CN111855657A - Sperm motility evaluation method based on energy gradient - Google Patents

Sperm motility evaluation method based on energy gradient Download PDF

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CN111855657A
CN111855657A CN202010711921.4A CN202010711921A CN111855657A CN 111855657 A CN111855657 A CN 111855657A CN 202010711921 A CN202010711921 A CN 202010711921A CN 111855657 A CN111855657 A CN 111855657A
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energy gradient
sperm motility
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optical trap
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张青川
徐探
吴尚犬
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University of Science and Technology of China USTC
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Abstract

The invention relates to a sperm motility evaluation method based on energy gradient, which is characterized in that a binary complementary mask plate and a plurality of grating phase images for generating single light spots are generated based on a randomized binary complementary mask strategy, and a single hologram for generating a dot matrix optical trap is synthesized; loading the obtained hologram on a spatial light modulator, and carrying out phase regulation on an emergent light field to obtain an optical trap array with energy gradient; and capturing a batch of sperms in the microfluidic channel according to the obtained optical trap array with the energy gradient, and observing and tracking the escape process of each captured sperm in each energy gradient optical trap, thereby completing sperm motility evaluation based on the energy gradient. The invention can quantitatively, quickly and simply measure the sperm motility.

Description

Sperm motility evaluation method based on energy gradient
Technical Field
The invention relates to the field of in-vitro sperm quality detection, in-vitro assisted reproduction and artificial insemination, in particular to a sperm motility evaluation method based on energy gradient, which is applied to the sperm motility detection in large batch.
Background
In the fields of breeding industry or assisted reproduction, the key to improving the efficiency of artificial insemination is to screen high-quality sperms. The assessment of sperm quality depends on a number of biomarkers including sperm specific volume, sperm density, osmolarity, seminal plasma pH, seminal plasma chemical composition, enzyme activity, ATP concentration, motility, morphology and ultrastructure, fertilization ability, etc. Sperm motility is often an important parameter for sperm quality measurement due to its ease of assessment. The evaluation of sperm motility is divided into a subjective method, a semi-quantitative method and a quantitative computer-aided method. Wherein, the subjective method is to divide the sperm motility into 5 grades according to the percentage of the motile sperm in the visual field: 1) level 0, i.e. no motion state; 2) grade 1, i.e. up to 25% of the sperm are moving; 3) grade 2, i.e. up to 50% of the sperm are moving; 4) grade 3, i.e. up to 75% of the sperm are moving; 5) grade 4, i.e. more than 75% of the sperm are moving. It follows that the subjective method relies on experienced evaluators, statistical results cannot be obtained, and measurements are unreliable. In order to increase the accuracy of sperm motility measurements, the percentage of initially motile sperm can be measured by semi-quantitative analysis of sperm motility by two or more independent observers, in particular, recorded sperm motility video is played at low speed on a screen with a grid, however this method is very time consuming. The quantitative Computer-Aided principle consists of a Computer-Aided Sperm Analysis system (CASA), which visualizes and digitizes Sperm images by a Computer and processes and analyzes the Sperm images, in a manner that, despite its high reproducibility, is complex and expensive. Therefore, there is a need to develop a quantitative, rapid, and simple method for measuring sperm motility.
Disclosure of Invention
The technical problem of the invention is solved: the method overcomes the defects of the prior art, and provides a sperm motility evaluation method based on energy gradient, which can accurately, quickly and simply quantitatively characterize the movement motility of a large batch of sperm by generating a plurality of optical trap arrays with energy gradient, thereby overcoming the defects of inaccuracy, time consumption, complexity and the like of the traditional characterization mode, getting rid of the requirements of professionals with rich experience, and reducing the operation requirements and the cost of sperm motility characterization.
The technical solution of the invention is as follows: a sperm motility evaluation method based on energy gradient is shown in figure 1 and comprises the following steps:
firstly, generating a binary complementary mask plate and a plurality of grating phase images for generating single light spots by using a computer based on a randomized binary complementary mask strategy, and synthesizing to obtain a single hologram for generating a dot matrix optical trap, namely a synthetic phase.
