CN114813519A - System for detecting active oxygen of single sperm and mitochondrial membrane potential of sperm - Google Patents
System for detecting active oxygen of single sperm and mitochondrial membrane potential of sperm Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Optical investigation techniques, e.g. flow cytometry
- G01N15/1434—Optical arrangements
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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Abstract
The invention discloses a single sperm active oxygen and sperm mitochondrial membrane potential detection system, which comprises a shell and a cabin door which are assembled, wherein a sample cell for placing a utensil for containing semen is arranged in the shell, an excitation mechanism capable of exciting fluorescence of a single sperm in the utensil is arranged above the sample cell, a detection mechanism for detecting the active oxygen and mitochondrial membrane potential of the excited sperm is arranged below the sample cell, and a controller for controlling the detection mechanism and the excitation mechanism to work is arranged in the shell; the outside of casing is provided with and is connected, conveniently looks over the display of testing result with the controller output. The invention adopts the exciting light probe and the photon-level optical detection module to realize the detection of the potential level of the active oxygen of a single sperm and the sperm mitochondrial membrane, and has the advantages of sensitivity, accuracy, automation and rapidness.
Description
Technical Field
The invention relates to the technical field of sperm analysis, in particular to a system for detecting active oxygen of a single sperm and a sperm mitochondrial membrane potential.
Background
Reactive Oxygen Species (ROS) are a group of oxygen-containing species produced by organisms during aerobic metabolism, and mainly include superoxide anions (O) 2 · - ) Hydrogen peroxide (H) 2 O 2 ) Hydroxyl radical (OH), active nitrogen (NO, etc.), has strong oxidizing power, is bilaterally used for sperms, and small amount of continuous active oxygen stimulation is beneficial to sperms to generateThe normal physiological functions, such as cell signal transduction, hormone generation, sperm capacitation, acrosome reaction, sperm motility and the like are exerted, but excessive active oxygen causes sperm oxidative stress, damages the sperm oxidative defense system, generates potential toxicity to the sperm, causes lipid peroxidation of sperm membrane and protein peroxidation in the sperm, damages of sperm DNA, damages of sperm mitochondria and the like, and seriously influences the functions of the sperm. The damaging effect of reactive oxygen species-mediated oxidative stress on sperm has been considered as one of the important causes of male infertility, and therefore, the detection of reactive oxygen species levels in male semen and sperm is an important means for diagnosis and adjuvant treatment of male infertility.
Mitochondria are energy production units in cells, energy substances ATP generated by the mitochondria can provide power for sperm movement, and energy required by metabolism of almost the whole sperm is provided by the mitochondria, so that the functional state of the mitochondria has significance for the movement and survival of the sperm. When mitochondria generate energy, electrochemical potential energy is stored in an inner membrane of the mitochondria, and Mitochondrial Membrane Potential (MMP) is formed on two sides of the inner membrane if the concentration of protons and other ions is asymmetrically distributed, wherein normal MMP is a prerequisite for maintaining the mitochondria to carry out oxidative phosphorylation and generate adenosine triphosphate, is important for maintaining the normal physiological function of cells, and researches show that the apoptosis of various cells under the action of different factors is accompanied with the reduction of MMP. In the field of germ cells, the normal level of mitochondrial MMP reflects the good ATP energy metabolism condition of sperm mitochondria, and the change or loss of MMP indicates that the synthesis of ATP energy required by sperm movement is obstructed and is an early signal for apoptosis of sperm cells.
At present, the methods for detecting the active oxygen of the sperms mainly comprise a chemiluminescence method and a fluorescence probe method, and the fluorescence probe method is mainly used for detecting the mitochondrial membrane potential of the sperms.
