CN109540889B - Novel sperm quality evaluation system - Google Patents
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- CN109540889B CN109540889B CN201811357545.2A CN201811357545A CN109540889B CN 109540889 B CN109540889 B CN 109540889B CN 201811357545 A CN201811357545 A CN 201811357545A CN 109540889 B CN109540889 B CN 109540889B
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Abstract
The invention discloses a novel sperm quality evaluation system. The invention realizes the large-visual-field multi-target detection of the sample by using the large-visual-field microscopic imaging system, realizes the high-resolution microscopic imaging of the sample by using the high-resolution microscopic imaging system, realizes the high-resolution imaging detection of the sample under the large visual field by combining the two microscopic imaging systems, is particularly suitable for the high-resolution morphological analysis of a large number of moving sperms under the large visual field in the human semen examination, and can simultaneously detect the indexes such as the concentration, the vitality, the morphology and the like of the viable sperms of the non-staining and non-marking semen sample.
Description
Technical Field
The invention belongs to the technical field of microscopic imaging for semen examination, and particularly relates to a novel sperm quality evaluation system.
Background
The sperm motility and morphology as the key parts in human semen examination are of great significance to reproductive and developmental research, especially to clinical reproductive medicine. The existing medical clinical detection adopts different sample preparation and microscopic detection methods to respectively detect the concentration, the activity and the morphology of sperms.
The sperm motility can be completed manually or with the assistance of a computer, and the quantitative parameters of the sperm motility can be provided with high accuracy by utilizing the computer-aided sperm analysis technology, thereby being beneficial to the normalization and the standardization of the semen examination. The computer aided sperm analysis technology utilizes low power microscopic imaging system, image acquiring, processing and analyzing system and records the continuous motion locus of sperm to make quantitative statistics and analysis of sperm activity and concentration. However, clinical studies have shown that it is not sufficient to rely on the movement speed of sperm alone in the quality assessment of sperm, and it is also necessary to combine the morphology and structure of sperm, especially the genetic function index. The sperm morphology measurement analysis needs to observe and analyze the morphology of static sperms through a smear staining research method, the sperm morphology examination in human semen examination needs to observe a smear staining sample under a high-power microscopic imaging system, the processing process of the sample can kill sperms, and the smear staining can cause certain influence on the sperm morphology due to factors such as osmotic pressure change and the like.
The movement speed of the sperms is extremely high, the speed is dozens of microns/second, and large-view field microscopic imaging is needed when the moving sperms and hundreds of targets are continuously observed and tracked; the size of the head of the sperm is 3 to 5 microns, the limit resolution of low-magnification imaging is above the micron level, high-resolution microscopic imaging cannot be realized, and the head morphology cannot be accurately measured and analyzed.
The field size of the imaging is inversely proportional to the magnification of the objective lens due to the limitation of the existing optical imaging design, the large field cannot be realized by low-power magnification imaging, the large field cannot be directly realized by high-power magnification imaging, the imaging method is suitable for unmoving samples by splicing and other methods, and is not suitable for sperm which are moving objects. The effective imaging area is limited by the micro-imaging detection element and the optical design, and the effective imaging area of the micro-image acquisition element is limited by the technology and the cost.
The processing method of smear dyeing is used for observing the shape of the sperms, only the information of one side of the sperms can be observed, and the three-dimensional structure information and the dynamic measurement result of the movement of the tail can be obtained by observing the rotating movement of the sperms.
The invention content is as follows:
the invention aims to provide a novel sperm quality evaluation system, which can be used for detecting high-resolution morphological analysis of a large number of moving sperms under a large visual field and simultaneously detecting indexes such as sperm concentration, motility and morphology of a non-staining and non-marking sperm sample.
The novel sperm quality evaluation system is used for detecting the vitality indexes of a large number of sperms and realizing high-resolution observation and evaluation of the shape of the sperms.
The system comprises a sperm sample stage which can move in three dimensions and be accurately controlled and two independent microscopic imaging systems: a large-visual-field microscopic imaging system and a high-resolution microscopic imaging system, wherein the two imaging systems can be adjusted to be in a confocal and coaxial state.
