CN218938136U - Single sperm active oxygen and sperm mitochondrial membrane potential detection system - Google Patents
Single sperm active oxygen and sperm mitochondrial membrane potential detection system Download PDFInfo
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- CN218938136U CN218938136U CN202220643979.4U CN202220643979U CN218938136U CN 218938136 U CN218938136 U CN 218938136U CN 202220643979 U CN202220643979 U CN 202220643979U CN 218938136 U CN218938136 U CN 218938136U
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Abstract
The utility model 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 accommodating a sperm container is arranged in the shell, an excitation mechanism capable of exciting fluorescence of a single sperm in the container is arranged above the sample cell, a detection mechanism for detecting 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 the display of being connected with the controller output, conveniently looks over the testing result. The utility model adopts the excitation light probe and the photon level optical detection module to realize the detection of the active oxygen of single sperm and the mitochondrial membrane potential level of sperm, and has the advantages of sensitivity, accuracy, automation and rapidness.
Description
Technical Field
The utility model relates to the technical field of sperm analysis, in particular to a single sperm active oxygen and sperm mitochondrial membrane potential detection system.
Background
Reactive oxygen species (Reactive oxygen species)en species, ROS) are a class of oxygenates produced by organisms in the aerobic metabolic process, mainly comprising superoxide anions (O 2 · - ) Hydrogen peroxide (H) 2 O 2 ) Hydroxyl free radicals (OH), active nitrogen species (NO and the like), has extremely strong oxidizing capability, has two sides for sperms, and a small amount of and continuous active oxygen stimulation is beneficial to the sperms to exert normal physiological functions, such as cell signal transduction, hormone generation, sperm capacitation, acrosome reaction, sperm motility and the like, but excessive active oxygen can cause sperm oxidative stress, damage the oxidation defense system of the sperms, and generate potential toxicity to the sperms, so that lipid peroxidation of sperm membranes, peroxidation of proteins in the sperms, damage of sperm DNA, damage of sperms mitochondria and the like are caused, and the functions of the sperms are seriously influenced. The damaging effects of active oxygen-mediated oxidative stress on sperm have been considered as one of the important causes of male infertility, and therefore, detection of active oxygen 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, and the energy substance ATP produced by the energy production units can provide power for sperm movement, and almost the whole energy required for sperm metabolism is also provided by the mitochondria, so that the functional state of the mitochondria has important significance for sperm movement and survival. When mitochondria generate energy, electrochemical potential energy is stored in an inner mitochondrial membrane, mitochondrial Membrane Potential (MMP) is formed on two sides of the inner mitochondrial membrane if the concentration of protons and other ions are asymmetrically distributed, normal MMP is a precondition for maintaining the oxidation and phosphorylation of mitochondria and generating adenosine triphosphate, and is critical for maintaining the normal physiological functions of cells, and research discovers that various cells undergo apoptosis under the action of different factors and have the decline of MMP. In the germ cell field, the normal level of mitochondrial MMP reflects that the sperm mitochondrial ATP energy metabolism is good, and the change or loss of MMP indicates that the ATP energy synthesis required by sperm motility is blocked and is also an early signal of sperm apoptosis, and various researches are carried out to evaluate the function of sperm through detection of MMP.
At present, the sperm active oxygen detection method mainly comprises a chemiluminescence method and a fluorescent probe method, and the sperm mitochondrial membrane potential detection mainly uses the fluorescent probe method.
In the detection process of the sperm active oxygen chemiluminescence method, luminol or luciferin is used as a chemiluminescent probe, the probe and active oxygen in semen can be degraded to emit light after reacting, and the generation of the sperm cell active oxygen can be quantitatively detected by using a chemiluminescent instrument (Luminometer), so that the sperm active oxygen chemiluminescence method has high sensitivity and specificity, but the technology is more suitable for evaluating the total (sperm and semen) active oxygen level of the semen and is not suitable for detecting the active oxygen level in single sperm. The detection of the active oxygen level in a single sperm can exclude larger background interference in the detection process, reflect the difference between the sperm and the sperm cells, and has more guiding significance and objectively evaluate the sperm quality compared with the semen total active oxygen evaluation.
