Method for controlling the state and movement of spermatozoa

Abstract

The application relates to a sperm posture and motion state control method. The control method comprises the following steps: S1: using two laser beams to act on the head of a sperm; S2: adjusting the positions and powers of the two laser beams acting on the head of the sperm; the sperm can be controlled at a selected position, and the posture (referring to the relative position and angle between the head of the sperm and an imaging focal plane) and motion state of the sperm are controlled to obtain images of sperms in different directions and different motion states, wherein the posture and motion state of the head of the sperm and the relative focal plane of the sperm tail are included. The sperm relative to the imaging plane is controlled in the posture and motion state by adjusting the positions and powers of the two laser beams acting on the head of the sperm; for example, the motion of the sperm tail in a plane parallel to the imaging focal plane is controlled by controlling the posture of the sperm, and the motion images of the head of the sperm and the tail in a three-dimensional space are observed and obtained for a long time.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of sperm movement control, and particularly relates to a method for controlling the posture and movement state of sperm. BACKGROUND

[0002] The motility of sperm (i.e., the movement ability of sperm) is a key factor for normal fertilization, and is a main standard for evaluating sperm quality in clinical practice. Sperm is mainly composed of a head containing genetic material and a flagellum. Sperm must pass through thousands of body lengths in the complex oviduct to reach the ovum, and the natural selection process in the body is also mainly realized in this process. The complex and tortuous epithelial tissue in the oviduct determines that the head of the sperm needs to interact with the epithelial surface and bypass the obstacles to reach the oocyte before fertilization.

[0003] Among them, the movement state of the head of the sperm plays an important role in successful fertilization. The movement state of the head of the sperm can affect the movement of the tail, and further affect the navigation and crossing ability of the sperm in the female reproductive tract. By receiving external mechanical sensory information, the head of the sperm can adjust the movement mode of the flagellum to adapt to different environmental conditions and overcome various barriers. When the sperm encounters obstacles such as mucus, viscous liquid or the surface of cells, these obstacles will exert resistance or produce mechanical stimulation. The head can perceive the mechanical stimulation and transmit relevant information to the flagellum, so as to adjust the activity of the flagellum. This adjustment can affect the swing frequency, amplitude and wavelength of the flagellum to adapt to different environmental conditions and overcome various barriers. Therefore, studying the movement state of the head of the sperm helps to further understand the behavior and adaptability of the sperm in the complex female reproductive tract, and provides a basis for developing more effective reproductive medical technology and treating infertility.

[0004] And the sperm obtains movement ability through the bending flagellum, and the flagellum exhibits different jumping modes under different external conditions, and this change is crucial for the sperm to complete fertilization, so observing the movement of the flagellum is of great significance for understanding the movement law of the flagellum and solving reproductive problems.

[0005] Because sperm has strong motility, it is easy to escape from the imaging field of view or the imaging focal plane during observation. For long-term observation of a single sperm, the traditional technique is to adhere the sperm head to a culture dish to constrain the movement of the sperm, so as to observe the head or flagellum of the sperm. However, this method limits the movement of the flagellum at the dish interface, and thus cannot reflect the real movement of the flagellum in three-dimensional space. Single-beam tweezer technology is an advanced technology for fixing and operating micron-level and nanometer-level objects by using the optical force generated by tightly focused laser. It is widely used in the fields of biomedical research and nanotechnology, and can non-contact capture and manipulate micro-objects such as cells and nanoparticles. The principle of optical tweezer is based on the optical force exerted on micro-objects by laser beams. When a focused laser beam irradiates a micro-object, photons are absorbed or reflected by the object, resulting in a change in photon momentum, thereby generating an optical force that can be used to fix, manipulate or suspend the micro-object. In recent years, optical tweezer has also been applied to sperm motility analysis. Usually, single-beam tweezers are used to fix sperm and measure parameters such as escape force and head rotation frequency to determine sperm motility. However, when using single-beam tweezer technology to manipulate and observe sperm, the sperm head in the optical trap can be considered as a three-axis ellipsoid. When the sperm head deviates from the center of the optical trap, the optical field will exert a torque on the sperm head in the optical trap. Since the long axis of the sperm head is parallel to the longitudinal axis of the sperm, the torque exerted by the optical field will make the long axis of the ellipsoid parallel to the direction of the optical axis, and the sperm cell is often captured vertically, which makes it difficult to clearly observe the flagellum of the sperm for a long time, thereby limiting the study of flagellum movement, see Figure 1 In addition, the rotation state of the sperm head cannot be controlled at will when the sperm is manipulated by the single-beam tweezer. SUMMARY

[0006] (I) Technical problems to be solved

[0007] In view of the above-mentioned shortcomings and deficiencies of the prior art, the present application provides a method for controlling the body state and movement state of sperm, which solves the technical problem that the traditional sperm imaging technology cannot reflect the real movement of a single sperm in three-dimensional space during long-term observation, and the technical problem that the single-beam tweezer technology cannot control the body state and movement state of sperm relative to the imaging plane for a long time.