Preferably, the large-field microscopic imaging system comprises a large-field microscopic imaging objective lens, a large-field microscopic imaging relay optical path and a large-field microscopic imaging photoelectric image detector which are sequentially connected.
Preferably, the high-resolution microscopic imaging system comprises a high-resolution microscopic imaging objective lens, a high-resolution microscopic imaging relay optical path and a high-resolution microscopic imaging photoelectric image detector which are sequentially connected.
Preferably, the sperm sample stage capable of three-dimensionally moving and accurately controlling can control the temperature of the sample cell thereon and can move the sperm sample arbitrarily according to the requirement;
preferably, the large-visual-field microscopic imaging system can rapidly shoot a sufficient number of sperms in a visual field, automatically output the position and speed information of each sperm in the visual field through image processing technology, multi-target tracking technology and the like, and count various vitality indexes of the sperms according to the requirements.
Preferably, when the high-resolution micro-imaging system collects morphological information in an imaging area of the large-view micro-imaging system, the high-resolution micro-imaging system collects position information of the sperms at the same time, and the position information corresponds to the position information of each sperm in the imaging area of the large-view micro-imaging system synchronously.
Preferably, the sperm sample stage capable of moving three-dimensionally and being controlled precisely can move the position of the sample stage rapidly according to the visual field size and the position information of the large-visual-field and high-resolution microscopic imaging systems, so that the sperms observed in the large-visual-field microscopic imaging systems sequentially move to the imaging area of the high-resolution microscopic imaging systems, the imaging of the high-resolution microscopic imaging systems in the imaging area of the large-visual-field microscopic imaging systems is further realized, all the sperms of all parts in the imaging area of the large-visual-field microscopic imaging systems are subjected to high-resolution microscopic imaging, and then all the sperms in the large-visual-field are subjected to morphological analysis.
Preferably, the novel sperm quality evaluation system has the advantages that the movement speed of any sperm can correspond to the morphological indexes of the sperm one by one, and meanwhile, the high-resolution microscopic imaging system can not repeatedly detect the same sperm when carrying out morphological analysis under the monitoring of the large-visual-field microscopic imaging system.
The invention discloses a novel sperm quality evaluation system, which comprises a large-visual-field microscopic imaging objective lens, a large-visual-field microscopic imaging relay optical path, a large-visual-field microscopic imaging photoelectric image detector, a high-resolution microscopic imaging objective lens, a high-resolution microscopic imaging relay optical path, a high-resolution microscopic imaging photoelectric image detector, an objective table and a computer graphic workstation, the large-field microscopic imaging objective lens and the high-resolution microscopic imaging objective lens are respectively arranged at the upper side and the lower side of the objective table, or the large-field microscopic imaging objective lens, the large-field microscopic imaging relay optical path and the large-field microscopic imaging photoelectric image detector are sequentially connected on the lower side and the upper side, the high-resolution microscopic imaging objective lens, the high-resolution microscopic imaging relay optical path and the high-resolution microscopic imaging photoelectric image detector are sequentially connected, and the large-field microscopic imaging photoelectric image detector and the high-resolution microscopic imaging photoelectric image detector are respectively connected with a computer graphic workstation through signals.
The large-field microscopic imaging objective lens refers to an objective lens of a large-field microscope, and the field of view of the objective lens is large, but the resolution is not high.
The high-resolution microimaging objective is an objective of a high-resolution microscope, and the resolution is high, but the field of view is small.
Imaging information acquired by the large-view-field microscopic imaging objective lens is transmitted to the large-view-field microscopic imaging photoelectric image detector through the large-view-field microscopic imaging relay light path, the large-view-field microscopic imaging photoelectric image detector converts the imaging information into digital picture information, and then the digital picture information is transmitted to the computer graphic workstation.
Imaging information acquired by the high-resolution microimaging objective lens is transmitted to the high-resolution microimaging photoelectric image detector through the high-resolution microimaging relay optical path, the high-resolution microimaging photoelectric image detector converts the imaging information into digital picture information, and then the digital picture information is transmitted to the computer graphic workstation.
The computer graphic workstation mainly realizes the control, transmission and processing of data acquisition of the photoelectric image detector, and realizes the general auxiliary computing functions of data analysis and storage and the like.