Luminol or lucigenin is used as a chemiluminescence probe in the process of detecting sperm active oxygen by a chemiluminescence method, the probe can be degraded to emit light after reacting with active oxygen in semen, a chemiluminescence instrument (Luminometer) can be used for quantitatively detecting the generation of sperm cell active oxygen, high sensitivity and specificity are achieved, but the technology is more suitable for the evaluation of the active oxygen level of semen totality (semen and semen) and is not suitable for the detection of the active oxygen level in single sperm. The detection of the reactive oxygen species level in a single sperm can eliminate larger background interference in the detection process, reflect the difference between the sperm and the sperm cells, and have more guiding significance and more objective evaluation of the sperm quality compared with the total reactive oxygen species evaluation of the semen.
The fluorescence probe method uses a permeable membrane fluorescent stain to stain sperms, and then uses a flow cytometer to detect, wherein, the detection of the active oxygen of the sperms uses a DCFH-DA probe, the active oxygen can be oxidized into DCF after penetrating the sperms cell membrane, the DCF can be stimulated to generate green fluorescence, the potential of the sperms mitochondrial membrane uses a JC-1 fluorescent probe, the JC-1 fluorescent probe can gather in the matrix of mitochondria to form polymer, and the red fluorescence is generated after the JC-1 fluorescent probe is stimulated. However, the flow cytometer has the following disadvantages in the detection process: 1) the detection of the fluorescence level is carried out by depending on the single sperm wrapped by the sheath fluid droplet, and the flowing shearing force of the sheath fluid droplet can cause the sperm to generate oxidative stress, thus causing inaccurate fluorescence detection; 2) the requirement of initial sample loading amount is met, the sample loss amount is large, the sperm sample can be generally subjected to other detection, and if the number of the sperms of a patient is small, the patient is not suitable for on-machine detection; 3) the flow cytometer detects a single flowing liquid drop, the single liquid drop is wrapped by sperms, if the single liquid drop deviates from a focal plane in the flowing process, the collected scattered fluorescence has errors, and the detection data is inaccurate; 4) the sample preparation is complex, the instrument needs to be calibrated, and the operation difficulty is high.
In summary, the existing detection technologies cannot accurately and quantitatively detect the level of active oxygen or mitochondrial membrane potential in a single sperm, mainly because the sperm has a small size, usually only 5 μm in diameter, and lacks an excitation light probe for directly performing fluorescence excitation on a single sperm.
Disclosure of Invention
The invention aims to provide a system for detecting the potential level of single sperm active oxygen and sperm mitochondrial membranes, which adopts an excitation light probe and a photon-level optical detection module to realize the detection of the potential level of the single sperm active oxygen and the sperm mitochondrial membranes.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows.
The system for detecting the active oxygen of a single sperm and the mitochondrial membrane potential of the sperm comprises a shell and a cabin door which are assembled, wherein a sample cell for containing a sperm vessel is arranged in the shell, an excitation mechanism capable of exciting the fluorescence of a target sperm in the vessel is arranged above the sample cell, a detection mechanism for collecting the fluorescence of the excited target sperm is arranged below the sample cell, and a controller for controlling the detection mechanism and the excitation mechanism to work is arranged in the shell.
According to the single sperm active oxygen and sperm mitochondrial membrane potential detection system, the sample cell is fixedly arranged in the middle of the shell, the middle of the sample cell is provided with the through hole, and the vessel is placed on the sample cell in a manner of covering the through hole.
In the single sperm active oxygen and sperm mitochondrial membrane potential detection system, the excitation mechanism comprises an excitation light source and an excitation light probe which are connected through a connecting wire, wherein the excitation light source is positioned through a partition board fixed on the upper part of the shell, and the excitation light probe is clamped through a probe clamp; the controlled end of the excitation light source is connected with the output end of the controller.
According to the single sperm active oxygen and sperm mitochondrial membrane potential detection system, the stepping motor module is fixedly arranged at the bottom of the shell, the probe clamp is arranged at the output end of the stepping motor module, and the controlled end of the stepping motor module is connected with the output end of the controller so as to change the positions of the probe clamp and the excitation light probe under the driving of the stepping motor module.
The detection mechanism comprises an optical detection module arranged below the sample cell, and the output end of the optical detection module is connected with the input end of the controller through a data line.