[0008] (II) Technical solutions

[0009] In order to achieve the above-mentioned purposes, the main technical solutions adopted by the present application include:

[0010] In a first aspect, the present application provides a method for controlling the body state and movement state of sperm, comprising:

[0011] S1: introducing two laser beams into a sample pool of a stage and acting on the sperm head of a single sperm;

[0012] S2: adjusting the position and power of the two beams of laser respectively acting on the sperm head;

[0013] the sperm head can be bound at a selected position, the long axis of the sperm head can be parallel to the focal plane, and the sperm head can be controlled at different rotating states to obtain images of the sperm head at different rotating states; or,

[0014] the sperm head can be bound at a selected position, the long axis of the sperm head can be parallel to the focal plane, and the sperm tail of the sperm can be controlled to move in a plane parallel to the focal plane to obtain a moving image of the sperm tail in three-dimensional space.

[0015] According to the present application, the step S1 further comprises:

[0016] After the laser in the laser generating assembly emits a single beam of laser, the single beam of laser is expanded by the first lens and the second lens in sequence, and then the expanded single beam of laser is divided into the two beams of laser with polarization perpendicular to each other by the half-wave plate and the first polarization beam splitter prism, the two beams of laser are reflected by the first mirror and the second mirror one by one, and then the two beams of laser are combined in space by the second polarization beam splitter prism, the combined two beams of laser are introduced into the sample cell of the objective table as control laser, and act on the sperm head of the single sperm;

[0017] The step S2 further comprises:

[0018] The mirror surface angles of the first mirror and the second mirror are adjusted to adjust the positions of the two beams of laser respectively acting on the sperm head;

[0019] The size of the output current of the laser in the laser generating assembly is adjusted, and the included angle between the fast axis of the half-wave plate and the single beam of laser is adjusted to adjust the power of the two beams of laser respectively acting on the sperm head.

[0020] According to the present application, the step S1 further comprises:

[0021] Two lasers in the laser generating assembly emit single beams of laser, which are expanded by the first lens and the second lens in sequence, and then the polarization directions are adjusted by the half-wave plate to form the two beams of laser with polarization perpendicular to each other, the two beams of laser are reflected by the first mirror and the second mirror one by one, and then the two beams of laser are combined in space by the second polarization beam splitter prism, the combined two beams of laser are introduced into the sample cell of the objective table to form two control laser traps and act on the sperm head of the single sperm;

[0022] The step S2 further comprises:

[0023] Adjusting the mirror angles of the first mirror and the second mirror to adjust the positions where the two laser beams act on the sperm heads, respectively;

[0024] Adjusting the powers of the single laser beams emitted by the two lasers to adjust the powers where the two laser beams act on the sperm heads, respectively.

[0025] According to the present application, the step S2 further comprises:

[0026] Firstly adjusting the positions where the two laser beams act on the sperm heads, respectively;

[0027] Then adjusting the powers where the two laser beams act on the sperm heads, respectively.

[0028] According to the present application, the step S1 further comprises:

[0029] After introducing the sperm sample into the sample pool of the stage, injecting the polystyrene ball solution into the sample pool of the stage, and introducing the two laser beams into the sample pool of the stage, the two laser beams capture the polystyrene balls, respectively.

[0030] The focal point distance of the two laser beams is calculated according to the gray centers of the two captured polystyrene balls, and the positions of the two laser beams in the sample pool are adjusted to make the initial focal point distance of the two laser beams in the sample pool be 1.5-3.5 μm.

[0031] Subsequently, the two control laser beams formed by the two laser beams with the initial focal point distance of 1.5-3.5 μm act on the sperm heads of the single sperm, respectively.

[0032] According to the present application, the target sperm to be controlled and observed in the sample pool is selected, and the stage is moved to drive the target sperm to move to the positions of the two laser beams with the initial focal point distance of 1.5-3.5 μm and be captured by the two laser beams, so that the two control laser beams formed by the two laser beams act on the sperm heads of the single sperm, respectively.

[0033] According to the present application, the step S1 further comprises:

[0034] Firstly, the illumination light emitted by the illumination assembly is projected into the sample pool of the stage after introducing the sperm sample into the sample pool of the stage, and the illumination light irradiated on the sperm is scattered by the sperm and imaged into the image collector.

[0035] Subsequently, the two laser beams are introduced into the sample pool of the stage.

[0036] The step S2 further comprises:

[0037] After adjusting the positions and powers of the two laser beams respectively acting on the sperm head, the image collector can collect sperm head images or sperm flagellum images and save image information of the sperm head images or sperm flagellum images into a computer.

[0038] According to the present application,

[0039] The illumination light emitted by the illumination light source in the illumination assembly is sequentially projected into the sample cell of the object table through the third lens and the condenser, and the illumination light irradiated on the sperm is scattered by the sperm and sequentially imaged into the image collector through the objective lens, the dichroic mirror and the imaging lens.

[0040] According to the present application, the method further comprises step S3: after obtaining the sperm head images or sperm flagellum images, sequentially comprising the following image processing steps:

[0041] S31: image smoothing and noise reduction are performed on the sperm head images or sperm flagellum images;

[0042] S32: the contrast of the sperm head images or sperm flagellum images is enhanced to improve the visibility of details;

[0043] S33: edge detection is performed on the sperm head images or sperm flagellum images to extract the contour and position information of the sperm head or sperm flagellum;

[0044] S34: the contour of the sperm head images or sperm flagellum images is binarized to convert into a black-and-white binary image;

[0045] S35: a threshold is set to separate the sperm head images or sperm flagellum images from the background;

[0046] S36: part of the connected domains in the sperm head images or sperm flagellum images is removed;

[0047] S37: skeletonization processing is performed on the remaining connected domains in the sperm head images or sperm flagellum images to extract the axis information of the sperm head or sperm flagellum.