And secondly, loading the synthesized phase obtained in the first step onto a spatial light modulator shown in fig. 4, and performing phase control on an emergent light field to obtain an optical trap array with N energy gradients.
And thirdly, capturing a batch of sperms in the microfluidic channel based on the optical trap array with N energy gradients obtained in the second step, and observing and tracking the escape process of each captured sperm in each energy gradient optical trap, thereby completing the sperm motility evaluation based on the energy gradients.
The first step is realized by the following specific steps: based on a randomized binary complementary mask strategy, i.e. according to the energy ratio r of N traps1:r2:…:rNN-1 thresholds th calculated by normalization1,th2,…,thN-1Then, the computer-generated initial matrix M is used to match all the thresholds (th)1,th2,…,thN-1) Comparing to obtain N binary complementary masks R1,R2,…,RNFor example, when the value of an element at a certain position in M belongs to [0, th1]When R is1The element value of the corresponding position in the group is set to 1, and the values of the remaining positions are set to 0; when the value of an element at a certain position in M belongs to [ th1,th2]When R is2The element value of the corresponding position in the group is set to 1, and the values of the remaining positions are set to 0; when the value of an element at a certain position in M belongs to [ thN-1,1]When R isNThe element value of the corresponding position in the group is set to 1, and the values of the remaining positions are set to 0; n binary complementary masks are obtained based on the method. Finally, a plurality of grating phases P for generating single point-like optical traps1,P2,…,PNMask plate R complementary to binary value1,R2,…,RNAnd correspondingly multiplying and summing to obtain a synthetic phase P.
The second step is realized by the following specific steps: and turning on a laser light source, collimating and beam-expanding a columnar light beam emitted by the laser light source, irradiating the columnar light beam onto a spatial light modulator, loading the synthetic phase P generated in the first step onto the spatial light modulator, changing the phase of an emitted light field due to the phase regulation and control effect of the spatial light modulator, adjusting the axial position of an objective lens, focusing a focus on the lower surface of a microfluidic channel, and generating an optical trap array with N energy gradients on a focal plane.
The third step is specifically realized by the following steps: injecting a sperm solution into the inlet end of the microfluidic channel, wherein the sperm with the weakest vitality can be captured by the optical traps with various energy gradients due to the attraction effect of the optical traps, and the sperm with the greatest vitality can only be captured by the optical trap with the strongest light intensity; and the injection of the culture solution at different constant speeds can be started at different stages after the injection of the sperm solution, so as to accelerate the escape process of each sperm captured by the energy gradient optical trap. And (4) observing and tracking the escape condition of a large quantity of captured sperms through video imaging to finish the evaluation process of the sperm motility.
Compared with the prior art, the invention has the advantages that: the optical tweezers technology endows researchers with the micro-nano control capability, and can capture, control and quantitatively detect a research object in a non-contact mode. Meanwhile, as a sensitive force sensing probe, the optical tweezers are rarely characterized by sperm motility. The invention provides a method for evaluating sperm motility through a generated optical trap with energy gradient, which can quickly and accurately determine the sperm motility, and has the advantages that the evaluation process is accelerated and the sperm motility is quantitatively evaluated by combining the constant-speed flow of microfluid, and the method has the following advantages: 1) the measurement is accurate, the repeatability is high, and the theoretical value can reach 100%; 2) the sperm motility can be measured qualitatively and quantitatively; 3) the requirements on the expertise and experience of operators are reduced; 4) the operation is simple and convenient, and the detection cost can be quite low.
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FIG. 1 is a flow chart of an embodiment of a sperm motility assessment method based on energy gradient;
FIG. 2 is a diagram of an embodiment of the present invention for evaluating sperm motility using an energy gradient optical trap;
FIG. 3 is a schematic diagram of acquisition of a synthetic phase for generating an optical trap with an energy gradient;
FIG. 4 is a schematic of the optical path for generating an energy gradient optical trap.
Detailed Description
The present invention will be described in detail below with reference to the accompanying drawings and examples.
