Sperm acrosome reaction detection method based on phase imaging

Abstract

The invention discloses a sperm acrosome reaction detection method based on phase imaging, which comprises the following steps: preparing two sperm smears, obtaining a phase diagram SP of sperms without acrosome reaction, and obtaining a phase diagram SPacrosome of sperms with acrosome reaction; obtaining a sperm nucleus average phase value SPt, a sperm acrosome average phase value SPd, a sperm nucleus average phase value SPtarosome and a sperm acrosome average phase value SPdacrosome; calculating a first difference value between the SPt and the SPd and a second difference value between the SPtasrosome and the SPdarosome, and calculating a second difference value between the SPtasrosome and the SPdarosome; calculating the phase ratio R of the sperm acrosome to the sperm nucleus and the phase ratio Racrosome of the sperm acrosome to the sperm nucleus; and through comparative analysis of the SPt, the SPd, the R, the SPtarosome, the SPdacrosome and the Racrosome, a distinguishing and judging rule of the sperms without the acrosome reaction and the sperms with the acrosome reaction is obtained. The sperm acrosome reaction detection method based on phase imaging has the advantages that vacuole detection can be carried out without fluorescence staining on sperms, unmarked quantitative measurement on the sperms is realized, the detection efficiency and accuracy are high, and the like.

Description

Technical Field

[0001] This invention relates to sperm detection technology, and in particular to a phase imaging-based method for detecting sperm acrosome reaction under dark-field imaging conditions, which tracks and detects sperm motility trajectories. Background Technology

[0002] Mammalian sperm are highly specialized cells that undergo morphological changes during spermatogenesis. The cytoskeleton of the subacrosomal region (SAR) lies between the inner acrosomal membrane and the nuclear envelope, while the cytoskeleton of the postacrosomal region (PAR) lies between the plasma membrane and the nuclear envelope. Together, they form the perinuclear theca (PT), which covers the outer surface of the nuclear membrane. The PT plays a crucial role in spermatogenesis, promoting proper sperm function, the establishment and maintenance of sperm head structure, capacitation, and the acrosomal reaction.

[0003] The acrosome is a sac-like organelle located in front of the sperm head cell nucleus, between the nucleus and plasma membrane. Situated in the anterior two-thirds of the sperm head, it is essentially a specialized lysosome derived from the Golgi apparatus. Enclosed by a single membrane, it is a flattened sac containing glycoproteins and various hydrolases, serving as a storage site for enzymes related to the acrosome reaction. The acrosome reaction refers to a series of changes that occur in the acrosome after sperm capacitation and encounter with the egg in the ampulla of the fallopian tube; specifically, it is the process by which the sperm releases acrosomal enzymes, dissolving the corona radiata and zona pellucida.

[0004] The acrosome reaction is a highly regulated exocytosis event that prepares the sperm for successful fertilization. After induction, acrosome exocytosis occurs via vesicles radiating throughout the sperm head, disrupting the stability of the acrosome vesicles and leading to the release of acrosin enzymes from within the acrosome. After acrosome detachment, the sperm can penetrate the outer layer of the oocyte and begin fertilization.

[0005] Therefore, the failure of sperm to complete the acrosome reaction is a relatively common cause of male infertility, and the integrity of the acrosome is a direct influencing factor in the clinical assessment of sperm fertilization capacity. However, clinically, the sperm acrosome reaction can only be detected through fluorescent staining, and samples tested cannot be used for subsequent developmental studies.

[0006] Label-free, non-contact quantitative phase imaging technology converts the phase information of the measured object into detectable changes in light intensity, and has become a valuable method for studying biological cells and tissues. Grating-based point diffraction phase imaging systems utilize an optical system structure where the object and reference beams share a common path, reducing interference from environmental mechanical vibrations and external media, improving the stability of interference fringes, and enabling the measurement of rapidly moving targets with single-frame imaging.

[0007] Therefore, sperm acrosome reaction detection helps determine the occurrence of the acrosome reaction based on the phase of the sperm nucleus and acrosome, thereby improving fertilization success rates and greatly benefiting the advancement of assisted reproductive technology. In current technologies, the detection of the sperm acrosome reaction requires fluorescent staining of the sperm. However, fluorescent labeling can easily damage the sperm being tested, rendering the tested sperm unusable for subsequent clinical developmental studies. Summary of the Invention

[0008] To overcome the shortcomings of the existing technologies, this invention provides a sperm acrosome reaction detection method based on phase imaging, which can effectively perform label-free quantitative measurement of the sperm head region and improve the efficiency and accuracy of sperm acrosome reaction detection.

[0009] The present invention adopts the following technical solution to solve the technical problem.

[0010] The present invention provides a method for detecting sperm acrosome reaction based on phase imaging, comprising the following steps:

[0011] Step 1: Set up the optical path for sperm acrosome reaction detection;

[0012] Step 2: Divide sperm from the same sperm source into two sperm samples, and prepare a first sperm smear and a second sperm smear respectively, and place them into the detection optical path;

[0013] Step 3: Obtain bright-field images of the first and second sperm smears, as well as two interference fringe images of the sperm-containing and sperm-free samples, respectively;

[0014] Step 4: Obtain the phase map (SP) of sperm that have not undergone acrosome reaction using two interference fringe images from the first sperm smear; obtain the phase map (SP) of sperm that have undergone acrosome reaction using two interference fringe images from the second sperm smear. acrosome ;

[0015] Step 5: Obtain the average phase value SPt of the sperm nucleus and the average phase value SPd of the sperm acrosome from the phase map SP of sperm that did not undergo acrosome reaction;

[0016] Phase mapping of sperm that have undergone acrosome reaction (SP) acrosome The average phase value SPt of sperm nuclei that have undergone acrosome reaction was obtained. acrosome and the average phase value of sperm acrosome SPd acrosome ;

[0017] Step 6: Calculate the first difference between SPt and SPd and SPt acrosome With SPd acrosome The second difference ;

[0018] Step 7: Calculate the acrosome-to-nucleus phase ratio R for sperm that have not undergone acrosome reaction and the acrosome-to-nucleus phase ratio R for sperm that have undergone acrosome reaction. acrosome ;

[0019] Step 8: Using the parameters SPt, SPd, , R, SPt acrosome SPd acrosome , and R acrosome Through comparative analysis, rules for distinguishing between sperm that have not undergone acrosome reaction and sperm that have undergone acrosome reaction were obtained.

[0020] The structural features of the sperm acrosome reaction detection method based on phase imaging of the present invention also lie in:

[0021] Furthermore, in step 1, the light source of the detection optical path includes a laser source for forming interference fringes and an array of light-emitting diodes (LEDs) for forming a bright field.