The detection mechanism also comprises a light path switching device fixedly arranged at the bottom of the shell, the top of the light path switching device is provided with an objective lens, and the objective lens is positioned below the sample cell and is opposite to the through hole; the optical detection module is arranged on one side of the light path switching device, the other side of the light path switching device is provided with a CCD camera, the controlled end of the light path switching device is connected with the output end of the controller, and the output end of the CCD camera is connected with the input end of the controller through a data line.
The single sperm active oxygen and sperm mitochondrial membrane potential detection system is characterized in that a bright field light source is fixedly arranged in the shell right above the vessel and below the partition plate through a bracket, and the controlled end of the bright field light source is connected with the output end of the controller.
In the single sperm active oxygen and sperm mitochondrial membrane potential detection system, the shell and the hatch are all light-proof shells.
In the single sperm active oxygen and sperm mitochondrial membrane potential detection system, the excitation light source is an LED single-wavelength excitation light source.
In the single sperm active oxygen and sperm mitochondrial membrane potential detection system, the display which is connected with the output end of the controller and is convenient for checking the detection result is arranged outside the shell; and four shock-absorbing gaskets are respectively arranged at the four feet of the bottom of the shell.
Due to the adoption of the technical scheme, the technical progress of the invention is as follows.
The invention completes the automatic identification and marking of sperms through the visual field of the CCD camera, sequentially positions the exciting light probe near a single sperm by combining the automatic control of the stepping motor module and the optical detection module, completes the fluorescent excitation and the acquisition of fluorescent signals, realizes the automatic detection of a plurality of single sperms, and improves the detection efficiency and the detection precision of single sperm active oxygen and mitochondrial membrane potential.
Drawings
FIG. 1 is a schematic structural view of the present invention;
wherein: 1. the device comprises a shell, a partition plate, a controller, an excitation light source, a connecting line, a probe clamp, a light path switching device, an optical detection module, a CCD camera, an objective lens, a sample cell, a vessel, a bright field light source, a stepping motor module, an excitation light probe, a display, a cabin door and a shock absorption gasket, wherein the partition plate is 2, the controller is 3, the excitation light source is 4, the connecting line is 5, the probe clamp is 6, the light path switching device is 7, the optical detection module is 8, the CCD camera is 9, the objective lens is 10, the sample cell is 11, the vessel is 12, the bright field light source is 13, the stepping motor module is 14, the excitation light probe is 15, the display is 16, the cabin door is 17, and the shock absorption gasket is 18.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.
The structure of the single sperm active oxygen and sperm mitochondrial membrane potential detection system is shown in figure 1, and the detection system comprises a shell 1 and a hatch 7 which are assembled, wherein the shell 1 and the hatch 7 form a detection cavity after being closed. In the invention, the shell 1 and the cabin door 17 are light-proof shells, so that the interference of external environment light on the single sperm detection can be blocked, and the detection precision is improved.
A sample cell 11 is arranged in the shell 1 and is used for placing a vessel 12 for containing semen; the sample cell 11 is fixedly arranged in the middle of the shell, a through hole is formed in the middle of the sample cell, and the vessel 12 is placed on the sample cell in a mode of covering the through hole.
An excitation mechanism is arranged above the sample cell 11 and can excite the fluorescence of target sperms stained by active oxygen or mitochondrial membrane potential in a vessel; a detection mechanism is arranged below the sample cell 11 and is used for collecting fluorescence and detecting active oxygen or mitochondrial membrane potential of the excited sperm; a controller 3 is arranged in the shell and used for coordinately controlling the detection mechanism and the excitation mechanism to work; and a display 16 is arranged outside the shell and is connected with the output end of the controller, so that an operator can conveniently check the detection result.
The excitation mechanism comprises a stepping motor module 14, an excitation light source 4, an excitation light probe 15 and a probe clamp 6, wherein the excitation light source 4 is connected with the excitation light probe 15 through a connecting wire 5, and the excitation light probe 15 is clamped through the probe clamp 6.