As shown in fig. 2, the method of the present invention includes the following steps:
first, based on the randomized binary complementary mask strategy, as shown in fig. 3, a binary complementary mask R is generated1,R2,R3,R4And four grating phase patterns P for generating a single light spot1,P2,P3,P4In which the grating phases P of different spatial frequencies1,P2,P3,P4The point-like light spots can be generated at four different specified positions in the camera view field; a binary complementary mask plate R1,R2,R3,R4And a grating phase pattern P1,P2,P3,P4The respective multiplications are summed to finally obtain a single composite phase P for generating an optical trap array with an energy gradient.
The specific implementation steps are as follows:
1) generating a randomized initial matrix M of size mxn, with elements in M obeying a uniform distribution between [0,1], according to the resolution of the spatial light modulator or the phase plate;
2) setting the number of optical traps N to 4 and the energy ratio between the traps r1:r2:…:rNCalculating N thresholds th by normalizationiI.e. by
Figure BDA0002596866460000031
i＝1,2,3；
3) As shown in fig. 3, the randomized initial matrix M is compared with the threshold values th obtained in the previous stepiN binary complementary masks R can be determinediDimension is the same as the randomized initial matrix M, and RiThere is no intersection between them, and the result of the union is a full matrix (a matrix with all 1 element values)). For example, when the element value at a position in the randomized initial matrix M belongs to [0, th1]When R is1The element value of the corresponding position is set to 1, and the element values of the other positions are set to 0; when the element value at a position in the randomized initial matrix M belongs to [ th1,th2]When R is2The element value of the corresponding position is set to 1, and the element values of the other positions are set to 0; when the element value at a position in the randomized initial matrix M belongs to [ th2,th3]When R is3The element value of the corresponding position is set to 1, and the element values of the other positions are set to 0; when the element value at a position in the randomized initial matrix M belongs to [ th3,1]When R is4The element value of the corresponding position is set to 1, and the element values of the other positions are set to 0;
1) generating N grating phases P for creating optical traps at specific locationsi；
2) A binary complementary mask plate RiAnd the grating phase PiMultiplying and summing correspondingly to obtain a synthetic phase P, mathematical expression
Figure BDA0002596866460000041
N＝4。
In FIG. 3, the randomized initial matrix M is generated by a computer whose elements obey [0,1]]Uniform distribution of the components; threshold thiIs by means of a user-set energy ratio r1,…,rNCalculated by a normalization operation, i.e.
Figure BDA0002596866460000042
1, 2, …, N-1; phase P of gratingiFor generating a single optical trap at a specified location; the synthetic phase P loaded on the spatial light modulator is formed by the binary complementary mask plate RiAnd the grating phase PiIs synthesized to obtain
Figure BDA0002596866460000043
N＝4。
In the second step, the synthetic phase P obtained in the first step is loaded on the spatial light modulator shown in FIG. 4,the phase of the emergent light field is adjusted to obtain four optical trap arrays with energy gradient as shown in FIG. 2, and the energy ratio is equal to the phase P corresponding to the grating in the first stepiBinary complementary mask plate RiThe ratio of squares of the number of effective pixels (the effective pixels refer to the binary complementary mask R)iA pixel with a middle element value equal to 1).