[0022] Furthermore, in step 1, the detection optical path includes a spatial filter PF.

[0023] Furthermore, the detection optical path includes a sample cell; the sample cell is a transparent plate with a grid.

[0024] Furthermore, in step 2, the process of obtaining the first sperm smear includes: a first sperm sample coating step and a first sperm sample fixation step.

[0025] Furthermore, in step 2, the process of obtaining the second sperm smear includes: an acrosome reaction step of the second sperm sample, a coating step of the second sperm sample, and a fixation step of the second sperm sample.

[0026] Furthermore, in the acrosome reaction step of the second sperm sample, calcium ion carrier A23187 was incubated in a water bath for 15 minutes to induce the acrosome reaction.

[0027] Furthermore, in step 4, the phase unwrapping method of the LS algorithm in the minimum p-norm is used to perform phase unwrapping.

[0028] Furthermore, the sperm acrosome reaction detection method based on phase imaging also includes the following two steps:

[0029] Step 9: After performing fluorescent staining on the first sperm smear that did not undergo acrosome reaction and the second sperm smear that did undergo acrosome reaction, fluorescent images of the first sperm smear and the second sperm smear were taken respectively.

[0030] Step 10: Based on the staining of sperm in the fluorescence image from Step 9, determine whether the sperm have undergone the acrosome reaction.

[0031] Furthermore, in step 9, the first sperm smear and the second sperm smear are immersed in peanut lectin fluorescent staining solution for staining treatment.

[0032] Compared with existing technologies, the beneficial effects of this invention are reflected in:

[0033] This invention discloses a method for detecting sperm acrosome reaction based on phase imaging, comprising the following steps:

[0034] Step 1: Set up the optical path for sperm acrosome reaction detection;

[0035] Step 2: Divide sperm from the same sperm source into two sperm samples, and prepare a first sperm smear and a second sperm smear respectively, and place them into the detection optical path;

[0036] Step 3: Obtain bright-field images of the first and second sperm smears, as well as two interference fringe images of the sperm-containing and sperm-free samples, respectively;

[0037] Step 4: Obtain the phase map (SP) of sperm that have not undergone acrosome reaction using two interference fringe images from the first sperm smear; obtain the phase map (SP) of sperm that have undergone acrosome reaction using two interference fringe images from the second sperm smear. acrosome ;

[0038] Step 5: Obtain the average phase value SPt of the sperm nucleus and the average phase value SPd of the sperm acrosome from the phase map SP of sperm that did not undergo acrosome reaction;

[0039] Phase mapping of sperm that have undergone acrosome reaction (SP) acrosome The average phase value SPt of sperm nuclei that have undergone acrosome reaction was obtained. acrosome and the average phase value of sperm acrosome SPd acrosome ;

[0040] Step 6: Calculate the first difference between SPt and SPd and SPt acrosome With SPd acrosome The second difference ;

[0041] Step 7: Calculate the acrosome-to-nucleus phase ratio R for sperm that have not undergone acrosome reaction and the acrosome-to-nucleus phase ratio R for sperm that have undergone acrosome reaction. acrosome ;

[0042] Step 8: Using the parameters SPt, SPd, , R, SPtacrosome SPd acrosome , and R acrosome Through comparative analysis, rules for distinguishing between sperm that have not undergone acrosome reaction and sperm that have undergone acrosome reaction were obtained.

[0043] This invention utilizes transmission diffraction phase microscopy to reconstruct the phase of live sperm cells without the need for sperm staining or fluorescent labeling. It can effectively perform label-free quantitative measurement of the acrosome region of sperm and detect whether the acrosome reaction has occurred.

[0044] The sperm acrosome reaction detection method based on phase imaging of the present invention has the advantages of detecting acrosome reaction without the need for sperm fluorescence staining, realizing label-free quantitative measurement of sperm, and having high detection efficiency and accuracy. Attached Figure Description

[0045] Figure 1 This is a diagram of the optical path and apparatus for obtaining high-contrast, label-free phase images of sperm using diffraction phase imaging.

[0046] Figure 2 This is a flowchart of obtaining sperm images using a microscopic imaging device;

[0047] Figure 3 The results are presented as statistical analyses of samples from five different individuals. The AI ​​group represents the results of the control group where no acrosome response occurred, and the AR group represents the results of the experimental group where an acrosome response was induced.

[0048] The present invention will be further described below through specific embodiments and in conjunction with the accompanying drawings. Detailed Implementation

[0049] See Figures 1-3 The present invention provides a method for detecting sperm acrosome reaction based on phase imaging, comprising the following steps:

[0050] Step 1: Set up the optical path for sperm acrosome reaction detection;

[0051] The schematic diagram of the detection optical path is as follows: Figure 1 As shown.

[0052] Step 2: Divide sperm from the same sperm source into two sperm samples, and prepare a first sperm smear and a second sperm smear respectively, and place them into the detection optical path;

[0053] Sperm samples were obtained and washed and purified to form a first sperm sample and a second sperm sample. The first sperm sample and the second sperm sample were then processed into smears to obtain a first sperm smear and a second sperm smear.

[0054] like Figure 2The procedure for obtaining sperm images involves having multiple participants provide fresh sperm. One participant's fresh sperm is used as the sperm sample. This sample is first washed and purified, then a portion of the sperm suspension is divided into two samples: a first sperm sample and a second sperm sample. The first sperm sample is directly processed into a smear to obtain a first sperm smear without acrosome reaction. The second sperm sample is first subjected to an acrosome reaction, and then processed into a smear to obtain a second sperm smear with an acrosome reaction.

[0055] Step 3: Obtain bright-field images of the first and second sperm smears, as well as two interference fringe images of the sperm-containing and sperm-free samples, respectively;

[0056] Obtain a bright-field image BI1 of the first sperm smear, and then obtain two interference fringe images of the first sperm smear; the two interference fringe images are respectively the first interference fringe image IF1 containing sperm samples and the second interference fringe image IF2 not containing sperm samples;

[0057] Obtain the bright field image BI2 of the second sperm smear, and then obtain two interference fringe images of the second sperm smear; the two interference fringe images are the third interference fringe image IF3 containing sperm samples and the fourth interference fringe image IF4 not containing sperm samples;

[0058] This invention discloses a sperm acrosome reaction detection method based on phase imaging. First, a sperm sample from a test subject is prepared into a smear and fixed. A bright-field image of the sperm is captured first. After labeling the sperm, an unlabeled phase image of the sperm is captured, which involves capturing both the interference fringe background image and the sample image. This allows for the acquisition of both the bright-field image and the phase image (the phase image obtained from the interference fringe background and the sample image) of the same sample. For the same sample, one bright-field image and two interference fringe images are acquired at a time, and multiple acquisitions are performed. Step 3, obtaining the image of the first sperm smear, specifically includes the following steps:

[0059] Step 31: Turn on the LED array in the detection optical path to obtain the bright field image BI1 of the first sperm smear;

[0060] The bright-field image BI1 contains sperm samples. Since this study requires comparison of multiple imaging modes (including bright-field, interference fringe, and fluorescence images), a bright-field image is first captured to mark sperm positions, facilitating subsequent acquisition of interference fringe and fluorescence images. Specifically, the sample cell S preferably uses a counting plate. The counting plate is a transparent plate with a grid, and the smear is a transparent glass slide. Therefore, the positions of sperm on the smear correspond to the grid, allowing for accurate sperm location and observation based on the grid positions. The sperm position can be represented by the grid coordinates of the sperm's main body (sperm head). Specifically, the sperm head is located at the grid position of its center: for example, the sperm coordinates in the second row, third column of the grid are (2, 3). Sperm whose heads cross the grid or whose location is inaccurate or inconvenient via the grid are not collected. In this way, the number of sperm suitable for image acquisition on the first sperm smear and the position of each sperm can be determined.

[0061] Step 32: Mark the position of the sperm using the bright field image BI1, move the first sperm smear so that the part containing the sperm is placed in the detection optical path, keep the position of the first sperm smear, and take the first interference fringe image IF1 of the sperm sample;

[0062] Step 33: Move the first sperm smear, and move it in the smallest possible direction so that the original position of the sperm sample is free of any sperm sample, that is, the detection optical path of the first interference fringe image IF1 is free of any sperm, and then collect the second interference fringe image IF2 which is free of sperm sample.

[0063] Through steps 31 to 33 above, for the first sperm smear, a portion of the sperm are selected and bright-field images and two interference fringe images are acquired one by one to obtain parameters of multiple sperm in the first sperm sample.

[0064] The specific process of obtaining the image of the second sperm smear in step 3 is the same as steps 31 to 33 above, except that the first sperm smear is replaced with the second sperm smear. The process involves taking a bright field image BI2 of the sperm that has undergone acrosome reaction, a third interference fringe image IF3 of the sperm that has undergone acrosome reaction, and a fourth interference fringe image IF4 of the sperm that does not contain a sperm sample.

[0065] On both the first and second sperm smears, besides the areas containing sperm, there are numerous blank areas. That is, where the smear overlaps with the counting plate, there are many blank spaces on the counting plate where no sperm are present. Since the area captured by the detection optical path is limited and cannot cover the entire sperm smear, by moving the smear, the area captured by the detection optical path can be either containing sperm or not. In practice, for the first sperm smear of one subject, a bright-field image BI1 of all sperm is captured at once; then, all sperm are marked on the grid of the bright-field image; finally, the number of sperm images to be collected is determined (e.g., images of 150 sperm are collected), and IF1 and IF2 images of each sperm are captured sequentially.

[0066] Step 4: Obtain the phase map (SP) of sperm that have not undergone acrosome reaction using two interference fringe images from the first sperm smear; obtain the phase map (SP) of sperm that have undergone acrosome reaction using two interference fringe images from the second sperm smear. acrosome ;

[0067] Performing a Fourier transform on the first interference fringe image IF1 yields a first enclosed phase image; performing a Fourier transform on the second interference fringe image IF2 yields a second enclosed phase image; subtracting the second enclosed phase image from the first enclosed phase image yields a first sample sperm phase image; the first enclosed phase is then calculated based on the first sample sperm phase image. For the first package phase Phase unrolling was performed (to obtain the true phase of the first sperm sample) to obtain the phase map SP of sperm that did not undergo acrosome reaction;

[0068] Performing a Fourier transform on the third interference fringe image IF3 yields the third enclosed phase image; performing a Fourier transform on the fourth interference fringe image IF4 yields the fourth enclosed phase image; subtracting the fourth enclosed phase image from the third enclosed phase image yields the second sample sperm phase image; the second enclosed phase image is then calculated based on the second sample sperm phase image. For the second package phase Phase unfolding was performed (to obtain the true phase of the second sperm sample) to obtain the phase map SP of sperm that had undergone acrosome reaction. acrosome ;

[0069] In step 4, the first interference fringe image IF1 (or the third interference fringe image IF3) includes sperm information and the background information of the first smear; the second interference fringe image IF2 (or the fourth interference fringe image IF4) only contains the background information of the first smear, therefore the difference between the two is the sperm information. That is, by subtracting the background information of the second interference fringe image IF2 (which does not contain sperm information) from the sperm information and the background information of the first smear in the first interference fringe image IF1 containing the first sperm sample, the first encapsulation phase of the first sperm sample is obtained. To solve for the unfolded phase (i.e. the true phase of the first sperm sample), that is, the sperm phase SP that did not undergo the acrosome reaction, including the average phase value SPt of the sperm nucleus and the average phase value SPd of the sperm acrosome in the sperm phase map SP that did not undergo the acrosome reaction;

[0070] The first encapsulation phase of sperm that did not undergo acrosome reaction is obtained by subtracting the first interference fringe image IF1 and the second interference fringe image IF2 from the first sperm smear. This allows for the detection of the phase difference between the sperm head nucleus that did not undergo an acrosome reaction and the background, as well as the phase difference between the acrosome region of sperm that did not undergo an acrosome reaction and the background.

[0071] In specific implementation, the transformation formula of the Fourier transform is shown in the following formula (1);

[0072] (1)

[0073] In formula (1), i is the imaginary unit, and e is the natural constant. F0(u,v) is the Fourier transform function, and A0 represents the DC information component of the 0th-order diffracted light. B0 and B1 both represent the frequency domain information of the sample carried by the +1st-order diffracted light; (u,v) represents the angular spectrum distribution. α represents the amplitude of the +1st-order diffracted light. This indicates the phase of the +1st order diffracted light; Representing complex amplitude information, the Fourier transform process includes a positive frequency component and a negative frequency component; among them, This represents the positive frequency component of the +1st order diffracted light. This represents the negative frequency component of the +1st order diffracted light; The complex amplitude index is represented by α, which represents the amplitude. Indicates phase, This represents the positive frequency information of the +1st order diffracted light. This represents the negative frequency information of the +1st order diffracted light.

[0074] The first interference fringe image IF1 and the second interference fringe image IF2 are subjected to Fourier transform by the formula (1) to obtain the first spectral information of the first interference fringe image IF1 and the second spectral information of the second interference fringe image IF2, thereby converting the spatial domain signal into the frequency domain signal. The first spectral information is subtracted from the second spectral information to obtain the sperm spectral information of the first sperm sample, that is, the spectral information of the sperm that did not undergo the acrosome reaction.