The upper part of the shell is fixedly provided with a partition plate 2, the controller 3 and the excitation light source 4 are both fixedly arranged on the partition plate, and the controlled end of the excitation light source 4 is connected with the output end of the controller 3.
The stepping motor module 14 is fixedly arranged at the bottom of the shell and can move along X, Y, Z three directions, and the moving precision of each direction is 50 nm. Probe anchor clamps 6 set up the output at step motor module 14, and step motor module 14's controlled end is connected with the output of controller, and step motor module 14 action is controlled to the controller to change probe anchor clamps 6 and excitation light probe 15's position, conveniently accurately carry out fluorescence to a plurality of sperms and arouse one by one.
In the embodiment, the excitation light source 4 is an LED single-wavelength excitation light source and is used for high-efficiency fluorescence excitation of a single sperm, and the generated excitation light wavelength 1 is 490nm and is used for fluorescence excitation of single sperm active oxygen; the generated excitation wavelength 2 is 585nm and is used for fluorescence excitation of the single sperm mitochondrial membrane potential; the excitation light source 4 outputs excitation light of a specific wavelength under the instruction of the controller.
The detection mechanism comprises a light path switching device 7, an optical detection module 8, a CCD camera 9 and an objective lens 10, wherein the light path switching device 7, the optical detection module 8, the CCD camera 9 and the objective lens 10 are all arranged below the sample cell 11.
The light path switching device 7 is fixedly arranged at the bottom of the shell, the objective lens 10 is arranged at the top of the light path switching device 7, and the objective lens 10 is positioned below the sample cell and is opposite to the through hole, so that the image and the fluorescence information in the vessel can be conveniently collected; the optical detection module 8 is arranged on one side of the optical path switching device 7, and the CCD camera 9 is arranged on the other side of the optical path switching device 7; the controlled end of the light path switching device 7 is connected with the output end of the controller, and the output ends of the CCD camera 9 and the optical detection module 8 are respectively connected with the input end of the controller 3 through data lines.
The optical path switching device 7 can perform azimuth conversion on light entering the objective lens, so that bright field light in a sperm imaging process enters the CCD camera 9, and fluorescence in a sperm detection process enters the optical detection module 8; the optical detection module 8 is used for collecting fluorescence signals, the photosensitive sensitivity is at the photon level, the minimum photosensitive sensitivity is 50 photons, and the photosensitive wave band is 230 nm-700 nm; the controller is sent into after the CCD camera gathers sperm distribution state, and the controller can carry out automatic identification and mark with the sperm in the CCD camera field of vision, conveniently controls excitation mechanism and fixes a position near single sperm and accomplish fluorescence signal collection one by one, realizes the automated inspection of a plurality of single sperms. In the present embodiment, the objective lens 10 includes 4 ×, 10 ×, 20 ×, 40 × and 60 × objective lens.
In order to improve the definition of the CCD camera, a bright field light source 13 is fixedly arranged in a shell which is positioned right above a vessel and below a partition plate through a bracket, the controlled end of the bright field light source 13 is connected with the output end of a controller 3, and the controller controls the on and off of the bright field light source. Meanwhile, in order to reduce the mechanical damage and detection fluctuation of the excitation light probe caused by vibration, damping gaskets 18 are respectively arranged at four foot positions at the bottom of the shell.
The workflow of the present invention is as follows.
Firstly, the sperm is carried out active oxygen fluorescent staining by using a DCFH-DA sperm active oxygen staining reagent, and the sperm is carried out mitochondrial membrane potential fluorescent staining by using a sperm mitochondrial membrane potential staining reagent JC-1 to respectively prepare sperm suspensions which are respectively dripped into the vessel 12.
Secondly, the focal length of the objective lens 10 is adjusted, in the sperm imaging process, the bright field light source 13 is turned on, the excitation light source 4 is turned off, the bright field light entering the objective lens 10 is converted to enter the CCD camera, and the sperm distribution is seen in the visual field; the display 16 is used for observing the sperms in the visual field of the CCD camera 9, so that the sperms can be displayed clearly, and the controller 3 can automatically identify and mark the position of the single sperms according to the visual field of the CCD camera 9.
