to the bottom and stabilizes, a coverslip is placed on top, and the bottom of the crucible is wiped with a lint-free cloth to prevent residual liquid. The reduced liquid volume decreases the drift of the oocyte during the cooling process.

[0092] III. Visual observation of the freeze-thaw process to control the cooling rate:

[0093] a) The cooling system mainly consists of a BCS196 cryogenic cold stage, an LNP95 automatic cooling system, a T95 temperature control system (Linkam Scientific Instruments, UK), and Linksys 32 (Linkam Scientific, UK) temperature control software. The imaging system uses a high-speed industrial camera with dimensions of 29mm×29mm×29mm, an effective pixel count of 300,000 (640×480), a frame rate of 815fps, an exposure time of 16μs~1s, and a signal-to-noise ratio of 38dB. All camera lenses use a standard C-type interface, with the other end connected to a computer's USB 3.0 interface via a data transmission cable.

[0094] b) Pour liquid nitrogen into the Dewar flask, and after the liquid nitrogen in the flask stops boiling, put on the sealing cap;

[0095] c) Turn on the LNP95 liquid nitrogen pump controller, T95 temperature controller, Linksys 32 computer software and microscope light source in sequence. Unscrew and remove the round cover on the cold stage. Visually inspect the surface of the silver block. Use a blower to blow away the dust to ensure that the surface is clean and dry.

[0096] d) Cover the round cap, insert the gas purification tube into the corresponding port of the cold stage, and insert the gas valve connector on the other side to connect the cavity with the outdoor environment. Manually adjust the liquid nitrogen flow rate to the maximum value in the software to purge dry nitrogen into the cold stage cavity, reduce the moisture content in the cavity, and prevent condensation and frost formation on the sample during freezing;

[0097] e) Rotate the coarse focal length of the microscope to lower the cold stage, maximizing the distance between the cold stage and the microscope objective. Open the round cover of the cold stage, and quickly place the quartz crucible into the sample feeder with tweezers. Check whether the crucible is stable and ensure that its bottom surface is in contact with the surface of the silver stage. Cover the round cover to seal the chamber.

[0098] f) Secure the nitrogen purge tube to the round cover of the cold stage, and adjust the purge tube outlet position so that it is directly aligned with the observation window on the round cover. Use the coarse and fine adjustment handwheels of the rotating microscope to focus the sample;

[0099] g) Set the cooling program for oocyte cryopreservation in the software as follows: maintain the initial temperature of the cold stage at 20℃; the cooling rate can be set in the range of 0.1-100℃ / min, lower to -80℃ and hold for 2 min; then raise the temperature to 20℃ at a certain rate, which can be set in the range of 0.1-100℃ / min.

[0100] h) Open the recording software and start the pre-set cooling program to conduct the experiment.

[0101] IV. Visual observation of the high-speed quenching process:

[0102] a) At room temperature, the thermal conductivity of sapphire is approximately 0.08 W / (m·K). Under the same conditions, the thermal conductivity of sapphire glass is superior to that of a quartz crucible. Therefore, during high-speed quenching, the quartz crucible is replaced with a sapphire glass sheet with a diameter of 7 mm and a thickness of 0.3 mm. The structure of the high-speed quenching platform is as follows: Figure 4 As shown;

[0103] b) In order to prevent the sapphire glass plate 3 from falling off the sampler during rapid movement, it needs to be reinforced. Apply a layer of solid glue evenly inside the steel ring of the sampler with a cotton swab and embed the sapphire glass plate 3 into the sampler.

[0104] c) Then place the sampler on a constant temperature table to dry overnight. After the solid glue has completely solidified, clean the sapphire glass plate 3 and the sampler steel ring with anhydrous ethanol to remove any remaining glue.

[0105] d) Install the prepared sample feeder onto the cold stage, and then connect the cold stage to the Dewar flask containing liquid nitrogen;

[0106] e) Before cooling down the silver block 6, place the sampler containing the oocyte sample on the silver block 6 for focusing, and then move the sampler to the temperature column 2.