[0048] (III) Beneficial effects

[0049] The beneficial effects of the present invention are as follows: the method for controlling the body and motion state of sperm of the present invention can introduce two laser beams into the sample pool of the stage to form two control laser beams acting on the sperm head of a single sperm. By adjusting the position and power of the two laser beams acting on the sperm head, the sperm head can be constrained to a selected position, with the long axis of the sperm head parallel to the focal plane, and the sperm head can be controlled to be in different rotational states, thereby obtaining images of the sperm head in different rotational states. The sperm head can also be constrained to a selected position, with the long axis of the sperm head parallel to the focal plane, thereby further controlling the movement of the sperm flagellum in a plane parallel to the focal plane, thereby obtaining an image of the movement of the sperm flagellum in three-dimensional space. Thus, the method of the present invention achieves control over individual sperm, including confining the sperm to a specific position. It can further control the orientation and position of the sperm head relative to the imaging plane, as well as the different rotational states of the sperm head. This allows for long-term observation of the complete form and motion patterns of the sperm head and flagellum under different motion states, facilitating the determination of sperm motility characteristics, improving the accuracy and reliability of the data, and providing an effective indicator for evaluating sperm quality. Furthermore, the method of applying two laser beams separately to the sperm head disperses the laser energy to two locations on the sperm head, reducing the laser's phototoxicity to the sperm, ensuring sperm motility during observation, and enabling long-term sperm observation. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 Schematic diagram of a method for controlling sperm posture and movement using single optical tweezers;

[0051] Figure 2 Schematic diagram of the method for controlling sperm posture and motility of the present invention;

[0052] Figure 3 Schematic diagram of a first control system for sperm posture and movement status according to the present invention;

[0053] Figure 4 is a schematic diagram of a second control system for sperm posture and movement status of the present invention;

[0054] Figure 5 This is a schematic diagram of the three-dimensional motion trajectory of the sperm flagellum obtained by the method for controlling the sperm body shape and motion state of the present invention.

[0055] [Description of Reference Numerals]

[0056] 1: sperm; 11: sperm head; 12: sperm flagellum;

[0057] 21: single laser beam; 22: two laser beams; 23: focal plane;

[0058] 31: stage; 32: objective lens; 33: dichroic mirror;

[0059] 41: laser; 42: first lens; 43: second lens;

[0060] 5: light splitting assembly; 51: half wave plate; 52: first polarization splitting prism;

[0061] 61: first mirror; 62: second mirror; 63: second polarization splitting prism;

[0062] 7: telescopic assembly; 71: fourth lens; 72: fourth mirror; 73: fifth lens;

[0063] 8: illumination assembly; 81: illumination light source; 82: third lens; 83: third mirror; 84: condenser;

[0064] 9: image acquisition assembly; 91: imaging lens; 92: image acquisition device; 93: computer. DETAILED DESCRIPTION

[0065] In order to better explain the present application, so as to be understood, the present application is described in detail by specific embodiments in combination with the accompanying drawings.

[0066] Embodiment 1

[0067] Referring to Figures 1-5 The sperm posture and motion state control method proposed by the embodiment of the present application comprises the following steps:

[0068] S1: Introducing two laser beams 22 into the sample cell of the stage 31 to form two control laser beams acting on the sperm head 11 of a single sperm 1.

[0069] S2: Adjusting the positions and powers of the two laser beams 22 acting on the sperm head 11:

[0070] The sperm head 11 can be bound at a selected position, and the long axis of the sperm head 11 is parallel to the focal plane 23 and the sperm head 11 is in different rotating states, so as to obtain sperm head images in different rotating states; or,

[0071] The sperm head 11 can be bound at a selected position, and the long axis of the sperm head 11 is parallel to the focal plane 23, so as to further control the sperm tail 12 of the sperm 1 to move in a plane parallel to the focal plane 23, so as to obtain the motion image of the sperm tail 12 in the three-dimensional space.

[0072] Therefore, the sperm body state and motion state control method can introduce the two laser beams 22 into the sample pool of the stage 31 to form two control lasers acting on the sperm head 11 of the single sperm 1. By adjusting the positions and powers of the two laser beams 22 acting on the sperm head 11, the torque generated by the rotation of the sperm tail 12 to swing the sperm head 11 can be reduced or eliminated when the sperm head 11 of the sperm 1 needs to be controlled and observed, so as to bind the sperm head 11 at a selected position, make the long axis of the sperm head 11 parallel to the focal plane 23, and make the sperm head 11 in different rotation states, so as to observe and obtain the images of the sperm head 11 in different rotation states on the focal plane 23 for a long time. When the sperm tail 12 of the sperm 1 needs to be observed, the torque applied to the sperm head 11 by the light field can be reduced or eliminated, so as to bind the sperm head 11 and the sperm tail 12 at a selected position, make the long axis of the sperm head 11 parallel to the focal plane 23, and control the sperm tail 12 to move in a plane parallel to the focal plane 23, so as to observe and obtain the motion images of the sperm tail 12 in three-dimensional space for a long time. Therefore, the sperm body state control method realizes the control of the body state of the single sperm 1, further controls the orientation and position of the sperm head 11 relative to the imaging plane, and controls different rotation states of the sperm head 11, so as to realize the long-time separate observation of the complete body state of the sperm head 11 and the sperm tail 12 in different motion states, so as to facilitate the determination of the motion characteristics of the sperm 1, improve the accuracy and reliability of the data, and provide an effective index for evaluating the quality of the sperm 1. At the same time, the two control lasers formed by the two laser beams 22 acting on the sperm head 11 can also disperse the laser energy to two positions of the sperm head 11, so as to reduce the phototoxicity of the laser to the sperm 1, ensure the vitality of the sperm 1 during observation, and realize the long-time observation of the sperm 1.