The object stage is a device for bearing the semen sample, and is provided with a constant temperature control device, so that the semen sample on the object stage can be kept at a constant temperature of 37 ℃.
Preferably, the system also comprises a large-field microscopic imaging auxiliary function system, and the large-field microscopic imaging auxiliary function system is connected with the large-field microscopic imaging relay optical path.
Preferably, the system also comprises a high-resolution micro-imaging auxiliary function system, and the high-resolution micro-imaging auxiliary function system is connected with the high-resolution micro-imaging relay optical path.
The large-field microscopic imaging auxiliary function system has the functions of providing auxiliary functions such as illumination, laser introduction and the like for large-field microscopic imaging and high-resolution microscopic imaging, for example, an additional system light source, wherein the luminous spectrum range of the illumination light source is mainly 400-700 nm, the nominal power is 150W, and the color temperature is 3450 Kelvin, so that the microscopic illumination function can be realized.
The high-resolution microscopic imaging auxiliary function system has the functions of providing auxiliary functions such as fluorescence excitation and spectrum detection for large-field microscopic imaging and high-resolution microscopic imaging, for example, the introduction of laser and spectrum detection are realized by additionally adding an additional dichroscope, and the killing, detection and the like of sperms can be realized by controlling the introduced laser.
The invention can use the novel sperm quality evaluation system to perform high-resolution imaging of a large number of moving sperms under a large visual field, and the specific method comprises the following steps:
placing a semen sample on an objective table, wherein a large-view field microimaging objective and a high-resolution microimaging objective are respectively arranged at the upper side and the lower side of the objective table, or the lower side and the upper side of the objective table, the view field range of the high-resolution microimaging objective is positioned in the view field range of the large-view field microimaging objective, the two view field ranges are clear, imaging information acquired by the large-view field microimaging objective and the high-resolution microimaging objective is transmitted to a computer graphic workstation through data, and the computer graphic workstation continuously acquires, observes and stores a plurality of target sperms, records and tracks the target sperms;
determining the initial positions of the target sperm and the target sperm according to the imaging information and the recorded result acquired by the large-visual-field microscopic imaging objective lens, then moving the target sperm from the visual field range of the large-visual-field microscopic imaging objective lens to the visual field range of the high-resolution microscopic imaging objective lens by moving the objective table, acquiring the imaging data (such as high-resolution morphological data) of the target sperm, recording the imaging information acquired by the visual field range of the large-visual-field microscopic imaging system and the visual field range of the high-resolution microscopic imaging system in a computer graphics workstation, and completing the acquisition work of the imaging data of the target sperm.
After the imaging data acquisition work of a certain target sperm is finished, determining the next target sperm to be detected in the field range of the large-view-field microscopic imaging system, repeating the steps, starting the next cycle acquisition until all the target sperm to be acquired in the field range of the large-view-field microscopic imaging system are acquired, and finishing the current acquisition.
And moving the object stage randomly or according to a statistical principle, changing the field range of the large-field microscopic imaging system, and starting to collect a new field until the number of the sperm information required by statistics is reached.
The novel sperm quality evaluation system is adopted for collection, and the method comprises the following specific steps:
the semen sample is placed on an object stage, a large-view field microscopic imaging objective lens and a high-resolution microscopic imaging objective lens are respectively arranged on the upper side and the lower side of the object stage, or the lower side and the upper side of the object stage, the view field range of the high-resolution microscopic imaging objective lens is positioned in the view field range of the large-view field microscopic imaging objective lens, the two view field ranges are clear, imaging information acquired by the large-view field microscopic imaging objective lens is transmitted to a large-view field microscopic imaging photoelectric image detector through a large-view field microscopic imaging relay light path, the large-view field microscopic imaging photoelectric image detector converts the imaging information into digital picture information, the digital picture information is transmitted to a computer graphic workstation, and the computer graphic workstation observes a plurality of target sperms through continuous acquisition and stores, records and tracks the sperms.