[0107] f) The temperature of the silver block 6 in the center of the cold stage was lowered to -180℃ by software settings, while the temperature column 2 was kept at 20℃.

[0108] g) Open the recording software, and then push the X-axis moving control lever 4 towards the cold stage. The movement of the fast push lever 1 will pull the sample feeder to move the sample from the warm column 2 to the silver block 6, thus completing the high-speed quenching loading of the sample.

[0109] h) Rapid cooling of the sample is achieved by rapidly moving the sample from the heated column 2 to the pre-cooled silver block 6 under a large temperature difference;

[0110] i) By moving the fast push rod 1 away from the cold stage, the sample can be moved from the silver block 6 to the warm column 2, thus unloading the sample.

[0111] V. Image Processing Operation Methods:

[0112] a) Export the recorded high-speed video frame by frame in Kinovea software. Divide the number of frames after the high-speed industrial camera is triggered by the camera’s time resolution of 815fps to determine the elapsed time after the trigger. Obtain the temperature information corresponding to each image based on the cooling rate used in each group of experiments.

[0113] b) Extract the complete intracellular ice growth process from the video captured by the high-speed camera and export the images frame by frame, selecting the previous frame image before the formation of intracellular ice as a mask.

[0114] c) Once extracellular ice crystals have formed, the oocyte no longer shifts during the entire process of intracellular ice formation. The Labelme annotation tool is used to segment the oocyte and background image.

[0115] d) Convert the generated JSON file into an image and use the obtained mask to batch process the key image sequences of intracellular ice.

[0116] VI. The process of determining intracellular ice formation sites:

[0117] a) To classify the radial distribution of intracellular ice formation sites, the oocyte's projected region is divided into internal and peripheral regions (e.g., ...). Figure 5 As shown in the figure, the inner region accounts for 90% of the total projected area of ​​the circle, while the opposite edge region accounts for 10% of the total projected area of ​​the circle.

[0118] b) Since the diameter of oocytes is affected by individual differences, a mask of the corresponding scale is first created when processing data. The mask is enlarged or reduced proportionally according to different cell sizes, so as to quickly identify the region of intracellular ice formation sites.

[0119] c) By exporting intracellular ice images from high-speed video recording frame by frame, the formation sites of intracellular ice can be determined based on the division of the oocyte projection region;

[0120] d) Intracellular ice sites of marginal region type (e.g. Figure 6a As shown in the figure, the ice crystal formation site appears at the boundary of the oocyte;

[0121] e) Intracellular ice sites of internal region type (e.g.) Figure 6b As shown in the figure, the site where intracellular ice crystals form is close to the cell edge but not in contact with it. The formation site is determined to be in the inner region of the projection by comparing the region boundary with the mask.

[0122] f) Intracellular ice sites with coexisting edge and interior regions (e.g., Figure 6c As shown in the image, ice crystals appear together at the boundary and inside of the oocyte.

[0123] VII. The process of determining the growth type and pattern of intracellular ice:

[0124] a) By analyzing the exported images of intracellular ice behavior in oocytes, observe the differences in intracellular ice behavior during the cooling process; according to the experimental images, two different types of intracellular ice exist during the oocyte cooling process (e.g., Figures 7a to 7i As shown in the figure, * indicates "dark" cells and → indicates "trembling" cells.

[0125] b) "Dark type" cells: These cells form intracellular ice 2-7 ms after contact with extracellular ice crystals. The ice crystals grow rapidly, and the cells eventually appear dark black.

[0126] c) "Shaking" cells: These cells do not immediately form ice crystals upon contact with extracellular ice crystals. Intracellular ice sites are formed 600–900 ms after the extracellular ice crystals have formed. Subsequently, the ice crystals grow rapidly, and eventually the ice crystals at the edge of the cell are lighter in color, while the color of the central region is the same as that of the dark ice crystals in the type of cells.