[0075] The first encapsulation phase of sperm that did not undergo acrosome response was analyzed using a phase unwrapping algorithm. Phase unwrapping was performed to obtain the phase map SP of sperm that did not undergo acrosome response; the second wrapped phase of sperm that had undergone acrosome response was obtained using a phase unwrapping algorithm. Phase unfolding was performed to obtain the phase map (SP) of sperm that had undergone acrosome reaction. acrosome The phase unwrapping algorithm can be summarized as follows: First, perform Fourier transforms on IF1 and IF2 (or IF3 and IF4) to obtain the spectra of the two interference fringe images. Then, apply windowed bandpass filtering to these two spectra. Subtract the two spectra of IF1 and IF2 (or IF3 and IF4) and use the arctangent function to unwrap the first (or second) wrapped phase containing only sperm. Finally, the LS algorithm is used to unfold the wrapped phase into continuous phase values. By subtracting this value from the background phase value, the true phase value of the sperm head is obtained.

[0076] In specific implementation, in step 4, the first sample spectrum image and the second sample spectrum image are processed by a window function bandpass filter, and the wrap-around phase is extracted.

[0077] For spectral analysis, only the spectrum of the +1st order diffracted light is taken. A window function bandpass filter is used to process and extract the encapsulated phase, precisely selecting and isolating the positive frequency information of the sperm sample containing the target phase information. .

[0078] Solving the first and second wrap phases using the arctangent function The calculation formula is shown in the following formula (2).

[0079] (2)

[0080] In formula (2), g is the encapsulated phase; g0 is the complex field containing the object light wave after the inverse Fourier transform, g r Let be the reference light wave field after the inverse Fourier transform, and (x, y) represent the spatial spectral position coordinates. Im and Re are respectively g0(x, y)·g r * The phase value of the imaginary and real parts of (x, y) (where "·" is the dot product operator for complex numbers / vectors) is obtained by using the arctangent function.

[0081] The processing procedure for the images of the second sperm smear is the same as that for the first sperm smear.

[0082] The second encapsulation phase map of the sperm that has undergone acrosome reaction is obtained by subtracting the interference fringe map IF3 and the interference fringe background map IF4 from the interference fringe map IF4. Then, the phase difference between the sperm head nucleus and the background of the sperm that has undergone acrosome reaction, and the phase difference between the acrosome region of the sperm that has undergone acrosome reaction and the background are detected.

[0083] Step 5: Obtain the average phase value SPt of the sperm nucleus and the average phase value SPd of the sperm acrosome of sperm that did not undergo an acrosome reaction by using the phase map SP of sperm that did not undergo an acrosome reaction (i.e., sum the values ​​of specific parts of the sperm head and divide by the area to obtain the average).

[0084] In the first sperm smear, a large number of sperm meet the requirements for image acquisition. For example, 150 sperm are selected from the first sperm smear and steps 2 to 4 above are performed to obtain the phase map SP for each sperm. In the phase map SP of each sperm, the sperm nucleus phase value SPn and the acrosome phase value Spa of that sperm are obtained. Thus, for the first sperm smear, 150 sperm nucleus phase values ​​SPn and 150 acrosome phase values ​​Spa can be obtained. The average value of the 150 sperm nucleus phase values ​​SPn is calculated to obtain the average sperm nucleus phase value SPt of the 150 sperm; this SPt value is used as the average sperm nucleus phase value SPt of all sperm in the first sperm smear, that is, the average sperm nucleus phase value SPt of the sperm source of the subject. The average acrosome phase value Spa of 150 sperm cells is calculated to obtain the average acrosome phase value SPd of 150 sperm cells. This SPd value is used as the average acrosome phase value SPd of all sperm cells in the first sperm smear, i.e., the average acrosome phase value SPd of the sperm from the subject of the test, which is the source of the sperm. In practice, the number of sperm cells collected can be adjusted according to the actual situation to increase the number of samples collected and make the calculation of the average value more accurate.

[0085] Therefore, the sperm nucleus average phase value SPt is obtained by calculating the sperm nucleus phase value after obtaining the phase of each sperm in the first sperm smear on the sperm phase map SP of sperm that have not undergone acrosome reaction, and then averaging the sperm nucleus phase values ​​of multiple sperm in the acquired image on the first sperm smear. The same applies to the sperm acrosome average phase value SPd.

[0086] Phase mapping of sperm that have undergone acrosome reaction (SP) acrosome The average phase value SPt of sperm nuclei that have undergone acrosome reaction was obtained. acrosome and the average phase value of sperm acrosome SPd acrosome ;

[0087] For the second sperm smear, a large number of sperm also met the requirements for image acquisition. Similar to the first sperm smear, the sperm nucleus phase value and sperm acrosome phase value of 150 sperm were calculated, and then two averages were calculated: the average sperm nucleus phase value SPt. acrosome and the average phase value of sperm acrosome SPd acrosome .

[0088] like Figure 3As shown, the first distinguishing feature between sperm that did not undergo an acrosome response and those that did is that the acrosome phase value SPa of sperm that did not undergo an acrosome response is significantly higher than that of sperm that did undergo an acrosome response. acrosome SPa > SPa acrosome Furthermore, the two values ​​differ significantly, and the statistical difference between them is Spa-SPa. acrosome It follows a normal distribution.

[0089] Step 6: Calculate the first difference between SPt and SPd and SPt acrosome With SPd acrosome The second difference ;

[0090] Calculate the first difference between the average phase value SPt of sperm nuclei and the average phase value SPd of sperm acrosomes in sperm that did not undergo an acrosome reaction. ;Right now: And SPt > SPd.

[0091] Calculate the average phase value SPt of sperm nuclei that have undergone acrosome reaction. acrosome The average acrosome phase value SPd of sperm that have already undergone acrosome reaction acrosome The second difference ;Right now: = SPt acrosome -SPd acrosome And SPt acrosome > SPd acrosome .

[0092] like Figure 3 As shown, the second distinguishing feature between sperm that have not undergone an acrosome reaction and those that have:

[0093] The difference between the sperm nucleus phase value SPn and the acrosome phase value Spa in sperm that did not undergo the acrosome reaction, Spa-SPn, ranges from approximately 0.88; simultaneously, the difference between the averages of these two values, i.e., the first difference... The value range should also be around 0.88;

[0094] The sperm nucleus phase value SPn of sperm that have already undergone acrosome reaction acrosome Spa with the phase value of the acrosome acrosome The difference Spa acrosome -SPn acrosome The value range is around 0.4; meanwhile, the difference between the averages of these two values, i.e., the second difference... The value range should also be around 0.4.