Determining the initial positions of a target sperm and the target sperm according to the imaging information acquired by the large-view field microimaging objective lens and the recorded result, moving the target sperm from the view field range of the large-view field microimaging objective lens to the view field range of the high-resolution microimaging objective lens by a moving objective table, acquiring the imaging data (such as high-resolution morphological data) of the target sperm, transmitting the imaging information acquired by the high-resolution microimaging objective lens to a high-resolution microimaging photoelectric image detector through a high-resolution microimaging relay optical path, converting the imaging information into digital picture information by the high-resolution microimaging photoelectric image detector, and transmitting the digital picture information to a computer graphic workstation to finish the acquisition of the imaging data of the target sperm.
Placing the semen sample on an objective table means that the semen sample waits for liquefaction at 37 ℃, uniformly mixing and flaking the sample according to a corresponding laboratory manual method and keeping stable temperature, placing the flaking semen sample on the objective table between a large-view microscopic imaging objective lens and a high-resolution microscopic imaging objective lens, and preheating and storing the specimen table in advance to keep the temperature constant at 37 ℃.
The transverse stroke of the objective table is 110 mm, the longitudinal stroke is 75 mm, the displacement resolution is 0.1 micron, the repetition precision is less than 1 micron, the maximum running speed is 7 mm/s, and the maximum load of the objective table is 5 kg.
The existing detection method is used for carrying out segmentation measurement on a sample, and processing is carried out through different processes to respectively obtain each piece of information of sperms, the motility (linear speed) and the form (head size and the like) of the sperms are measured by cleavage statistics, the measurement results cannot be correlated, and the relationship between the form size and the forward speed cannot be confirmed. The invention has two microscopic imaging systems with different imaging ranges and resolutions, the two microscopic imaging systems simultaneously perform co-location imaging on the detected sample, the sample does not need special treatment such as smear dyeing marks and the like, thereby keeping the sperm activity and the fertilization capability, and the detected sample can be used for scientific research, clinical medical treatment (in vitro fertilization) and the like through sorting.
The invention has the following beneficial effects:
1. the invention realizes the morphological detection and statistics of the live sperms in the semen, but does not need dyeing detection, thereby the invention keeps the sperm activity and the fertilization capability, and can be used for scientific research, clinical medical treatment (in vitro fertilization) and the like after sorting.
2. The present invention can detect the activity and the shape of a great amount of sperms in semen sample at the same time, and is convenient for scientific research and clinical statistical analysis.
3. The invention can detect the dynamic information of the moving sperm shape and the tail moving state.
4. The invention can accurately measure the vitality (curve speed, linear speed and the like) and the shape (head size, head shape, cytoplasm droplets and the like) of a single live sperm, and the superposition statistics can more accurately reflect the sperm quality.
5. The invention realizes the 3D information acquisition of the sperms, and the information such as the top and head shapes of the sperms, the vacuole positions and the like can be popularized and used for the injection of the single sperms, provides more accurate clinical guidance for the diagnosis of the diseases of the test tube infants, and can realize the research of the single cells of the sperms with high throughput by combining the proteomics detection and the single cell sequencing of the sperms in the basic research.
Description of the drawings:
FIG. 1 is a schematic view of field-of-view range imaging planes of a large-field microscopic imaging system and a high-resolution microscopic imaging system;
FIG. 2 is a schematic side view of an acquisition of a large field of view microscopy imaging system and a high resolution microscopy imaging system;
FIG. 3 is a schematic diagram of the structure of the novel sperm quality evaluation system;
FIG. 4 is a photograph of sperm collected by a large field microscopic imaging system;
FIG. 5 is a photograph of sperm collected by the high resolution microscopic imaging system;
wherein 1, a large-field microscopic imaging objective lens; 2. a relay optical path for large-field microscopic imaging; 3. a large-field microscopic imaging accessory function system; 4. a large-field microscopic imaging photoelectric image detector; 5. a high resolution microimaging objective lens; 6. a high-resolution microscopic imaging relay optical path; 7. a high-resolution microscopic imaging accessory function system; 8. a high-resolution microscopic imaging photoelectric image detector; 9. an object stage; 10. a computer graphics workstation; 11. a semen sample;
101. the field of view range of the large-field microscopic imaging system; 102. a field of view range of the high resolution microscopic imaging system; 103. a target sperm large visual field starting position; 104. a target sperm high-resolution observation position; 105. a target sperm movement trajectory;
201. a field-of-view side of the large-field microscopic imaging system; 202. a field-of-view side of the high-resolution microscopic imaging system; 203. and (3) observing the side surface of the position of the target sperm with high resolution.