[0127] d) The ice crystal growth pattern of "dark" cells is characterized by "diffusion" (e.g., Figure 8a (As shown). The formation sites of "diffused" ice crystals are distributed in both the edge and interior regions, and as the temperature decreases, they spread throughout the cell in a point-like diffusion manner.

[0128] e) The ice crystal growth pattern of "trembling" cells is characterized by "aggregation" (e.g., Figure 8b(As shown). The formation sites of "aggregated" ice crystals are only distributed in the edge region of the cell. In the early stage of growth, the ice crystals converge towards the center of the cell with the entire cell outline as the interface for ice front movement. The ice front moves relatively quickly and then fills the entire cell.

[0129] 8. The calculation process of intracellular ice growth rate (e.g.) Figure 9 As shown):

[0130] a) Use Python to program each pixel of the image to be processed individually, select a threshold, and compare its gray value with the set threshold to obtain a binarized image;

[0131] b) Threshold segmentation is expressed using function (1):

[0132] T = T[x, y, p(x, y), f(x, y)] (1)

[0133] Where x and y represent the horizontal and vertical coordinates of the pixel, p(x,y) represents the local feature of the pixel, and f(x,y) represents the gray value of the pixel. The image after thresholding is defined as Equation (2):

[0134]

[0135] Pixels marked with a grayscale value of 1 correspond to key pixels, i.e., intracellular ice regions, while pixels marked with a grayscale value of 0 correspond to the background. The number of pixels occupied by ice crystals is counted to calculate the intracellular ice formation area.

[0136] The growth rate V of intracellular ice crystals is measured by measuring the instantaneous surface area A of intracellular ice. i Intracellular ice area A in the previous frame b The calculated value is t, which is the time interval between two adjacent frames. Therefore, the intracellular ice growth rate can be obtained according to equation (3):

[0137]

[0138] In this embodiment, the intracellular ice growth rate was calculated as follows: Figure 10 As shown.

[0139] In summary, this technical solution addresses the issue of oocyte and embryo crushing by placing a C-shaped ring at the bottom of the quartz crucible; it addresses microscope defocusing by reducing the volume of oocyte and embryo droplets; and it replaces conventional low-frame-rate cameras with a high-speed industrial camera mounted on a cryogenic microscope, enabling high-speed imaging of the freeze-thaw process. The high-speed industrial camera achieves visual monitoring of the oocyte freeze-thaw process while maintaining pixel count and frame rate. A high-speed quenching and cooling device is integrated into the cryogenic stage to achieve ultra-rapid sample cooling and simulate the vitrification process. Using Kinovea software, the recorded high-speed video is exported frame by frame, and image masks are created. Python programming is then used to process each pixel of the image to achieve single-point processing. Based on the processed images, analytical methods for the initial site of intracellular ice, its growth pattern, and its growth rate are designed.

[0140] This technical solution proposes a sample preparation method for visual observation of the freeze-thaw process of oocytes and embryos. This sample preparation method can effectively avoid problems such as oocyte crushing and microscopic defocusing during the observation process: on the one hand, a C-shaped ring with an outer diameter of 14 mm, an inner diameter of 13 mm, and a thickness of 50 μm is placed at the bottom of the crucible; on the other hand, to address the defocusing problem, the tip of the oocyte transfer needle is arranged in a liquid column-air column-liquid column pattern, using the siphon effect to slow down the liquid flow rate, controlling the oocyte at the tip of the oocyte transfer needle, and gently blowing the oocyte into the droplet with the smallest volume.

[0141] This technical solution proposes a method for visually observing the freeze-thaw process by controlling the cooling rate. Using a high-speed industrial camera suitable for oocytes and embryos, the growth of ice crystals during the cooling process can be observed. In this embodiment, the high-speed industrial camera has an effective pixel count of 300,000 (640×480), a frame rate of 815fps, an exposure time of 16μs to 1s, and a signal-to-noise ratio of 38dB. Under these parameters, sufficient images can be obtained to record the formation and growth of ice crystals while ensuring high image clarity.