The fluorescent probe method uses a permeable fluorescent dye to dye sperms, and then uses a flow cytometry to detect, wherein the detection of sperm active oxygen uses a DCFH-DA probe, after penetrating the sperm cell membrane, the sperm active oxygen can be oxidized into DCF, green fluorescence can be stimulated, the sperm mitochondrial membrane potential uses a JC-1 fluorescent probe, polymers can be formed by aggregation in the mitochondrial matrix, and red fluorescence can be generated after the stimulation. But the flow cytometer has the following disadvantages in the detection process: 1) Depending on the detection of fluorescence level of individual sperms wrapped by the sheath liquid drops, the flow shear force of the sheath liquid drops can cause oxidation stress of the sperms, so that the fluorescence detection is inaccurate; 2) The method has the advantages that the initial sample loading quantity is required, the sample loss is large, the sperm sample can be detected otherwise, and if the number of sperms of a patient is rare, the method is not suitable for on-machine detection; 3) The flow cytometer detects single flowing liquid drop, sperm is wrapped in the single liquid drop, and if the single liquid drop deviates from a focal plane in the flowing process, the collected scattered fluorescence has errors, so that the detection data is inaccurate; 4) The sample preparation is complicated, and the instrument needs to be calibrated, and the operation degree of difficulty is big.
In summary, the existing detection technology cannot accurately and quantitatively detect the level of active oxygen or mitochondrial membrane potential in a single sperm, mainly because the size of the sperm is too small, usually only 5 μm in diameter, and an excitation light probe for directly carrying out fluorescence excitation on the single sperm is lacked.
Disclosure of Invention
The utility model aims to solve the technical problem of providing a single sperm active oxygen and sperm mitochondrial membrane potential detection system, which adopts an excitation light probe and a photon level optical detection module to realize the detection of the single sperm active oxygen and sperm mitochondrial membrane potential level.
In order to solve the technical problems, the technical scheme adopted by the utility model is as follows.
The utility model provides a single sperm active oxygen and sperm mitochondrial membrane potential detecting system, includes casing and hatch door that matches the dress, be provided with the sample cell of placing splendid attire semen household utensils in the casing, the top of sample cell is provided with the excitation mechanism that can excite the fluorescence of target sperm in the household utensils, and the below of sample cell is provided with the detection mechanism that carries out fluorescence collection to the target sperm after the excitation, is provided with the controller that controls detection mechanism and excitation mechanism work in the casing, the outside of casing is provided with and is connected, conveniently looks over the display of testing result with the controller output.
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 through hole is formed in the middle of the sample cell, and the vessel is placed on the sample cell in a mode of covering the through hole.
The single sperm active oxygen and sperm mitochondrial membrane potential detection system 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 baffle plate fixed on the upper part of a 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 drive of the stepping motor module.
The single sperm active oxygen and sperm mitochondrial membrane potential detection system 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 single sperm active oxygen and sperm mitochondrial membrane potential detection system also comprises an optical path switching device fixedly arranged at the bottom of the shell, wherein an objective lens is arranged at the top of the optical path switching device, and is positioned below the sample cell and is opposite to the position of the through hole; the optical detection module is arranged on one side of the optical path switching device, the CCD camera is arranged on the other side of the optical path switching device, the controlled end of the optical 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.
According to the single sperm active oxygen and sperm mitochondrial membrane potential detection system, the bright field light source is fixedly arranged in the shell right above the vessel and below the partition plate through the bracket, and the controlled end of the bright field 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 shell and the cabin door are both light-proof shells.
The single sperm active oxygen and sperm mitochondrial membrane potential detection system is characterized in that the excitation light source is an LED single-wavelength excitation light source.
According to the single sperm active oxygen and sperm mitochondrial membrane potential detection system, the damping gaskets are respectively arranged at the four feet at the bottom of the shell.
By adopting the technical scheme, the utility model has the following technical progress.
According to the utility model, the automatic identification and labeling of sperms are completed through the CCD camera visual field, the automatic control of the stepping motor module and the optical detection module is combined, the excitation light probe is sequentially positioned near a single sperm, the fluorescence excitation and the collection of fluorescence signals are completed, the automatic detection of a plurality of single sperms is realized, and the detection efficiency and detection precision of the active oxygen and mitochondrial membrane potential of the single sperm are improved.
Drawings
FIG. 1 is a schematic diagram of the structure of the present utility model;
wherein: 1. the device comprises a shell, a baffle plate, a controller, an excitation light source, a connecting wire, 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 stepper motor module, an excitation light probe, a display, a cabin door and a shock absorption gasket.
Detailed Description
The utility model will be described in further detail with reference to the drawings and the detailed description.
The single sperm active oxygen and sperm mitochondrial membrane potential detection system has a structure shown in figure 1 and comprises a shell 1 and a cabin door 7 which are matched, wherein the shell 1 and the cabin door 7 form a detection cavity after being sealed. In the utility model, the shell 1 and the cabin door 17 are both light-proof shells, so that the interference of external environment light on single sperm detection can be prevented, and the detection precision is improved.
A sample pool 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 pool 11 and can excite the fluorescence of target sperms dyed by active oxygen or mitochondrial membrane potential in the 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 the detection mechanism and the excitation mechanism are coordinated to control to work; the display 16 is arranged outside the shell and connected with the output end of the controller, so that operators 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 and the excitation light probe 15 are connected 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 baffle plate 2, the controller 3 and the excitation light source 4 are fixedly arranged on the baffle plate, and the controlled end of the excitation light source 4 is connected with the output end of the controller 3.