[0073] Further, to realize the imaging of the sperm 1, the step S1 further includes:

[0074] After the sperm sample is introduced into the sample pool of the stage 31, the illumination light emitted by the illumination assembly 8 is projected into the sample pool of the stage 31, and the illumination light irradiated on the sperm 1 is scattered by the sperm 1 and then imaged into the image collector 92. Then, the two laser beams 22 are introduced into the sample pool of the stage 31.

[0075] Specifically, the illumination light emitted by the illumination light source 81 in the illumination assembly 8 is projected into the sample pool of the stage 31 through the third lens 82 and the condenser lens 84 in sequence, and the illumination light irradiated on the sperm 1 is scattered by the sperm 1 and then imaged into the image collector 92 through the objective lens 32, the dichroic mirror 33 and the imaging lens 91 in sequence.

[0076] After adjusting the positions and power of the two laser beams 22 acting on the sperm head 11, the image information of the actual observation required for the relative position and motion state of the sperm 1 to the imaging focal plane can be collected by the image collector 92, and then saved to the computer 93.

[0077] Further, in order to ensure accurate experimental control, the initial relative position of the two laser beams 22 needs to be calibrated, therefore, step S1 further includes:

[0078] After introducing the sperm sample into the sample pool of the stage 31, injecting the polystyrene ball solution into the sample pool of the stage 31, and introducing the two laser beams 22 into the sample pool of the stage 31, the two laser beams 22 capture the polystyrene balls. Then, the focal point distance of the two laser beams 22 is calculated by the gray center of the captured two polystyrene balls, and the positions of the two laser beams 22 in the sample pool of the stage 31 are adjusted so that the initial focal point distance of the two laser beams 22 in the sample pool of the stage 31 is 1.5-3.5 μm.

[0079] Subsequently, the two control laser beams formed by the two laser beams 22 with an initial focal point distance of 1.5-3.5 μm act on the sperm head 11 of the single sperm 1 respectively.

[0080] Specifically, the target sperm to be controlled and observed in the sample pool is selected, and the stage 31 is moved to drive the target sperm to move to the two laser beams 22 with an initial focal point distance of 1.5-3.5 μm and be captured by the two laser beams 22, so that the two control laser beams formed by the two laser beams 22 act on the sperm head 11 of the single sperm 1 respectively.

[0081] Further, the adjustment of the positions and power of the two laser beams 22 acting on the sperm head 11 includes two methods:

[0082] The first adjustment method is:

[0083] Step S1 further includes:

[0084] After the laser 41 in the laser generating assembly emits a single laser beam 21, the single laser beam 21 is expanded by the first lens 42 and the second lens 43 in sequence, and then the expanded single laser beam 21 is divided into two laser beams 22 with perpendicular polarizations by the half-wave plate 51 and the first polarization beam splitter prism 52 in sequence. The two laser beams 22 are reflected by the first mirror 61 and the second mirror 62 respectively, and then combined in space by the second polarization beam splitter prism 63. The combined two laser beams 22 are introduced into the sample pool of the stage 31 to form two control laser beams and act on the sperm head 11 of the single sperm 1.

[0085] Step S2 further includes:

[0086] Adjusting the mirror angles of the first mirror 61 and the second mirror 62 to adjust the positions where the two control lasers act on the sperm head 11 respectively;

[0087] Adjusting the output current of the laser 41 in the laser generating assembly and adjusting the included angle between the fast axis of the half-wave plate 51 and the single laser 21 to adjust the powers of the two lasers 22 acting on the sperm head 11 respectively.

[0088] The second adjusting method is:

[0089] The step S1 further comprises:

[0090] The single lasers 21 emitted by the two lasers 41 in the laser generating assembly are expanded by the first lens 42 and the second lens 43 in sequence and then pass through the half-wave plate 51 to adjust the polarization directions, so as to form two lasers 22 with perpendicular polarizations, which are reflected by the first mirror 61 and the second mirror 62 respectively and then combined in space by the second polarization beam splitter prism 63, and the combined two lasers 22 are introduced into the sample cell of the objective table 31 to form two optical traps and act on the sperm head 11 of the single sperm 1.