[0142] This technical solution proposes a visualization observation method for high-speed quenching process. By modifying the cold stage, the visualization observation of ultra-high cooling rate is realized. On the one hand, a sapphire glass plate with a thermal conductivity of about 0.08 W / (m·K), a diameter of 7 mm and a thickness of 0.3 mm is used as a carrier, and the sapphire glass plate is fixed on the sample feeder with solid glue.

[0143] On the other hand, before cooling the silver block, the sampler carrying the oocyte sample is placed on the silver block for focusing, and then the sampler is moved to the heating column. The temperature of the silver block at the center of the cooling stage is lowered to -180℃ by software settings, and the heating column is kept at 20℃. Then, by pushing the quick push handle in the X-axis direction towards the cooling stage, the movement of the quick push handle pulls the sampler to move the sample from the heating column to the silver block, completing the high-speed quenching loading of the sample.

[0144] This technical solution proposes a method for processing images obtained during the freeze-thaw process of oocytes and embryos. It involves using the Labelme annotation tool to segment the oocyte and embryo background images. The generated JSON files are then converted into images, and the obtained masks are used for batch processing of key intracellular ice image sequences. Finally, Python is used to programmatically process each pixel of the image to be processed individually.

[0145] This technical solution proposes an analytical method for the formation sites of intracellular ice in oocytes and embryos. Through research, it was found that there are three types of intracellular ice formation sites: edge region type, internal region type, and coexistence of edge and internal regions. The edge region type is ice crystal formation site that appears at the boundary of the oocyte. The internal region type is determined by comparing the region boundary with a mask to determine that the formation site is in the internal region of the projection. The coexistence of edge and internal regions is ice crystal formation site that appears at both the boundary and internal region of the oocyte.

[0146] This technical solution proposes a method for determining the growth type and pattern of intracellular ice in oocytes. Research reveals that during intracellular ice growth, oocytes can be categorized into "dark-type cells" and "trembling-type cells," with intracellular ice growth patterns of "diffusion" and "aggregation," respectively. Specifically, "dark-type" cells begin forming intracellular ice 2–7 ms after contact with extracellular ice crystals; "trembling-type" cells do not immediately form ice crystals after contact, but rather after 600–900 ms of extracellular ice crystal formation, resulting in intracellular ice sites.

[0147] Ice crystals can form at both the edge and interior of the cell. As the temperature decreases, they spread throughout the cell in a point-like manner, which is called "diffusion-type" growth. Ice crystals can also form at only the edge of the cell. In the early stages of growth, they converge towards the center of the cell using the entire cell outline as the interface for ice front movement. The ice front moves relatively quickly, which is called "aggregation-type" growth.

[0148] This technical solution also proposes a method for analyzing the growth rate of intracellular ice. The binary oocyte image obtained by Python programming is used to identify the area change of intracellular ice in the oocyte through grayscale marking, and then the growth rate of intracellular ice under different cooling rates is obtained through mathematical relationships.