Thirdly, capturing a batch of sperms in the microfluidic channel based on the four optical trap arrays with energy gradients obtained in the second step, observing and tracking the escape process of each captured sperm in each energy gradient optical trap, thereby completing the sperm motility evaluation based on the energy gradients, since the loaded synthetic phase P is capable of generating an array of optical traps at a given location, the location of the optical traps in the camera's field of view is also determined, and when the camera is used to record the capture and escape process of a single sperm in its field of view, the position of individual sperm in the field of view can be obtained by a particle tracking algorithm, such as the centroid method, by comparing the position of each optical trap to the position of each sperm, the residence time of a single sperm in the optical trap can be obtained, and if the single sperm stays at the optical trap of each energy gradient, the single sperm can be captured by the optical trap. Specifically, if a single sperm can be captured by the four energy gradient optical traps of fig. 2, the sperm is labeled with a motility rating of 1; labeling a sperm motility rating of 2 if a single sperm can be captured by the energy gradient optical trap 2-4 of figure 2; labeling a sperm motility rating of 3 if a single sperm can be captured by the energy gradient optical trap 3-4 of figure 2; if a single sperm can only be captured by the optical trap 4 with the strongest energy, then the mirror viability grade is marked as 4; based on this for qualitative characterization of each sperm motility in the visual field. In addition, a speed threshold value of each sperm escaping from the visual field is determined through the constant-speed flow of the external injection culture solution, the speed threshold value is used as a quantitative basis for the sperm motility characterization, if a single sperm can escape from the optical trap at a set speed minimum value (10 mu m/s), the speed is used as an evaluation index of the sperm motility, if a single sperm can escape from the trap at a set speed maximum value (100 mu m/s), the speed is used as the evaluation index of the sperm motility, and meanwhile, the speed interval is set to be 10 mu m/s to quantitatively estimate the sperm escaping threshold value in the speed range of 10-100 mu m/s, so that the sperm motility evaluation based on the energy gradient is completed.
In order to realize the representation of the motility of mass sperms by using the energy gradient optical trap, the invention introduces an energy gradient trap generation method, the principle of which is shown in figure 3, and the grating phases P of four different spatial frequencies1、P2、P3、P4Mask plate R complementary to binary value1、R2、R3、R4The resultant phases P are obtained by multiplication and summation, and after loading them onto the spatial light modulator, the four optical traps with energy gradients illustrated in fig. 3 will be generated in the objective focal plane, which can be generalized to more optical traps with energy gradients based on the same principle.
To generate multiple optical traps with energy gradient experimentally, based on the basic structure of holographic optical tweezers, as shown in fig. 4, a captured laser is collimated and spread, and then irradiated onto a Spatial Light Modulator (SLM), and a desired synthetic phase is loaded
Figure BDA0002596866460000051
N-4, four optical traps with energy gradient are generated in focal plane of objective lens, and position of single optical trap is determined by corresponding grating phase PiWhile the energy ratio between the traps satisfies r1:r2:…:rNI.e. a binary complementary mask R1、R2、R3、R4Is the ratio of the squares of the effective pixel numbers of (a). Because the interaction results of the sperms with different motility activities and the optical traps with different energy gradients are different, for example, the sperms with weaker motility can be captured by the optical traps with lower energy and can also be captured by the traps with higher energy; and the sperm with stronger motility can only be captured by a high-energy trap. Then after designing a weak to strong array of optical traps (in FIG. 4, the trap energy is gradually increased from top to bottom, and the trap energy is uniform in the horizontal direction), the array passes through the microfluidic channel in FIG. 4Injecting a quantitative sperm sample into the inlet, recording the capture and escape conditions of the sperm by the array trap by using a camera, and classifying the sperm into a plurality of grades according to the capture result of a single sperm by the strong and weak trap, for example, classifying the sperm into 4 grades in fig. 4, namely, grade 1, grade 2, grade 3 and grade 4, wherein the corresponding sperm motility is increased from the weakest to the strongest; specifically, the least motile sperm can be captured by the level 1 (weakest) optical trap, while the most motile sperm can only be captured by the level 4 (strongest) optical trap. Thus, the array optical trap with gradient energy can rapidly classify the motility level of the sperm. In addition, the captured sperm can be impacted by injecting fluid with proper flow rate through the microfluidic channel, and the motility level of the sperm can be determined more quickly because the sperm with lower motility first escape in the process of increasing the flow rate.
The schematic diagram of the experimental optical path is shown in fig. 4, wherein the collimated and expanded laser beam is reflected to the aperture behind the objective lens by the dichroic mirror after being subjected to the phase control action of the spatial light modulator (on which the synthetic phase P is loaded), and is used for generating an optical trap array with an energy gradient on the focal plane of the objective lens; the illumination light source is used for illuminating the microfluidic channel, so that experiment observation and recording are facilitated, the illumination light source penetrating through the microfluidic channel is collected by the objective lens, then transmits the dichroic mirror, is reflected to the sleeve lens by the reflector, and images the sperm sample on the camera; the sleeve lens and objective lens are matched to achieve the desired imaging magnification.