[0095] Step 7: Calculate the acrosome-to-nucleus phase ratio R for sperm that have not undergone acrosome reaction and the acrosome-to-nucleus phase ratio R for sperm that have undergone acrosome reaction. acrosome ;

[0096] Based on the average phase value SPt of the sperm nucleus and the average phase value SPd of the sperm acrosome of sperm that did not undergo an acrosome reaction, the phase ratio R between the acrosome and the sperm nucleus of sperm that did not undergo an acrosome reaction is calculated; where R = SPd / SPt.

[0097] R is the average value of multiple sperm in the first sperm sample. Here, Spa is the acrosome phase value of a specific sperm in the first sperm smear, and SPd is the average acrosome phase value of a certain number x sperm collected from the first sperm smear, i.e., the average acrosome phase value of x sperm is SPd = (SPd1 + SPd2 + SPd3 + ... + SPd...). x ) / x; where SPd x This represents the acrosome phase value of the x-th sperm.

[0098] Based on the average phase value SPt of sperm nuclei that have undergone acrosome reaction acrosome The average acrosome phase value SPd of sperm that have already undergone acrosome reaction acrosome Calculate the phase ratio R between the acrosome and the sperm nucleus in sperm that have undergone acrosome reaction. acrosome Among them, R acrosome = SPd acrosome / SPt acrosome .

[0099] and R acrosome It is calculated as the average of multiple sperm from the second sperm sample. Among them, Spa... acrosome SPd represents the acrosome phase value of a sperm in a second sperm smear. acrosome SPd is the average acrosome phase value of a certain number x sperm collected from the second sperm smear. acrosome =(SPd acrosome1 +SPd acrosome2 + SPd acrosome3 +…..+ SPd acrosomex ) / x; where SPd acrosomex This represents the acrosome phase value of the x-th sperm.

[0100] Step 8: Using the parameters SPt, SPd, , R, SPt acrosome SPd acrosome , and Racrosome Through comparative analysis, rules for distinguishing between sperm that have not undergone acrosome reaction and sperm that have undergone acrosome reaction were obtained.

[0101] The distinction judgment rule includes three distinguishing features: a first distinguishing feature, a second distinguishing feature, and a third distinguishing feature. A detailed comparison of these three distinguishing features is provided below. Based on extensive statistical data, SPt, SPd, , R, SPt acrosome SPd acrosome , and R acrosom There are certain patterns, which can be summarized as the first distinguishing feature, the second distinguishing feature, and the third distinguishing feature. These three distinguishing features can then be used as a basis for comprehensively judging whether sperm have undergone the acrosome reaction.

[0102] The present invention provides a method for detecting sperm acrosome reaction based on phase imaging, which uses laser to directly irradiate sperm cells and inserts a transmission grating into the microscope imaging port to form multi-order diffraction light: 0th order diffraction light and 1st order diffraction light.

[0103] The 0th-order diffracted light passes through a pinhole at the focal plane of the L1 lens of the 4f system to remove object light information and serves as the reference light; the 1st-order diffracted light passes completely and serves as the object light; the 1st-order diffracted light, serving as the object light, propagates through the L2 lens of the 4f system and forms an interference fringe pattern on the camera target surface at a certain angle, which is then captured. Due to the different thicknesses and refractive indices of different parts of the sperm cell, and the certain optical path difference with the background, the interference fringes change. The spectrum of the sperm cell fringe pattern is obtained using Fourier transform, and then the object spectrum of the 1st-order diffracted light is obtained through windowed bandpass filtering. Simultaneously, the background fringe pattern without sperm is acquired. The two are subtracted, and the arctangent function is used to solve for the encapsulated phase pattern containing only sperm. Finally, the LS algorithm is used for encapsulated phase unwrapping. Measurements are taken for sperm with and without acrosome reaction, and the phase difference between the sperm head nucleus and the acrosome region and the background is detected.

[0104] The phase difference between the sperm head nucleus and the sperm acrosome region, whether or not the acrosome response has occurred. , The phase ratios R0 and R1 of the sperm head nucleus and acrosome region, respectively, with or without the presence of an acrosome reaction. acrosome This allows for size comparisons, providing label-free, high-contrast quantitative phase imaging. It can directly image rapidly swimming live sperm, reconstructing the phase values ​​of the sperm acrosome, and based on... , With R, R acrosome The analysis focused on the state of sperm acrosome reaction.

[0105] In specific implementation, in step 1, the light source of the detection optical path includes a laser source for forming interference fringes and an array of light-emitting diodes (LEDs) for forming a bright field.

[0106] like Figure 1 This is a diagram of the imaging optical path and device of the present invention. The DPM imaging source is a 638nm laser with a wavelength of 638nm. The LED array is a programmable LED array. This LED array consists of 92 LEDs and is controlled by a microcontroller unit on a personal computer.

[0107] The first laser beam emitted by the 638nm laser source is reflected by mirror M1 and then passes through the center of the LED lamp disk. Next, the first laser beam and the LED beam pass through the sample cell S and the smear within the sample cell, entering the objective lens MO. Then, the first laser beam and the LED beam pass through dichroic mirrors DM and are reflected by mirror M4 to the tube lens TL before reaching the beam splitter BS. At the beam splitter BS, the LED beam is reflected to camera Camera1, which acquires a bright-field image.

[0108] Then, the first laser beam emitted by the 638nm laser source passes through the fractional mirror BS and reaches the interference optical path of the DPM microscopy system. It passes through the 4f system (including lens L1, spatial filter PF and lens L2), forming interference fringes, which are then captured by the camera Camera2.

[0109] The second laser beam emitted from the 488nm laser source is reflected twice by the color filter F and the reflectors M2 and M3, and then reflected by the dichroic mirror DM to the rear pupil of the objective lens MO. After passing through the objective lens MO, it excites fluorescence imaging. The fluorescence imaging then passes sequentially through the dichroic mirror DM, the reflector M4, and the tube lens TL before entering the fractional mirror BS.

[0110] In summary, bright-field imaging and fluorescence imaging are directly reflected to Camera 1 via the fractional mirror (BS) for imaging. Figure 1 The portion of the optical path above the central mirror M4 is based on a commercial microscope (Olympus, IX73), while the portion of the optical path to the left of mirror M4 (i.e., the 4f system) is a modified version of the commercial microscope.

[0111] In specific implementation, in step 1, the detection optical path includes a spatial filter PF.