Then, the controller 3 controls the stepping motor module 14 and the optical detection module 8 to coordinate to position the excitation light probe 15 near a single sperm one by one, so as to complete fluorescence excitation and fluorescence signal acquisition, realize automatic detection of a plurality of single sperm and obtain an average fluorescence value A. In the process of single sperm detection, the bright field light source 13 is closed, the excitation light source 4 is opened, and the fluorescence entering the objective lens 10 is converted and enters the optical detection module 8 for detection.
The fluorescence value A represents the active oxygen level of the sperm or the mitochondrial membrane potential level of the sperm in the tested sample, if the fluorescence value is abnormal, namely the active oxygen level of the sperm is higher than a normal threshold value or the mitochondrial membrane potential of the sperm is lower than the normal threshold value, the sperm in the sample is in an abnormal state, and the examination of other clinical indexes in a hospital is recommended.
As above, the invention realizes the fluorescence excitation of single sperm by arranging the excitation light probe, avoids the interference of semen and other sperms to the detection, and can accurately and sensitively detect the active oxygen and the mitochondrial membrane potential aiming at the single sperm; the light-shading shell is arranged, so that the interference of external stray light on the detection of single sperm is reduced; damping gaskets are arranged at four feet of the bottom of the shell, so that damage and detection fluctuation of the exciting light probe caused by vibration are reduced; the optical detection module with photon-level photosensitivity is used for signal acquisition of fluorescence generated by a single sperm, so that the resolution of the detection of active oxygen and mitochondrial membrane potential of the single sperm is improved; by arranging the nanometer precision stepping motor module, the precise position control of the exciting light probe is realized, so that the exciting light probe can stably touch the vicinity of a single sperm, and the fluorescence excitation deviation of the single sperm caused by the position deviation is avoided; through setting up light path auto-change over device, realized the two-way light path control that sperm observed and single sperm detected, avoided the imaging process to cause the influence to detecting.
Combining the present invention with other sperm detection techniques, it can be seen that the present invention has the following advantages.
The method has the advantages that the background interference is small, as described in the working process, the fluorescence excitation can be carried out on a single sperm, the fluorescence interference of semen or other sperms cannot be caused in the detection process, each detection probe is positioned on the single sperm, the background error cannot be generated, the influence of fluid shearing force does not exist, the oxidative stress of the sperms cannot be caused in the detection process, the detection precision is greatly improved, and the quality of the sperms is ensured.
Secondly, the invention has high resolution, the general flow cytometry cannot sensitively detect the real fluorescence difference between sperms, the final obtained result is to count the number of sperms exceeding the fluorescence threshold, the detection of the invention is the real fluorescence value of a single sperm, the final obtained result is the fluorescence value after a plurality of single sperms are averaged, not only can sensitively detect the difference between the single sperms with different qualities, but also can accurately evaluate the active oxygen level of the sperms or the mitochondrial membrane potential in different samples.
Thirdly, the sample demand of the invention is small, the flow cytometer and the chemiluminescence apparatus have high requirements for the number of sperms, the number of sperms of patients with severe oligospermia is small clinically, flow analysis or chemiluminescence analysis can not be carried out, and the invention is not beneficial to the sample reservation of other sperm inspection projects.
The above embodiments are only for illustrating the technical solutions of the present invention, and those skilled in the art will understand that the technical solutions of the present invention can be modified or equivalently replaced within the scope of the claims of the present invention.
Claims (10)
1. The system for detecting the active oxygen of the single sperm and the mitochondrial membrane potential of the sperm is characterized in that: the semen collection device comprises a shell (1) and a cabin door (7) which are assembled, wherein a sample pool (11) for placing a semen container (12) is arranged in the shell (1), an excitation mechanism capable of exciting fluorescence of target sperms in the container is arranged above the sample pool (11), a detection mechanism for collecting the fluorescence of the excited target sperms is arranged below the sample pool (11), and a controller (3) for controlling the detection mechanism and the excitation mechanism to work is arranged in the shell.