[0091] The step S2 further comprises:

[0092] Adjusting the mirror angles of the first mirror 61 and the second mirror 62 to adjust the positions where the two control lasers act on the sperm head 11 respectively;

[0093] Adjusting the powers of the single lasers 21 emitted by the two lasers 41 to adjust the powers of the two lasers acting on the sperm head 11 respectively.

[0094] Preferably, in the above two control methods, the positions where the two lasers 22 act on the sperm head 11 are adjusted first, and then the powers of the two lasers 22 acting on the sperm head 11 are adjusted.

[0095] In actual use, the positions and powers of the two lasers 22 acting on the sperm head 11 can be adjusted to obtain the image information of the body shape and motion state of the sperm 1 relative to the imaging focal plane as required in actual observation:

[0096] When the sperm head 11 needs to be controlled and observed, the positions of the two lasers 22 acting on the sperm head 11 are adjusted first to make the long axis of the sperm head 11 parallel to the focal plane, and then the powers of the two lasers 22 acting on the sperm head 11 are adjusted to control the sperm head 11 to be in different rotating states (when the power is increased, the rotating speed of the sperm head 11 can be reduced until it stops rotating).

[0097] When the sperm flagellum 12 needs to be controlled and observed: first, adjust the positions of the two laser beams 22 acting on the sperm head 11 so that the long axis of the sperm head 11 is parallel to the focal plane, and then adjust the power of the two laser beams 22 acting on the sperm head 11 to constrain the sperm head 11 at the selected position, thereby being able to control the movement of the sperm flagellum 12 in the plane parallel to the focal plane.

[0098] It should be noted that due to the differences in size and activity of different sperm 1, the combination of the positions and powers of the two laser beams 22 acting on the sperm head 11 is different in order to achieve the same posture or movement state.

[0099] Further, the sperm posture and movement state control method provided by the embodiment further includes the following steps:

[0100] S3: After obtaining the sperm head image or sperm flagellum image, the following image processing steps are included in sequence:

[0101] S31: Image smoothing and noise reduction are performed on the sperm head image or sperm flagellum image;

[0102] S32: The contrast of the sperm head image or sperm flagellum image is enhanced to improve the visibility of details;

[0103] S33: Edge detection is performed on the sperm head image or sperm flagellum image to extract the contour and position information of the sperm head 11 or sperm flagellum 12;

[0104] S34: The contour of the sperm head image or sperm flagellum image is binarized to convert it into a black and white binary image;

[0105] S35: A threshold is set to separate the sperm head image or sperm flagellum image from the background;

[0106] S36: Part of the connected domain in the sperm head image or sperm flagellum image is removed;

[0107] S37: Skeletonization processing is performed on the remaining connected domain in the sperm head image or sperm flagellum image to extract the axis information of the sperm head 11 or sperm flagellum 12.

[0108] Therefore, after image processing of the obtained sperm head image or sperm flagellum image, the axis information of the sperm head 11 and sperm flagellum 12 can be obtained, and the position data of the sperm head 11 and sperm flagellum 12 can be obtained, so as to further calculate and obtain the dynamic parameters of the sperm head 11 and sperm flagellum 12, such as the swing frequency, amplitude, and curvature of the sperm flagellum 12. The above parameter information can be used as an index for evaluating the activity of sperm 1, and provides reference data for further studying and understanding the movement behavior and function of sperm 1.

[0109] Specifically, in step S31, the image is smoothed and noise is reduced by using Gaussian filtering or other filtering methods. In step S33, edge detection is performed by using Sobel or Canny algorithm. In step S36, some connected domains are removed, which can exclude irrelevant regions caused by noise or other interference factors. In step S37, the skeletonization processing can obtain the axis information of the sperm head 11 or the sperm flagellum 12, so as to facilitate subsequent measurement and analysis.

[0110] Embodiment 2

[0111] Based on the embodiment 1, the embodiment provides a sperm posture control system applying the sperm posture and motion state control method in the embodiment 1.

[0112] The control system includes a laser generating assembly and a stage 31.

[0113] The laser generating assembly is configured to form two laser beams 22. The two laser beams 22 form two control laser beams acting on the sperm head 11 of the target sperm in the sample cell of the stage 31, so as to reduce or eliminate the torque generated by the rotation of the sperm flagellum 12 to swing the sperm head 11, bind the sperm head 11 at a selected position, and make the long axis of the sperm head 11 parallel to the focal plane 23 and the sperm head 11 in different rotation states, so as to obtain and observe the sperm head image of the complete posture of the sperm head 11 in different rotation states; or reduce or eliminate the torque applied to the sperm head 11 by the light field when the sperm head 11 deviates from the center of the control laser beam, bind the sperm head 11 and the sperm flagellum 12 at a selected position, and make the long axis of the sperm head 11 parallel to the focal plane 23 and control the sperm flagellum 12 to move in a plane parallel to the focal plane 23, so as to obtain and observe the image of the sperm flagellum 12 moving in the three-dimensional space.

[0114] Further, the control system includes the following two setting modes:

[0115] The first setting mode of the control system is that the control system includes the laser generating assembly, the light splitting assembly 5, the first reflecting mirror 61 and the second reflecting mirror 62 arranged in sequence along the transmission direction of the laser. The laser generating assembly includes a laser 41. Along the transmission direction of the laser, the light splitting assembly 5 includes a half-wave plate 51 and a first polarization light splitting prism 52 in sequence.