[0112] During the optical path modification, a transmission grating beam splitter G is placed at the focal plane of the microscope's tube lens TL. Due to the periodicity of the transmission grating beam splitter G, multi-order sample diffracted light is generated at different angles. Therefore, interference occurs when different orders of light are collected. The multi-order diffracted light modulated by the transmission grating beam splitter G passes through a 4f system. The focal lengths of the cemented doublet lenses L1 and L2 are both 50mm, allowing the beam to be focused onto the back focal plane of L1 and filtered by a spatial filter PF with a pinhole. In the 4f system, the focal plane of lens L1 filters the 0th order diffracted light with PF (pinhole size 50 μm), allowing only the DC component to pass through, resulting in a uniform reference light wave; the +1st order diffracted light passes completely through the pinhole of the spatial filter PF as the object light wave. At this time, the 0th order reference wave and the +1st order object light wave form interference fringes at a certain angle on the camera surface Camera 2 and are collected. Bright-field imaging and fluorescence imaging are acquired by the high-sensitivity camera Camera 1. Camera 2 only collects the first laser beam from the 638nm laser source to obtain the interference fringe image.

[0113] In specific implementation, step 2, the process of obtaining the first sperm smear includes: a first sperm sample coating step and a first sperm sample fixation step.

[0114] In practice, the first sperm sample and the second sperm sample are stored in a test tube; then two glass slides and two coverslips are taken out; a coverslip is placed on one of the glass slides to form a smear, thus obtaining two smears: the first smear and the second smear.

[0115] The smear preparation process for the first sperm sample is as follows:

[0116] Step 211: First sperm sample coating step; Take a glass slide, coat the first sperm sample onto the glass slide; Place the first coverslip on the first glass slide coated with the first sperm sample to obtain the first sperm smear containing the first sperm sample, that is, the first sperm smear that has not undergone acrosome reaction;

[0117] Step 212: First sperm sample fixation step; air-dry the first sperm smear; place the first sperm smear that has not undergone acrosome reaction in a sample cell containing 95% (V / V) ethanol for 30 minutes to fix it, air-dry it, and then place it on the sample cell S of the detection optical path for imaging.

[0118] In specific implementation, step 2, the process of obtaining the second sperm smear includes: the acrosome reaction step of the second sperm sample, the coating step of the second sperm sample, and the fixation step of the second sperm sample.

[0119] The smear preparation process for the second sperm sample is as follows:

[0120] Step 221: Second sperm sample acrosome reaction step; For the second sperm sample, calcium ion carrier A23187 was incubated in a water bath at 37°C for 15 minutes to induce the acrosome reaction, and the second sperm sample that has been sent for acrosome reaction was obtained.

[0121] Step 222: Second sperm sample coating step; Take another glass slide, coat the second sperm sample that has undergone acrosome reaction onto the glass slide; Place another coverslip on the glass slide coated with the second sperm sample that has undergone acrosome reaction, to obtain a first sperm smear containing the first sperm sample, i.e., the first sperm smear that has not undergone acrosome reaction, and obtain a second sperm smear that has undergone acrosome reaction.

[0122] Step 223: Second sperm sample fixation step; air-dry the second sperm smear; place the second sperm smear that has undergone acrosome reaction in a sample cell containing 95% (V / V) ethanol for 30 minutes to fix it, air-dry it, and then place it on the sample cell S of the detection optical path for imaging.

[0123] In specific implementation, the sample cell S is preferably a counting plate. The counting plate is a transparent plate with a grid, which can be used to locate sperm on the smear, thereby determining the accurate location of the sperm and making observations.

[0124] In specific implementation, during the acrosome reaction step of the second sperm sample, calcium ion carrier A23187 is incubated in a water bath for 15 minutes to induce the acrosome reaction.

[0125] In practice, for the second sperm sample, calcium ion carrier A23187 was incubated in a water bath at 37°C for 15 minutes to induce the acrosome reaction and obtain a sperm acrosome reaction sample.

[0126] In the process of processing sperm samples, since most normal sperm cannot spontaneously undergo acrosome reaction, the same batch of samples were divided into two groups: a control group (no special treatment) and a group treated with calcium ion carrier A23187. The calcium ion carrier in the A23187 stock solution induces calcium ion influx in the acrosome region of sperm, changing the resting potential to an action potential, receiving information, releasing acrosomal enzymes, and thus inducing the acrosome reaction.

[0127] Steps 3 through 6 were used to image different male sperm samples, and the differences in phase values ​​for acrosome reaction assessment were statistically analyzed. Taking acrosome phase as an example, sperm that had or had not undergone an acrosome reaction were divided into two categories: sperm that had not undergone an acrosome reaction and sperm that had undergone an acrosome reaction. The acrosome phase values ​​of the two types of sperm samples were statistically analyzed and compared with the corresponding sperm fluorescence labeling results of phase imaging. It was found that the acrosome phase results of sperm that had not undergone an acrosome reaction could be basically distinguished.

[0128] like Figure 3 Statistical analysis of multiple sperm samples from different sources was conducted using the Kolmogorov-Smirnov test (KS test) to identify the acrosome phase, nucleus-acromion phase difference, and phase ratio in sperm that had undergone acrosome reaction. The results showed that the p-values ​​were all less than 0.001 in the significance analysis and less than 0.05 in the normality test, indicating significant differences and conformity to a normal distribution. The advantage of the KS test lies in its ability to capture the distributional differences in sample statistical results and its intuitive visualization and interpretation.

[0129] In specific implementation, step 4 uses the LS algorithm in the minimum p-norm to unwrap the phase and perform phase unwrapping.

[0130] The phase unwrapping method uses the Least Squares (LS) algorithm in the least p-norm to unwrap the phase and perform phase unwrapping model.

[0131] In specific implementation, the phase expansion model is shown in the following formula (3);

[0132] (3)

[0133] In formula (3), Operators representing Euclidean norms It is the gradient operator; the L2 norm fidelity term represents the measurement of the package phase in formula (2). Phase to be determined The absolute continuous phase can be obtained by taking the extreme value of the sum of squared errors between them. .

[0134] The LS (Least Squares) algorithm has strong noise robustness, effectively handling the problem of discontinuities in the true phase of sperm, and ensuring the smoothness and fidelity of sperm balance. Finally, statistical analysis is performed on the phase between each sperm nucleus and acrosome.

[0135] In practice, the sperm acrosome reaction detection method based on phase imaging also includes the following two steps:

[0136] Step 9: After performing fluorescent staining on the first sperm smear that did not undergo acrosome reaction and the second sperm smear that did undergo acrosome reaction, fluorescent images of the first sperm smear and the second sperm smear were taken respectively.

[0137] After collection, the sample cells (after drying and evaporation of all ethanol solution) were immersed in peanut lectin fluorescent dye (PNA-FITC) and stained in a refrigerator at 4 degrees Celsius in the dark for more than 1 hour. Each slide was washed with deionized water, and the sperm staining was observed and photographed using excitation light of 450-490 nm. All collected data were statistically analyzed.

[0138] Step 10: Based on the staining of sperm in the fluorescence image from Step 9, determine whether the sperm have undergone the acrosome reaction.