The specific implementation mode is as follows:
for further understanding of the features and technical means of the present invention, as well as the specific objects and functions attained by the present invention, the advantages and spirit of the present invention will be further understood by reference to the following detailed description and the accompanying drawings.
Example 1:
as shown in fig. 3, the novel sperm quality evaluation system of this embodiment includes a large-field microscopic imaging objective lens 1, a large-field microscopic imaging relay optical path 2, a large-field microscopic imaging auxiliary function system 3, a large-field microscopic imaging photoelectric image detector 4, a high-resolution microscopic imaging objective lens 5, a high-resolution microscopic imaging relay optical path 6, a high-resolution microscopic imaging auxiliary function system 7, a high-resolution microscopic imaging photoelectric image detector 8, a stage 9 and a computer graphics workstation 10, wherein the large-field microscopic imaging objective lens 1 and the high-resolution microscopic imaging objective lens 5 are respectively disposed at the upper and lower sides of the stage, the large-field microscopic imaging objective lens 1, the large-field microscopic imaging relay optical path 2, the large-field microscopic imaging photoelectric image detector 4 are sequentially connected, the high-resolution microscopic imaging objective lens 5, the high-resolution microscopic imaging relay optical path 6, and the high-resolution microscopic imaging photoelectric image detector 8 are sequentially connected, the large-field microscopic imaging photoelectric image detector 4 and the high-resolution microscopic imaging photoelectric image detector 8 are respectively in signal connection with a computer graphic workstation 10. And the large-field microscopic imaging auxiliary function system 3 is connected with the large-field microscopic imaging relay optical path 2. And the high-resolution microscopic imaging auxiliary function system 7 is connected with the high-resolution microscopic imaging relay optical path 6.
The large-field microscopic imaging auxiliary function system can be a system light source, and the light-emitting spectral range of the illumination light source is mainly 400-700 nm, the nominal power is 150W, and the color temperature is 3450 Kelvin.
The large-field microscopic imaging objective lens 1: the magnification of the objective lens is 20, the NA is 0.25, the working distance is 25 mm, and the transmittance of 400-700 nm is more than 90%.
The high-resolution microimaging objective 5: the magnification of the objective lens is 100, the NA is 1.4, the working distance is 0.15 mm, and the transmittance of 400-700 nm is more than 80%.
The exposure time of the large-field microscopic imaging photoelectric image detector 4 is less than 1 millisecond, and the microsecond exposure time can be accurately controlled.
The transverse stroke of the object stage 9 is 110 mm, the longitudinal stroke is 75 mm, the displacement resolution is 0.1 micron, the repetition precision is more than 1 micron, the maximum running speed is 7 mm/s, and the maximum load of the object stage is 5 kg.
And a beam splitter in the relay optical path 2 of the large-field microscopic imaging, wherein the beam splitter substrate is fused silica, the two-dimensional size is 25 mm in width, 36 mm in length and 1 mm in thickness, the reflectivity and the transmissivity are between 10% and 90% in the range of 350 to 1100 nm of the spectrum, and the ratio of the reflectivity to the average value of the transmissivity is between 9: 1 and 9: 1.
The method for performing high-resolution imaging of a large number of moving sperms under a large visual field by using the novel sperm quality evaluation system comprises the following steps:
and after the semen sample is liquefied at 37 ℃, uniformly mixing and flaking the semen sample according to a corresponding laboratory manual method and keeping the temperature at stable 37 ℃ to obtain a flaking semen sample. Placing the sperm sample 9 on an objective table 9 between a large-field microscopic imaging objective lens and a high-resolution microscopic imaging objective lens, and preheating the objective table 9 in advance to keep the temperature constant at 37 ℃.