The stepper motor module 14 is fixedly arranged at the bottom of the shell and can move along the X, Y, Z three directions, and the moving precision of each direction is 50nm. The probe clamp 6 is arranged at the output end of the stepping motor module 14, the controlled end of the stepping motor module 14 is connected with the output end of the controller, and the controller controls the stepping motor module 14 to act so as to change the positions of the probe clamp 6 and the excitation light probe 15, thereby facilitating the accurate fluorescence excitation to a plurality of sperms 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 single sperms, the generated excitation light wavelength 1 is 490nm and is used for fluorescence excitation of active oxygen of the single sperms; the excitation wavelength 2 is 585nm, and is used for fluorescence excitation of the mitochondrial membrane potential of the single sperm; the excitation light source 4 outputs excitation light of a specific wavelength under the instruction of the controller.
The detection mechanism comprises an optical path switching device 7, an optical detection module 8, a CCD camera 9 and an objective lens 10, and the optical path switching device 7, the optical detection module 8, the CCD camera 9 and the objective lens 10 are 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 position of the through hole, so that the image and 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 light path switching device 7 can perform azimuth conversion on light entering the objective lens, so that bright field light in the sperm imaging process enters the CCD camera 9, and fluorescence in the sperm detection process enters the optical detection module 8; the optical detection module 8 is used for collecting fluorescent signals, the photosensitivity is at the photon level, the minimum photosensitivity is 50 photons, and the photosensitivity wave band is 230-700 nm; the CCD camera collects the sperm distribution state and then sends the sperm distribution state to the controller, the controller can automatically identify and mark the sperm in the field of view of the CCD camera, the excitation mechanism is conveniently controlled to be positioned near the single sperm successively, fluorescence signal collection is completed, and automatic detection of a plurality of single sperm is realized. In this embodiment, the objective lens 10 includes 4×,10×, 20×, 40×, and 60× objective lens.
In order to improve the definition of the CCD camera, the bright field light source 13 is fixedly arranged in the shell right above the vessel and below the partition plate through the bracket, the controlled end of the bright field light source 13 is connected with the output end of the controller 3, and the controller controls the bright field light source to be turned on or off. Meanwhile, in order to reduce mechanical damage and detection fluctuation of the excitation light probe caused by vibration, shock absorption gaskets 18 are respectively arranged at the four feet of the bottom of the shell.
The workflow of the present utility model is as follows.
First, sperm cells were stained with DCFH-DA sperm cell reactive oxygen species using a reactive oxygen species fluorescent dye, and sperm cells were stained with sperm cell mitochondrial membrane potential dye JC-1 using a mitochondrial membrane potential dye, respectively, to prepare sperm cell suspensions, each of which was added dropwise to vessel 12.
Secondly, the focal length of the objective lens 10 is regulated, in the sperm imaging process, the bright field light source 13 is turned on, the excitation light source 4 is turned off, bright field light entering the objective lens 10 is converted into a CCD camera, and sperm distribution is seen in the field of view; the sperm in the field of view of the CCD camera 9 is observed through the display 16, so that the sperm is clearly displayed, and the controller 3 can automatically identify and mark the single sperm position according to the field of view of the CCD camera 9.
Then, the controller 3 controls the stepping motor module 14 and the optical detection module 8 to coordinate operation, the excitation light probe 15 is positioned near a single sperm successively, fluorescence excitation and fluorescence signal acquisition are completed, automatic detection of a plurality of single sperm is realized, and an average fluorescence value A is obtained. In the single sperm detection process, the bright field light source 13 is turned off, the excitation light source 4 is turned on, and fluorescence entering the objective lens 10 is converted into the optical detection module 8 for detection.
The fluorescence value A represents the level of sperm active oxygen or the level of sperm mitochondrial membrane potential in the tested sample, and if the fluorescence value is abnormal, namely the level of sperm active oxygen is higher than a normal threshold value or the level of sperm mitochondrial membrane potential is lower than a normal threshold value, the sperm in the sample is in an abnormal state, and other clinical indexes are recommended to be checked in a hospital.
As above, the fluorescence excitation of single sperms is realized by arranging the excitation light probe, the interference of semen and other sperms on detection is avoided, and the detection of active oxygen and mitochondrial membrane potential can be accurately and sensitively carried out on the single sperms; by arranging the light-shielding shell, the interference of external stray light on single sperm detection is reduced; the vibration-reducing gaskets are arranged at the four feet at the bottom of the shell, so that the damage and detection fluctuation of the excitation light probe caused by vibration are reduced; the optical detection module with photon-level photosensitivity is used for collecting signals of fluorescence generated by single sperms, so that the resolution of single sperm active oxygen and mitochondrial membrane potential detection is improved; by arranging the stepping motor module with nanometer precision, the accurate position control of the excitation light probe is realized, so that the excitation 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; by arranging the light path switching device, the bidirectional light path control of sperm observation and single sperm detection is realized, and the influence of the imaging process on detection is avoided.