[0139] In specific implementation, in step 9, the first sperm smear and the second sperm smear are immersed in peanut lectin fluorescent staining solution for staining treatment.

[0140] In practice, sperm smears are immersed in peanut lectin fluorescent staining solution. After staining, fluorescence images of the corresponding sperm samples are obtained to observe their acrosome reaction status. For sperm that have not undergone an acrosome reaction, the entire acrosome region of sperm with intact acrosomes is stained. For sperm that have undergone an acrosome reaction, only the equatorial zone or post-acrosomal region is stained (i.e., only partial staining). By determining whether the acrosome region of the sperm is fully stained or only partially stained, it is possible to accurately determine whether an acrosome reaction has occurred. The fourth distinguishing feature between sperm that have not undergone an acrosome reaction and those that have is: the acrosome region of sperm that have not undergone an acrosome reaction is fully stained after staining, while the acrosome region of sperm that have undergone an acrosome reaction is only partially stained (only the equatorial zone or post-acrosomal region is stained, and the anterior acrosome region is unstained).

[0141] The acrosome reaction is determined by staining, and the results are compared with those in steps 1 to 8 above to verify the accuracy of the determinations in steps 1 to 8. (Fluorescent staining is a traditional method and the current gold standard for clinical identification, so it is used as the verification standard for several distinguishing features of this invention.)

[0142] The judgment rules in steps 1 to 8 were further verified by the results of fluorescent staining: the phase difference between the nuclear phase and the acrosome phase of sperm that have undergone acrosome response is significantly higher than that of sperm that have not undergone acrosome response.

[0143] In phase imaging, a coherent laser is used as the illumination source, and the collimated beam enters the optical microscope for imaging. A blazed grating placed on the microscope's imaging plane, due to its periodicity, generates multi-order sample diffraction light at different angles. To ensure the two beams satisfy the interference conditions, the 0th and +1st order diffracted beams are typically selected as the object beam and reference beam, respectively. Because the aperture of lens L1 in the 4f system is limited, only a portion of the diffracted beams can pass through. A spatial filter with a physical mask is placed at the Fourier surface of lens L1. The +1st order diffracted beam passes through a pinhole, allowing only the DC component to pass through, resulting in a uniform plane wave, which serves as the reference beam for the interference path. The 0th order beam passes completely and serves as the object beam. The two beams pass through lens L2, and the interference field fringes are captured by a CCD camera at its back focal plane.

[0144] In the process of acquiring quantitative phase data, the light intensity acquired by the CCD camera is first subjected to Fourier transform to obtain the spectral distribution of the hologram. Only the +1 order spectrum is taken in the spectral analysis to recover the required phase. This is then processed by windowed bandpass filtering and utilizing off-axis holography. Figure 3 The characteristic of term spectral separation is that it directly filters out zero-order terms and conjugate terms, resulting in a spectral distribution containing only the object light wave. Finally, an inverse Fourier transform is performed to obtain a complex field containing only the object light wave. Considering system measurement errors, various background noises, and the additional phase factor generated by sample magnification through the objective lens, since only the fundamental frequency and no other spectral components are present after filtering, resulting in a wrapper containing only phase information, only the reference light wave field without sample information needs to be taken. The phase wrapper containing sample information is directly subtracted from the phase wrapper of the reference light wave, and the LS phase unwrapping algorithm is used to perform phase expansion to obtain the accurate two-dimensional phase distribution of the sperm head.

[0145] like Figure 3 The results are presented as statistical analyses of samples from five different individuals (sperm samples from different test subjects). Figure 3 In the table, (a), (b), and (c) represent the average phase value of the sperm acrosome, the phase difference between the sperm head nucleus and the sperm acrosome region, and the phase ratio between the sperm head nucleus and the sperm acrosome region, respectively. These parameters are distributed across five different sample groups (AI and AR). The AI ​​group represents samples that did not undergo an acrosome reaction, and the AR group represents samples that did. Sample N corresponds to the same sample group, and N can be 1, 2, 3, 4, or 5. The following distinguishing features are observed:

[0146] (1) First distinguishing feature: The statistical analysis of each N is about 150-160 samples (150 sperm were collected from two sperm smears of each person). It can be clearly seen that the acrosome phase value of the AI ​​group sperm is significantly higher than that of the AR group sperm that has undergone acrosome reaction, and the results conform to the normal distribution.

[0147] (2) Second distinguishing feature: The phase difference between the sperm nucleus and the acrosome in sperm that have undergone acrosome reaction (i.e., the difference between the average values ​​of these two values ​​is the second difference). The phase difference between the sperm nucleus and acrosome in sperm that did not undergo an acrosome reaction is around 0.4, and the difference between the average of these two values ​​is the first difference. It's only around 0.88;

[0148] (3) Third distinguishing feature: The acrosome-to-nuclear phase ratio R of sperm that has undergone acrosome reaction acrosome The ratio of the acrosome to the nucleus phase in sperm that did not undergo an acrosome reaction is around 0.78, while the ratio of the acrosome to the nucleus phase in sperm that did not undergo an acrosome reaction is only around 0.53.

[0149] The p-value of the results for the control group that did not undergo the acrosome reaction was obtained using the KS test (p-value is...). Figure 3 The asterisks marking each sample indicate significant differences (three stars represent significant differences). The acrosome results showed significant differences between sperm samples with and without an acrosome reaction.

[0150] In addition, the judgment of the first to third distinguishing feature points is verified by the fourth distinguishing feature.

[0151] Experiments have shown that the accuracy of using the above three distinguishing features to determine whether sperm has undergone the acrosome reaction is basically the same as that of the traditional fourth distinguishing feature method.

[0152] A cell counting chamber was used to fix and record sperm samples, which facilitated the analysis of multimodal imaging results of different sperm. For the reconstructed phase image of the entire sperm, including the sperm head, since the acrosome region of the sperm accounts for about 40-70% of the head, the acrosome and head of the sperm were segmented by image processing software using bright-field fluorescently labeled images. After smoothing, cropping, scaling, and registration, the images were matched with the phase images of the corresponding sperm to obtain the average phase value between the sperm nucleus and the acrosome. The average value of the nucleus is obtained by subtracting the phase value between the sperm head and the acrosome. For the same individual sample, there is a certain difference in the average phase value between the nucleus and acrosome of sperm samples that have undergone acrosome reaction and those that have not. This is because the sperm head cell nucleus contains highly concentrated genetic material. However, the phase difference between the nucleus and acrosome of sperm samples that have undergone acrosome reaction is significantly higher than that of sperm samples that have not undergone acrosome reaction. This result was further verified by fluorescent staining. After statistically analyzing approximately 150 to 160 samples from five different individuals, it was found that there was a large difference in the phase between the nucleus and acrosome of sperm samples that have undergone acrosome reaction and those that have not. The p-value of the KS test showed a significant difference, and the statistical results conformed to a normal distribution. Therefore, phase imaging can be used as a new method to assist in the judgment of sperm acrosome reaction.