[0116] After the single laser beam 21 is emitted by the laser 41, the single laser beam 21 is expanded by the lens group, and then is divided into two laser beams 22 with polarization directions perpendicular to each other by the half-wave plate 51 and the first polarization beam splitter prism 52 in turn. The two laser beams 22 are reflected by the first mirror 61 and the second mirror 62 in turn, and then are combined in space by the second polarization beam splitter prism 63. The combined two laser beams 22 are used as control laser, and are introduced into the sample cell of the objective table 31 by the objective lens to form two control laser beams acting on the sperm head 11 of the single sperm 1.

[0117] In use, the first mirror 61 and the second mirror 62 are rotated to adjust the included angle between the mirror surface of the first mirror 61 and the second mirror 62 and the corresponding laser beam, and then the positions of the two laser beams 22 acting on the sperm head 11 are adjusted. The size of the output current of the laser 41 in the laser generating assembly is adjusted, and the included angle between the fast axis of the half-wave plate 51 and the single laser beam 21 is adjusted, so that the power of the two laser beams 22 acting on the sperm head 11 is adjusted. Specifically, the size of the output current of the laser 41 is adjusted to adjust the total output power of the emitted single laser beam, and the half-wave plate 51 is rotated to adjust the included angle between the fast axis of the half-wave plate 51 and the polarization direction of the single laser beam 21, so that the power ratio of the two laser beams 22 is changed to adjust the power of the two control laser beams formed by the two laser beams 22 and acting on the sperm head 11. More specifically, the included angle between the half-wave plate 51 and the transmission direction of the single laser beam 21 is 0-90°.

[0118] Specifically, the first lens 42 and the second lens 43 are arranged along the transmission direction of the laser in turn between the laser 41 and the beam splitting assembly 5.

[0119] After the single laser beam 21 is emitted by the laser 41 in the laser generating assembly, the single laser beam 21 is expanded by the first lens 42 and the second lens 43 in turn, and then the expanded single laser beam 21 is divided into two laser beams 22 with polarization directions perpendicular to each other by the half-wave plate 51 and the first polarization beam splitter prism 52 in turn.

[0120] More specifically, the first lens 42 and the second lens 43 are confocal convex lenses, and the curved surfaces of the first lens 42 and the second lens 43 face in opposite directions.

[0121] Preferably, the diameter of the expanded single laser beam 21 is 6-8mm.

[0122] The second setting mode of the control system is that the control system comprises a laser generating assembly, a first mirror 61 and a second mirror 62 arranged along the transmission direction of the laser in turn. The laser generating assembly comprises two lasers 41. The laser generating assembly comprises two lasers 41, two first lenses 42, two second lenses 43 and two half-wave plates 51.

[0123] The single laser beams 21 emitted by the two lasers 41 in the laser generating assembly are expanded by the first lens 42 and the second lens 43 in sequence, and then the polarization directions of the single laser beams 21 are adjusted by the half-wave plate 51 to form two laser beams 22 with perpendicular polarization directions. The two laser beams 22 are reflected by the first mirror 61 and the second mirror 62 in sequence, and then the two laser beams 22 are combined in space by the second polarization beam splitter prism 63. The combined two laser beams 22 are introduced into the sample cell of the objective stage 31 to form two control laser beams acting on the sperm head 11 of the single sperm 1.

[0124] In use, the mirror angles of the first mirror 61 and the second mirror 62 are adjusted to adjust the positions where the two control laser beams act on the sperm head 11. The powers of the single laser beams 21 emitted by the two lasers 41 are adjusted to adjust the powers of the two control laser beams acting on the sperm head 11.

[0125] Preferably, the lasers 41 in the above two control system setting modes are continuous lasers with a wavelength of 1064 nm.

[0126] Preferably, in the above two control system setting modes, the power adjustment range of each of the two laser beams 22 is 80-200 mw, and the power adjustment ranges of the two laser beams 22 are consistent to effectively balance the forces of the two control laser beams formed by the two laser beams 22 acting on the sperm head 11, so that the sperm 1 can be stably bound at a selected position, the long axis of the sperm head 11 is parallel to the focal plane 23, and the sperm head 11 is controlled to be in different rotating states, or the long axis of the sperm head 11 is parallel to the focal plane 23, and the sperm tail 12 is controlled to move in a plane parallel to the focal plane 23.

[0127] In the above two control system setting modes, by adjusting the powers and positions of the two control laser beams formed by the two laser beams 22 acting on the sperm head 11, the body state of the sperm head 11 and the sperm tail 12 can be adjusted: the positions of the sperm head 11 and the sperm tail 12 can be fixed, the long axis of the sperm head 11 can be controlled to be parallel to the focal plane 23, the sperm head 11 can be controlled to be in different rotating states, and the sperm tail 12 can be controlled to move in a plane parallel to the focal plane 23. Preferably, due to the large individual differences in the size and shape of the sperm head 11 of the sperm 1, the distance between the two control laser beams formed by the two laser beams 22 acting on the sperm head 11 is 1.5-3 μm, so that the two control laser beams formed by the two laser beams 22 can effectively capture different sperms 1.

[0128] Specifically, the first mirror 61 and the second mirror 62 are respectively installed on control tables that can accurately control the mirror angles thereof.