2. A single sperm reactive oxygen species and sperm mitochondrial membrane potential detection system as described in claim 1 wherein: the sample cell (11) is fixedly arranged in the middle of the shell, a through hole is formed in the middle of the sample cell, and the vessel (12) is placed on the sample cell in a mode of covering the through hole.
3. A single sperm reactive oxygen species and sperm mitochondrial membrane potential detection system as described in claim 1 wherein: the excitation mechanism comprises an excitation light source (4) and an excitation light probe (15) which are connected through a connecting wire (5), wherein the excitation light source (4) is positioned through a partition plate (2) fixed on the upper part of the shell, and the excitation light probe (15) is clamped through a probe clamp (6); the controlled end of the excitation light source (4) is connected with the output end of the controller (3).
4. A single sperm reactive oxygen species and sperm mitochondrial membrane potential detection system as described in claim 1 wherein: the probe clamp is characterized in that a stepping motor module (14) is fixedly arranged at the bottom of the shell, the probe clamp (6) is arranged at the output end of the stepping motor module (14), and the controlled end of the stepping motor module (14) is connected with the output end of the controller so as to change the positions of the probe clamp (6) and the exciting light probe (15) under the driving of the stepping motor module (14).
5. A single sperm reactive oxygen species and sperm mitochondrial membrane potential detection system as described in claim 1 wherein: the detection mechanism comprises an optical detection module (8) arranged below the sample cell (11), and the output end of the optical detection module (8) is connected with the input end of the controller (3) through a data line.
6. A single sperm reactive oxygen species and sperm mitochondrial membrane potential detection system according to claim 5 wherein: the detection mechanism further comprises a light path switching device (7) fixedly arranged at the bottom of the shell, an objective lens (10) is arranged at the top of the light path switching device (7), and the objective lens (10) is positioned below the sample cell and is opposite to the through hole; the optical detection module (8) is arranged on one side of the light path switching device (7), and the other side of the light path switching device (7) is provided with a CCD camera (9); the controlled end of the light path switching device (7) is connected with the output end of the controller, and the output end of the CCD camera (9) is connected with the input end of the controller (3) through a data line.
7. A single sperm reactive oxygen species and sperm mitochondrial membrane potential detection system as described in claim 1 wherein: a bright field light source (13) is fixedly arranged in the shell right above the vessel and below the partition plate through a support, and the controlled end of the bright field light source (13) is connected with the output end of the controller (3).
8. A single sperm reactive oxygen species and sperm mitochondrial membrane potential detection system as described in claim 1 wherein: the shell (1) and the cabin door (17) are all light-proof shells.
9. A single sperm reactive oxygen species and sperm mitochondrial membrane potential detection system as described in claim 3 and wherein: the excitation light source (4) is an LED single-wavelength excitation light source.
10. A system for detecting single sperm reactive oxygen species and sperm mitochondrial membrane potential according to any one of claims 1 to 9 wherein: the outside of casing is provided with and is connected, conveniently look over display (16) of testing result with the controller output, the bottom four feet position of casing is provided with shock attenuation gasket (18) respectively.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210291639.4A CN114813519A (en) | 2022-03-24 | 2022-03-24 | System for detecting active oxygen of single sperm and mitochondrial membrane potential of sperm |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210291639.4A CN114813519A (en) | 2022-03-24 | 2022-03-24 | System for detecting active oxygen of single sperm and mitochondrial membrane potential of sperm |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114813519A true CN114813519A (en) | 2022-07-29 |
Family
ID=82530795
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210291639.4A Pending CN114813519A (en) | 2022-03-24 | 2022-03-24 | System for detecting active oxygen of single sperm and mitochondrial membrane potential of sperm |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114813519A (en) |
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2022
- 2022-03-24 CN CN202210291639.4A patent/CN114813519A/en active Pending
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