The large-field microscopic imaging objective lens 1 and the high-resolution microscopic imaging objective lens 5 are respectively arranged at the upper side and the lower side of the objective table. The field of view range 102 of the high resolution microimaging objective is located within the field of view range 101 of the large field of view microimaging objective (fig. 1). The method comprises the following steps: the field of view range 102 of the high-resolution micro-imaging system is located in the middle of the field of view range 101 of the large-field micro-imaging objective, and the side 203 of the high-resolution observation position of the target sperm in the semen sample 11 is located on the imaging focal plane of the field of view side 202 of the high-resolution micro-imaging objective and the field of view side 201 of the large-field micro-imaging objective.
Adjusting parameters such as illumination intensity and the position of a semen sample to enable imaging of a field range 101 of a large-view micro imaging system and a field range 102 of a high-resolution micro imaging system to be clear, transmitting imaging information acquired by a large-view micro imaging objective lens 1 to a large-view micro imaging photoelectric image detector 4 through a large-view micro imaging relay optical path 2, converting the imaging information into digital picture information by the large-view micro imaging photoelectric image detector, transmitting the digital picture information to a computer graphic workstation 10, and observing a plurality of targets through continuous acquisition and storing, recording and tracking by the computer graphic workstation; imaging information acquired by the high-resolution microscopic imaging objective lens 5 is transmitted to the high-resolution microscopic imaging photoelectric image detector 8 through the high-resolution microscopic imaging relay optical path 6, the high-resolution microscopic imaging photoelectric image detector 8 converts the imaging information into digital picture information, and then the digital picture information is transmitted to the computer graphic workstation 10.
According to the imaging information and the recorded result collected by the large-visual-field microscopic imaging objective lens, the starting positions 103 of the target sperm and the target sperm are determined, the target sperm is rapidly moved from a target sperm large-visual-field starting position 103 to a target sperm high-resolution observation position 104 (located in a visual field range of a high-resolution microimaging objective) according to a target sperm moving track 105 through a horizontal rapid moving object stage 9, imaging data (such as high-resolution morphological data) of the target sperm are collected, in the collection process, real-time information of the target sperm high-resolution observation position 104 is recorded by utilizing a visual field range 101 of a large-visual-field microimaging system and a visual field range 102 of a high-resolution microimaging system, by rapidly moving the object stage 304, the target sperm high-resolution observation position 104 is ensured to be positioned in the ideal imaging range of the field of view range 102 of the high-resolution microscopic imaging system, and the integrity of the high-resolution morphological data acquisition process is ensured.
The imaging data-image information collected by the field range 101 of the large-field microscopic imaging objective lens and the field range 102 of the high-resolution microscopic imaging objective lens are transmitted and recorded in the computer graphic workstation 10, and are analyzed, so that the collection work of the imaging data of the target sperm is completed.
And determining the next target sperm to be detected in the field of view range 101 of the large-field microscopic imaging system according to the principles of statistics and the like, repeating the steps, starting the next cycle of acquisition, and ending the current acquisition until all target sperm to be acquired in the field of view range 101 of the large-field microscopic imaging system.
And moving the object stage 9 randomly or according to a statistical principle, changing the field range 101 of the large-field microscopic imaging system, and starting to collect a new field until the number of the sperms information required by statistics is reached.
The image of the sperm collected by the large-field microscopic imaging system is shown in fig. 4, and the image of the sperm collected by the high-resolution microscopic imaging system is shown in fig. 5.
From the above, it can be seen that the system and method of the present invention employs two microscopic imaging systems of different imaging ranges and resolutions, which simultaneously co-localize imaging of a test sample. By coupling the imaging information of the large-visual-field microscopic imaging system and the high-resolution microscopic imaging system, the continuous movement track of the sperms can be obtained, the vitality, the concentration and the like of the sperms, the morphology and the structure of the sperms can be quantitatively counted and analyzed, particularly, the kit has genetic function indexes, can detect dynamic information of the form of a moving sperm and the state of tail movement, can accurately measure the activity (curve speed, linear speed and the like) and the form (head size, head form, cytoplasm droplets and the like) of a single live sperm, can reflect the sperm quality more accurately by superposition statistics, realizes the 3D information acquisition of the sperm, can be popularized and used for the injection of the single sperm by the information of the top, the head form, the vacuole position and the like of the sperm, provides more accurate clinical guidance for the disease diagnosis of the test tube infant, and provides sperm cell proteomics and single cell sequencing in basic research. And the sample does not need to be dyed for detection, so the invention maintains the sperm activity and the fertilization capability, and can be used for scientific research and clinical treatment (in vitro fertilization) after sorting. The system and the method can also simultaneously detect the vitality and the shape of a large number of sperms of the semen sample at one time, and are convenient for scientific research and clinical statistical analysis.