Claims

1. An oocyte and embryo freeze-thaw process visual observation system, characterized by, The low-temperature microscope includes a high-speed industrial camera, a high-speed quenching platform for high-speed quenching loading of the oocyte sample, and an image processor. The high-speed quenching process specifically includes: The quartz crucible is replaced by a sapphire glass sheet; A layer of solid glue is evenly coated inside the steel ring of the sample feeder, and the sapphire glass sheet is embedded in the sample feeder to prevent the sapphire glass sheet from falling off the sample feeder during rapid movement; Then place the sample feeder on the constant temperature table overnight to dry, and after the solid glue is completely solidified, clean the residual glue on the sapphire peeling sheet and the steel ring of the sample feeder with anhydrous ethanol; Install the treated sample feeder to the cold table, and then connect the cold table with the dewar bottle filled with liquid nitrogen; Before the silver block is cooled, the sample feeder loaded with the oocyte sample is placed on the silver block for focusing, and then the sample feeder is moved to the warm column; The temperature of the silver block at the center of the cold table is reduced to -180℃, and the warm column is always kept at 20℃; By pushing the X-axis fast push handle towards the cold table, the fast push handle moves to pull the sample feeder to move the sample from the warm column to the silver block, completing the high-speed quenching loading of the sample; The high-speed industrial camera is used to collect video images of the oocyte and embryo freeze-thaw process in real time and transmit them to the image processor; 2. The system according to claim 1, wherein the system is characterized by: The image processor is used to process and analyze the video images of the oocyte and embryo freeze-thaw process to determine the formation site of intracellular ice, the growth pattern of intracellular ice, and the growth rate of intracellular ice.

3. A method for visualizing observation of a freezing and thawing process of an oocyte and an embryo, characterized by, The low-temperature microscope is equipped with a C-shaped ring at the bottom of the quartz crucible to prevent the oocyte and embryo from being crushed. The method includes the following steps: S1, preparing an oocyte sample; S2, for the oocyte sample, performing a freeze-thaw process and / or a high-speed quenching process loading, and collecting video images in real time by a high-speed industrial camera; S3, preprocessing the video images, and then respectively judging the formation site of intracellular ice, judging the growth type and pattern, and calculating the growth rate to obtain corresponding observation results; The step S2 specifically includes the following steps: S21, transferring the oocyte sample to the quartz crucible using an oocyte transfer needle and then covering a cover glass; S22, performing a controllable cooling rate freeze-thaw process loading and / or a high-speed quenching process loading, and simultaneously collecting corresponding video images in real time by a high-speed industrial camera; The high-speed quenching process in step S22 specifically includes: The quartz crucible is replaced by a sapphire glass sheet; A layer of solid glue is evenly coated inside the steel ring of the sample feeder, and the sapphire glass sheet is embedded in the sample feeder to prevent the sapphire glass sheet from falling off the sample feeder during rapid movement; Then place the sample feeder on the constant temperature table overnight to dry, and after the solid glue is completely solidified, clean the residual glue on the sapphire peeling sheet and the steel ring of the sample feeder with anhydrous ethanol; Install the treated sample feeder to the cold table, and then connect the cold table with the dewar bottle filled with liquid nitrogen; Before the silver block is cooled, the sample feeder loaded with the oocyte sample is placed on the silver block for focusing, and then the sample feeder is moved to the warm column; The temperature of the silver block at the center of the cold table is reduced to -180℃, and the warm column is always kept at 20℃; By pushing the X-axis fast push handle to the cold stage direction, the fast push handle movement will pull the sample feeder to realize the movement of the sample from the warm column to the silver block, and complete the high-speed quenching loading of the sample.

4. The method according to claim 3, wherein the method is characterized by, The specific process of the step S21 is as follows: first, the front end of the oocyte transfer needle tube is arranged in a liquid column-gas column-liquid column mode, the liquid flow speed is slowed down by using the siphon effect, the oocyte is controlled at the tip of the oocyte transfer needle, and then the oocyte is gently blown into a liquid drop in the C-shaped ring at the bottom of the quartz crucible with a minimum volume, after the oocyte is stably settled at the bottom, a cover glass is covered, and the bottom surface of the quartz crucible is wiped with a dust-free cloth to prevent residual liquid.