[0129] Further, the control system further comprises a telescope assembly 7 between the laser generating assembly and the objective lens 32, and an objective lens 32 between the telescope assembly 7 and the objective lens 32. The telescope assembly 7 comprises a fourth lens 71 and a fifth lens 73 in the transmission direction of the laser.

[0130] The fourth lens 71 and the fifth lens 73 are used to receive the two beams of laser 22 after spatially combined by the second polarization beam splitter 63, and enter the sample pool of the objective lens 32 to act on the sperm head 11.

[0131] Specifically, the objective lens 32 is arranged between the fifth lens 73 and the objective lens 31. The fourth lens 71 and the fifth lens 73 image the laser at the second mirror 62 to the back focal plane of the objective lens 32.

[0132] Specifically, the telescope assembly 7 further comprises a fourth mirror 72 between the fourth lens 71 and the fifth lens 73. The fourth mirror 72 is used to reflect the two beams of laser 22 passing through the fourth lens 71 to the fifth lens 73.

[0133] Further, the control system further comprises an illumination assembly 8 and an image acquisition assembly 9. After the sperm sample is introduced into the sample pool of the objective lens 31, the illumination light emitted by the illumination assembly 8 is projected into the sample pool of the objective lens 31, and the illumination light irradiated on the sperm 1 is imaged into the image acquisition assembly 9 after being scattered by the sperm 1.

[0134] Specifically, the illumination assembly 8 comprises an illumination light source 81, a third lens 82, a second mirror 83 and a condenser 84.

[0135] The illumination light emitted by the illumination light source 81 is projected into the sample pool of the objective lens 31 in turn through the third lens 82, the second mirror 83 and the condenser 84. The illumination light irradiated on the sperm 1 in the sample pool of the objective lens 31 is imaged into the image acquisition assembly 9 after being scattered by the sperm 1.

[0136] Preferably, the illumination light source 81 is an LED light source.

[0137] Further, the control system further comprises a dichroic mirror 33. The dichroic mirror 33 is located between the telescope assembly 7 and the objective lens 32, and between the image acquisition assembly 9 and the objective lens 32. The dichroic mirror 33 can separate beams according to wavelength.

[0138] In actual use, the two beams of laser 22 emitted by the telescope assembly 7 are introduced into the sample pool of the objective lens 31 in turn through the dichroic mirror 33 and the objective lens 32. The image reflected by the objective lens 32 is transmitted to the image acquisition assembly 9 through the dichroic mirror 33.

[0139] Specifically, the condenser 84 and the objective lens 32 are located on both sides of the objective lens 31 respectively. The condenser 84 is used to converge the illumination light on the sperm 1.

[0140] Further, the image acquisition assembly 9 comprises an imaging lens 91 for receiving and transmitting the image formed by the objective lens 32, and an image collector 92 for receiving the image transmitted by the imaging lens 91.

[0141] Preferably, the image collector 92 is a CCD camera.

[0142] Specifically, the image acquisition assembly 9 further comprises a computer 93 connected with the image collector 92. The image collector 92 sends the sperm head image or sperm flagellum image collected by it to the computer 93 in the form of digital signal, and the computer 93 can convert the digital signal obtained by it into the sperm head image or sperm flagellum image, and then through image processing, obtain the image of the posture of the sperm head 11 and the sperm flagellum 12, and can be saved, realizing the visualization observation of the sperm head 11 and the sperm flagellum 12.

[0143] From the above, it can be seen that the control system of the posture and motion state of sperm of the present application has simple structure, low cost, and is convenient to operate.

[0144] In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "embodiment", "example", "specific example" or "some examples" means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are contained in at least one embodiment or example of the present application. In the present specification, the illustrative description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any appropriate manner in any one or more embodiments or examples. In addition, the person skilled in the art can combine and combine the different embodiments or examples described in the present specification and the features of the different embodiments or examples without contradiction.

[0145] Although the embodiments of the present application have been shown and described above, it should be understood that the above embodiments are exemplary and should not be construed as limiting the present application, and those skilled in the art can modify, modify, replace and modify the above embodiments within the scope of the present application.

Claims

1. A method for controlling sperm morphology and motility, characterized in that: include: S1: two laser beams (22) are introduced into the sample pool of the stage (31) and act on the sperm head (11) of a single sperm (1); S2: Adjust the positions and powers of the two laser beams (22) acting on the sperm head (11) respectively: The sperm head (11) can be constrained at a selected position, the long axis of the sperm head (11) is parallel to the focal plane (23), and the sperm head (11) is controlled to be in different rotational states to obtain images of the sperm head in different rotational states; or The sperm head (11) can be constrained at a selected position, and the long axis of the sperm head (11) can be parallel to the focal plane (23), and the sperm flagellum (12) of the sperm (1) can be controlled to move on a plane parallel to the focal plane (23) to obtain a moving image of the sperm flagellum (12) in a three-dimensional space.