Claims (6)
1. A novel sperm quality evaluation system is characterized in that the system is used for detecting the vitality index of a large amount of sperms and realizing the high-resolution detection and evaluation of the shape of the sperms;
the system comprises a sperm sample stage which can move in three dimensions and be controlled accurately and two independent imaging systems: the system comprises a large-visual-field microscopic imaging system and a high-resolution microscopic imaging system, wherein the two imaging systems can be adjusted to be in confocal and coaxial states;
the large-field microscopic imaging system comprises a large-field microscopic imaging objective lens, a large-field microscopic imaging relay optical path and a large-field microscopic imaging photoelectric image detector which are sequentially connected;
the high-resolution microscopic imaging system comprises a high-resolution microscopic imaging objective lens, a high-resolution microscopic imaging relay light path and a high-resolution microscopic imaging photoelectric image detector which are sequentially connected;
the sperm sample stage capable of moving in three dimensions and being accurately controlled can control the temperature of the sample cell on the sperm sample stage and can move sperm samples randomly according to requirements;
the large-visual-field microscopic imaging system can rapidly shoot a sufficient number of sperms in a visual field, automatically output the position and speed information of each sperm in the visual field through an image processing technology and a multi-target tracking technology, and count all vitality indexes of the sperms according to the requirements;
the high-resolution microscopic imaging system acquires morphological information in an imaging area of the large-view microscopic imaging system, simultaneously acquires position information of sperms, and synchronously corresponds to the position information of each sperm in the imaging area of the large-view microscopic imaging system.
2. The system of claim 1, wherein the sperm sample stage capable of moving in three dimensions and being precisely controlled can rapidly move the position of the sample stage according to the visual field size and position information of the large-visual-field microscopic imaging system and the high-resolution microscopic imaging system, so that the sperm observed in the large-visual-field microscopic imaging system can be sequentially moved to the imaging area of the high-resolution microscopic imaging system, thereby realizing the scanning of the imaging of the high-resolution microscopic imaging system in the imaging area of the large-visual-field microscopic imaging system, performing high-resolution microscopic imaging on all the sperm in each part of the imaging area of the large-visual-field microscopic imaging system, and further performing morphological analysis on all the sperm in the large visual field.
3. The system of claim 1, wherein the speed of any sperm movement corresponds to its morphological criteria, and the high resolution microscopic imaging system does not repeatedly detect the same sperm under the surveillance of the wide field microscopic imaging system.
4. The novel sperm quality evaluation system of claim 1 comprising a large field of view microimaging objective, a large field of view microimaging relay optics, a large field of view microimaging optoelectronic image detector, a high resolution microimaging objective, a high resolution microimaging relay optics, a high resolution microimaging optoelectronic image detector, a sample stage and a computer graphics workstation, the large-field microscopic imaging objective lens and the high-resolution microscopic imaging objective lens are separately arranged on two sides of the sample stage, the large-field microscopic imaging objective lens, the large-field microscopic imaging relay optical path and the large-field microscopic imaging photoelectric image detector are sequentially connected, the high-resolution microscopic imaging objective lens, the high-resolution microscopic imaging relay optical path and the high-resolution microscopic imaging photoelectric image detector are sequentially connected, and the large-field microscopic imaging photoelectric image detector and the high-resolution microscopic imaging photoelectric image detector are respectively connected with a computer graphic workstation through signals.
5. The system of claim 4, further comprising a wide-field microscopy imaging subsystem, wherein the wide-field microscopy imaging subsystem is connected to the relay optical path.
6. The system for evaluating sperm quality of claim 4 further comprising a high resolution micro imaging sub-functionality system, wherein the high resolution micro imaging sub-functionality system is coupled to the high resolution micro imaging relay optical path.
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