The above examples are provided only for the purpose of describing the present invention, and are not intended to limit the scope of the present invention. The scope of the invention is defined by the appended claims. Various equivalent substitutions and modifications can be made without departing from the spirit and principles of the invention, and are intended to be within the scope of the invention.

Claims (4)

1. A sperm motility evaluation method based on energy gradient is characterized by comprising the following steps:
firstly, generating a binary complementary mask plate and a plurality of grating phase images for generating a single light spot based on a randomized binary complementary mask strategy, and synthesizing to obtain a single hologram for generating a dot matrix optical trap, namely a synthesized phase;
secondly, loading the synthetic phase obtained in the first step onto a spatial light modulator, and carrying out phase regulation on an emergent light field to obtain an optical trap array with N energy gradients;
thirdly, capturing a batch of sperms in the microfluidic channel according to the optical trap array with the N energy gradients obtained in the second step; the concrete implementation is as follows:
recording the capturing and escaping processes of a batch of sperms in the microfluidic channel by using a camera, acquiring the position of each sperm in a camera view field by using a particle tracking algorithm, comparing the positions of N energy gradient optical traps with the position of each sperm, acquiring the residence time of each sperm in each optical trap, if each sperm in the view field stays at the set N energy gradient optical traps, indicating that the batch of sperms in the camera view field can be captured by the optical traps, and if a single sperm in the batch of sperms can be captured by the optical traps with various energy gradients, the weakest sperm motility is evaluated, the sperm motility grade is marked as 1, if a single sperm can only be captured by the optical trap with the strongest energy, the sperm motility grade is marked as N, thereby qualitatively characterizing the motility of each sperm in the visual field, wherein the value of N is set according to the number of sperms in the field of view of the camera and the resolution of the spatial light modulator in principle; in addition, the speed threshold value of each sperm escaping from the camera visual field is determined through the constant-speed flow of the external injection culture solution, the speed threshold value is taken as the quantitative basis of the sperm motility characterization, and if a single sperm is at the set minimum speed value v1The speed is used as the evaluation index of sperm motility when the sperm can escape from the optical trap; if the single sperm is at the set maximum velocity vXEscape from the trap, and the speed is used as an evaluation index of sperm motility; when the range of velocity values is v1To vXAnd determining a speed threshold value of the batch of sperm escaping, and taking the threshold value as a sperm motility evaluation index, thereby completing sperm motility evaluation based on the energy gradient.
2. An energy gradient-based sperm motility assessment method according to claim 1, wherein: in the first step, the randomized binary complementary mask strategy is implemented as: according to the energy ratio r of N traps1:r2:…:rNN-1 thresholds th calculated by normalization1,th2,…,thN-1Obeying [0,1] using computer generated element values]With uniformly distributed randomized initial matrix M and its element values are compared with all threshold values thiComparing to obtain N binary complementary masks RiFinally, a plurality of grating phases P for generating single point-like optical trapsiComplementary mask R with binary valuesiMultiply and sum to obtain a single composite phase P, wherein
Figure FDA0002596866450000011
i＝1,2,…,N。
3. An energy gradient-based sperm motility assessment method according to claim 1, wherein: the second step is realized by the following specific steps: and turning on a laser light source, collimating and beam-expanding a columnar laser beam emitted by the laser light source, irradiating the columnar laser beam onto a spatial light modulator, loading the synthetic phase P generated in the first step onto the spatial light modulator, changing the phase of an emitted light field due to the phase regulation and control effect of the spatial light modulator, adjusting the axial position of an objective lens, focusing the focus of the objective lens to the lower surface of a microfluidic channel, and generating an optical trap array with an energy gradient on the focal plane of the objective lens.
4. An energy gradient-based sperm motility assessment method according to claim 1, wherein: in the third step, the particle tracking algorithm acquires the centroid position of each sperm in the field of view by using a centroid method.
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