The utility model has the following advantages by combining the utility model with other sperm detection techniques.
Firstly, the background interference is small, as described in the workflow, the fluorescent excitation can be carried out on single sperms, the fluorescent interference of semen or other sperms can not be received in the detection process, each detection probe is positioned on the single sperms, the background error can not be generated, the influence of the fluid shearing force is avoided, the oxidation stress of the sperms can not be caused in the detection process, the detection precision is greatly improved, and the quality of the sperms is ensured.
Secondly, the utility model has high resolution, the normal flow cytometry analyzer can not sensitively detect the real fluorescence difference between sperms, the final result is the number of sperms exceeding the fluorescence threshold, the detection of the utility model is the real fluorescence value of a single sperm, the final result is the fluorescence value after the average of a plurality of single sperms, the difference between single sperms with different masses can be sensitively detected, and the level of active oxygen of sperms or the mitochondrial membrane potential in different samples can be accurately evaluated.
Thirdly, the utility model has small sample demand, the flow cytometer and the chemiluminescent instrument have high requirements on the number of sperms, the number of sperms of a patient with serious oligospermia is small in clinic, the flow analysis or the chemiluminescent analysis cannot be carried out, the sample reservation of other sperm test projects is not facilitated, the utility model has no special sample preparation demand, and the sample quantity can be reserved to the maximum extent.
The above embodiments are only for illustrating the technical solutions of the present utility model, and it should be understood by those skilled in the art that modifications or equivalent substitutions can be made to the technical solutions of the present utility model within the scope of the claims of the present utility model.
Claims (10)
1. A single sperm active oxygen and sperm mitochondrial membrane potential detection system, characterized in that: including casing (1) and hatch door (17) that match the dress, be provided with in casing (1) and place sample cell (11) of splendid attire semen household utensils (12), the top of sample cell (11) is provided with the excitation mechanism that can excite the fluorescence of target sperm in the household utensils, and the below of sample cell (11) is provided with carries out fluorescence collection's detection mechanism to the target sperm after the excitation, is provided with in the casing controller (3) of control detection mechanism and excitation mechanism work, the outside of casing is provided with and is connected, conveniently looks over display (16) of testing result with the controller output.
2. The single sperm-reactive oxygen species and sperm mitochondrial membrane potential detection system of 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. The single sperm-reactive oxygen species and sperm mitochondrial membrane potential detection system of 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 baffle plate (2) fixed at 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. The single sperm-reactive oxygen species and sperm mitochondrial membrane potential detection system of claim 1, wherein: the bottom of the shell is fixedly provided with a stepping motor module (14), 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 excitation light probe (15) under the driving of the stepping motor module (14).
5. The single sperm-reactive oxygen species and sperm mitochondrial membrane potential detection system of 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. The single sperm-reactive oxygen species and sperm mitochondrial membrane potential detection system of claim 5, wherein: the detection mechanism further comprises an optical path switching device (7) fixedly arranged at the bottom of the shell, an objective lens (10) is arranged at the top of the optical path switching device (7), and the objective lens (10) is positioned below the sample cell and is opposite to the position of the through hole; the optical detection module (8) is arranged on one side of the optical path switching device (7), and a 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 end of the CCD camera (9) is connected with the input end of the controller (3) through a data line.
7. The single sperm-reactive oxygen species and sperm mitochondrial membrane potential detection system of claim 1, wherein: a bright field light source (13) is fixedly arranged in the shell right above the vessel and below the partition board through a bracket, and the controlled end of the bright field light source (13) is connected with the output end of the controller (3).
8. The single sperm-reactive oxygen species and sperm mitochondrial membrane potential detection system of claim 1, wherein: the shell (1) and the cabin door (17) are both light-proof shells.
9. A single sperm-reactive oxygen species and sperm mitochondrial membrane potential detection system as described in claim 3, wherein: the excitation light source (4) is an LED single-wavelength excitation light source.
10. The single sperm-reactive oxygen species and sperm mitochondrial membrane potential detection system of claim 1, wherein: damping gaskets (18) are respectively arranged at the four feet at the bottom of the shell.
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| CN202220643979.4U CN218938136U (en) | 2022-03-24 | 2022-03-24 | Single sperm active oxygen and sperm mitochondrial membrane potential detection system |
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| CN202220643979.4U CN218938136U (en) | 2022-03-24 | 2022-03-24 | Single sperm active oxygen and sperm mitochondrial membrane potential detection system |
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