[0153] In this invention, sperm acrosomes are identified using stained bright-field images. Since the sperm acrosome can be distinguished regardless of its integrity, whether the acrosome region or the post-acromial region is stained after fluorescent staining, the acrosome can be differentiated. Then, the average phase of the sperm head nucleus and the average phase of the sperm acrosome are obtained from the phase image, and the phase values ​​are evaluated using a least-squares (LS) phase unwrapping algorithm. Specifically, the spectral information of the image is obtained through stripe samples and background images, and the wrapped phase is extracted using a window function bandpass filter.

[0154] The working principle of the sperm acrosome reaction detection method based on phase imaging of this invention is as follows: Based on interferometric diffraction phase imaging technology, without the need for fluorescent staining, it utilizes the change in optical path difference caused by changes in refractive index and thickness to determine whether the sperm has undergone an acrosome reaction through statistical analysis of the sperm head phase. The interferometric phase imaging method can directly perform high-contrast, label-free quantitative phase imaging of sperm, quantitatively obtain the phase value of the sperm head, directly analyze the sperm head structure, and verify the feasibility of the method using fluorescent labeling results. Furthermore, the occurrence of the acrosome reaction can be determined based on the phase between the sperm nucleus and the acrosome.

[0155] This invention provides a phase imaging-based method for detecting sperm acrosome reaction, primarily addressing the label-free imaging problem in detecting acrosome reaction in live sperm samples. In the diffraction phase imaging system, a coherent laser is used as the illumination source. After irradiating live sperm cells, the multi-order diffraction of the grating output light field is combined with a spatial filter to separate the reference light field and the object light field, thereby obtaining the sperm cell interferogram. The Fourier transform method is used to extract phase information, and the minimum p-norm phase unwrapping algorithm is used to obtain the absolute continuous phase of the sperm. Since the sperm acrosome reaction releases acrosomal enzymes, there is a difference in acrosome thickness between sperm that have undergone acrosome reaction and those that have not, which in turn leads to a change in phase value, thus revealing the occurrence of the sperm acrosome reaction. Using the phase imaging method for quantitative phase imaging of live sperm to detect the occurrence of the sperm acrosome reaction has the following advantages: (1) it avoids the damage to sperm caused by traditional fluorescent staining and labeling; (2) this method can be used for label-free rapid imaging of sperm, improving detection efficiency.

[0156] This invention combines a phase imaging system and features single-frame detection, rapid detection, high stability, high contrast, and quantitative analysis.

[0157] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

[0158] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for detecting sperm acrosome reaction based on phase imaging, characterized in that, Includes the following steps: Step 1: Set up the optical path for sperm acrosome reaction detection; Step 2: Divide sperm from the same sperm source into two sperm samples, and prepare a first sperm smear and a second sperm smear respectively, and place them into the detection optical path; Step 3: Obtain bright-field images of the first and second sperm smears, as well as two interference fringe images of the sperm-containing and sperm-free samples, respectively; Step 4: Obtain the phase map (SP) of sperm that have not undergone acrosome reaction using two interference fringe images from the first sperm smear; obtain the phase map (SP) of sperm that have undergone acrosome reaction using two interference fringe images from the second sperm smear. acrosome ; Step 5: Obtain the average phase value SPt of the sperm nucleus and the average phase value SPd of the sperm acrosome from the phase map SP of sperm that did not undergo acrosome reaction; Phase mapping of sperm that have undergone acrosome reaction (SP) acrosome The average phase value SPt of sperm nuclei that have undergone acrosome reaction was obtained. acrosome and the average phase value of sperm acrosome SPd acrosome ; Step 6: Calculate the first difference between SPt and SPd and SPt acrosome With SPd acrosome The second difference ; Step 7: Calculate the acrosome-to-nucleus phase ratio R for sperm that have not undergone acrosome reaction and the acrosome-to-nucleus phase ratio R for sperm that have undergone acrosome reaction. acrosome ; Step 8: Using the parameters SPt, SPd, , R, SPt acrosome SPd acrosome , and R acrosome Through comparative analysis, rules for distinguishing between sperm that have not undergone acrosome reaction and sperm that have undergone acrosome reaction were obtained.

2. The method for detecting sperm acrosome reaction based on phase imaging according to claim 1, characterized in that, In step 1, the light source of the detection optical path includes a laser source for forming interference fringes and an array of light-emitting diodes (LEDs) for forming a bright field.

3. The method for detecting sperm acrosome reaction based on phase imaging according to claim 1, characterized in that, In step 1, the detection optical path includes a spatial filter PF.

4. The method for detecting sperm acrosome reaction based on phase imaging according to claim 1, characterized in that, The detection optical path includes a sample cell; the sample cell is a transparent plate with a grid.

5. The method for detecting sperm acrosome reaction based on phase imaging according to claim 1, characterized in that, In step 2, the process of obtaining the first sperm smear includes: a first sperm sample coating step and a first sperm sample fixation step.

6. The method for detecting sperm acrosome reaction based on phase imaging according to claim 1, characterized in that, In step 2, the process of obtaining the second sperm smear includes: an acrosome reaction step of the second sperm sample, a coating step of the second sperm sample, and a fixation step of the second sperm sample.

7. The method for detecting sperm acrosome reaction based on phase imaging according to claim 6, characterized in that, In the acrosome reaction step of the second sperm sample, calcium ion carrier A23187 was incubated in a water bath for 15 minutes to induce the acrosome reaction.

8. The method for detecting sperm acrosome reaction based on phase imaging according to claim 1, characterized in that, In step 4, the phase unwrapping method of the LS algorithm in the minimum p-norm is used to perform phase expansion, obtaining the phase maps SP of sperm that have not undergone acrosome response and SP of sperm that have undergone acrosome response. acrosome .

9. The method for detecting sperm acrosome reaction based on phase imaging according to claim 1, characterized in that, It also includes the following two steps: Step 9: After performing fluorescent staining on the first sperm smear that did not undergo acrosome reaction and the second sperm smear that did undergo acrosome reaction, fluorescent images of the first sperm smear and the second sperm smear were taken respectively. Step 10: Determine whether the sperm have undergone the acrosome reaction based on the staining of the sperm in the fluorescence image from Step 9.

10. The method for detecting sperm acrosome reaction based on phase imaging according to claim 9, characterized in that, In step 9, the first sperm smear and the second sperm smear are immersed in peanut lectin fluorescent staining solution for staining treatment.

Images