5. The method according to claim 3, wherein the method is characterized by: The process of pre-processing the video image in the step S3 is as follows: In the Kinovea software, the recorded high-speed video is exported frame by frame, the frame number after the high-speed industrial camera is triggered is divided by the time resolution of the camera to determine the time elapsed after the triggering, and according to the cooling rate used in each group of experiments, the temperature information corresponding to each picture is obtained; The complete intracellular ice growth process is intercepted from the video shot by the high-speed camera, and the images are exported frame by frame, and the previous frame image of intracellular ice formation is selected as a mask; When extracellular ice crystals have formed, the oocyte no longer moves during the entire process of intracellular ice formation, and the Labelme labeling tool is called to perform image segmentation on the oocyte and the background; The generated json file is converted into an image, and the obtained mask is used to batch process the key image sequence of intracellular ice.

6. The method according to claim 5, wherein the method is characterized by, The process of judging the formation site of intracellular ice in the step S3 is as follows: In order to classify the radial distribution of intracellular ice formation sites, the projection area of the oocyte is divided into an internal region and an edge region, the internal region accounts for 90% of the total projection area of the circle, and the edge region accounts for 10% of the total projection area of the circle; Due to the influence of individual differences on the size of the oocyte diameter, a mask with a corresponding proportion is prepared when processing data, and the mask is enlarged or reduced in proportion according to different cell sizes, so that the region of the intracellular ice formation site can be quickly identified; By exporting the intracellular ice images recorded by the high-speed video frame by frame, the projection area of the oocyte is divided to determine the formation site of intracellular ice: 1) The intracellular ice site of the edge region type, the ice crystal formation site appears at the boundary of the oocyte; 2) The intracellular ice site of the internal region type, the intracellular ice crystal formation site is close to the cell edge but does not contact, and the formation site is determined to be in the internal region of the projection by comparing the region boundary through the mask; 3) The intracellular ice site of the coexistence type of edge and internal region, the ice crystal appears at the boundary and internal region of the oocyte.

7. The method according to claim 5, wherein the method is characterized by, The process of judging the growth type and mode in the step S3 is as follows: By analyzing the exported oocyte intracellular ice behavior image information, the differences in intracellular ice behavior of the oocyte during the cooling process are observed; according to the experimental images, it is determined that there are two different types of intracellular ice in the oocyte during the cooling process: "dark black type" cells and "flashing type" cells; The "dark black type" cell forms intracellular ice after 2-7 ms of contact with extracellular ice crystals, and the ice crystals grow rapidly, finally the cell appears dark black; the ice crystal growth mode of the "dark black type" cell is "diffusion", and the formation sites of the "diffusion" ice crystals are distributed in the edge region and the internal region, and with the decrease of temperature, the entire cell is filled with the point-like diffusion form; The "shaking type" cell does not immediately produce ice crystals after contact with extracellular ice crystals, and the intracellular ice sites are formed after 600-900 ms of extracellular ice crystal formation, and then the ice crystals grow rapidly, finally the color of the edge ice crystals of the cell is lighter, and the color of the central region is consistent with that of the dark black type cell; the ice crystal growth mode of the "shaking type" cell is "gather", and the formation sites of the "gather" ice crystals are only distributed in the edge region of the cell, and in the initial stage of growth, the entire cell contour is the ice front moving interface to the cell center, and the ice front moving speed is relatively fast, and then the entire cell is filled.

8. The method according to claim 5, wherein the method is characterized by, The growth rate calculation process in the step S3 is specifically: Each pixel point of the image to be processed is processed, a threshold value is selected, the gray value is compared with the set threshold, and a binary image is obtained. The thresholding function is: , where x, y are horizontal and vertical coordinates of the pixel respectively, p(x, y) is the local feature of the pixel, f(x, y) is the pixel gray value, and the image after thresholding is defined as: , Wherein, the pixel marked with 1 corresponds to the key pixel, i.e. intracellular ice region, and the pixel marked with 0 corresponds to the background. The number of pixels occupied by ice crystals is counted, the intracellular ice growth rate is obtained by calculating the intracellular ice occurrence area as follows: , where V is the growth rate of intracellular ice crystals, A i is the instantaneous area of intracellular ice in the current frame image, A b is the intracellular ice area of the previous frame image, and t is the time interval between two adjacent frame images.

Images