2. The method for controlling sperm morphology and motility according to claim 1, wherein: The step S1 further includes: After the laser (41) in the laser generating assembly emits a single laser beam (21), the single laser beam (21) is first expanded by a first lens (42) and a second lens (43) in sequence. The expanded single laser beam (21) is then separated into two laser beams (22) with mutually perpendicular polarizations by a half-wave plate (51) and a first polarization beam splitter prism (52). The two laser beams (22) are reflected one by one by a first reflector (61) and a second reflector (62) and then spatially combined by a second polarization beam splitter prism (63). The combined two laser beams (22) are used as control lasers and are introduced into a sample pool of the stage (31) through an objective lens and act on the sperm head (11) of a single sperm (1). The step S2 further includes: Adjusting the mirror angles of the first reflector (61) and the second reflector (62) to adjust the positions at which the two laser beams (22) act on the sperm head (11); The output current of the laser (41) in the laser generating assembly is adjusted, and the angle between the fast axis of the half-wave plate (51) and the single laser beam (21) is adjusted to adjust the power of the two laser beams (22) acting on the sperm head (11).

3. The method for controlling sperm morphology and motility according to claim 1, wherein: The step S1 further includes: The single laser beams (21) emitted by the two lasers (41) in the laser generating assembly are first expanded by the first lens (42) and the second lens (43) in sequence, and then adjusted in polarization direction by the half-wave plate (51) to form the two laser beams (22) with mutually perpendicular polarizations. The two laser beams (22) are reflected one by one by the first reflector (61) and the second reflector (62) and then spatially combined by the second polarization beam splitter prism (63). The two combined laser beams (22) are introduced into the sample pool of the stage (31) to form two control laser beams and act on the sperm head (11) of the single sperm (1); The step S2 further includes: Adjusting the mirror angles of the first reflector (61) and the second reflector (62) to adjust the positions at which the two laser beams (22) act on the sperm head (11); The power of the single laser beam (21) emitted by the two lasers (41) is adjusted to adjust the power of the two laser beams (22) acting on the sperm head (11) respectively.

4. The method for controlling sperm morphology and motility according to claim 2 or 3, wherein: The step S2 further includes: First, the positions of the two laser beams (22) acting on the sperm head (11) are adjusted respectively; The powers of the two laser beams (22) acting on the sperm head (11) are then adjusted.

5. The method for controlling sperm morphology and motility according to claim 1, wherein: The step S1 further includes: After the sperm sample is introduced into the sample pool of the stage (31), a polystyrene ball solution is injected into the sample pool of the stage (31), and then the two laser beams (22) are introduced into the sample pool of the stage (31), and the polystyrene balls are captured respectively by the two laser beams (22); The focal distance of the two laser beams (22) is calculated from the grayscale gravity centers of the two captured polystyrene balls, and the positions of the two laser beams (22) in the sample cell are adjusted so that the initial focal distance of the two laser beams (22) in the sample cell is 1.5 to 3.5 μm; Subsequently, two control lasers formed by the two laser beams (22) with an initial focal distance of 1.5 to 3.5 μm act on the sperm head (11) of a single sperm (1) respectively.

6. The method for controlling sperm morphology and motility according to claim 5, wherein: A target sperm to be controlled and observed in the sample pool is selected, and the stage (31) is moved to drive the target sperm to move to the two laser beams (22) with an initial focal distance of 1.5 to 3.5 μm and be captured by the two laser beams (22), so that the two control lasers formed by the two laser beams (22) act on the sperm head (11) of the single sperm (1) respectively.

7. The method for controlling sperm morphology and motility according to claim 1, wherein: The step S1 further includes: After the sperm sample is introduced into the sample pool of the stage (31), the illumination light emitted by the illumination component (8) is projected into the sample pool of the stage (31), and the illumination light irradiated on the sperm (1) is scattered by the sperm (1) and then imaged into the image collector (92); Subsequently, the two laser beams (22) are introduced into the sample cell of the stage (31); The step S2 further includes: After adjusting the positions and powers of the two laser beams (22) acting on the sperm head (11), the image collector (92) can collect sperm head images or sperm flagella images, and save image information of the sperm head images or sperm flagella images in a computer (93).

8. The method for controlling sperm morphology and motility according to claim 7, wherein: The illumination light emitted by the illumination light source (81) in the illumination assembly (8) is sequentially projected into the sample pool of the stage (31) through the third lens (82) and the condenser lens (84). The illumination light irradiated onto the sperm (1) is scattered by the sperm (1) and then sequentially imaged into the image collector (92) through the objective lens (32), the dichroic mirror (33) and the imaging lens (91).

9. The method for controlling sperm morphology and motility according to claim 1, wherein: The process also includes step S3: after obtaining the sperm head image or the sperm flagellum image, the following image processing steps are sequentially included: S31: performing image smoothing and noise reduction on the sperm head image or the sperm flagellum image; S32: enhancing the contrast of the sperm head image or the sperm flagellum image to improve detail visibility; S33: performing edge detection on the sperm head image or the sperm flagellum image to extract the outline and position information of the sperm head (11) or the sperm flagellum (12); S34: performing binarization processing on the outline of the sperm head image or the sperm flagellum image to convert it into a black and white binary image; S35: setting a threshold to separate the sperm head image or the sperm flagellum image from the background; S36: removing part of the connected domain in the sperm head image or the sperm flagellum image; S37: Skeletonize the remaining connected domains in the sperm head image or the sperm flagellum image to extract the axis information of the sperm head (11) or the sperm flagellum (12).

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