CN118131466A - Dark field microscope for sperm motility analysis and analysis method - Google Patents
Dark field microscope for sperm motility analysis and analysis method Download PDFInfo
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Abstract
The invention discloses a dark field microscope for sperm motility analysis and an analysis method, wherein the dark field microscope comprises a shell, an illumination light source, a two-dimensional moving object stage, an objective lens component, an imaging lens, an axial electric control platform, a reflecting mirror component, a CCD camera and a control module; the light path is designed in multiple reflection, so that the miniaturization target is achieved, and meanwhile, the manufacturing cost is reduced; the annular LED array is adopted for illumination, so that any illumination color and illumination brightness can be adjusted, and dark field imaging of illumination with different wavelengths is realized; the microscope is matched with an independently written LabVIEW sperm movement analysis program LabCASA, and a program page is provided with a microscope control panel and a sperm analysis panel, so that real-time imaging and real-time tracking of sperm movement tracks can be realized, and sperm motility can be analyzed. The system has clear imaging, high contrast of sperm images, reduced error of sperm motility detection, portable system size, low cost, and suitability for remote areas or resource limited areas.
Description
Technical Field
The invention relates to the technical fields of optical microscopic imaging and sperm detection, in particular to a dark field microscope for sperm motility analysis and an analysis method.
Background
Sperm analysis is a common examination item of people with reproductive dysfunction, and by detecting related parameters such as sperm morphology, sperm concentration, sperm motility and the like, the sperm quality is evaluated, and a corresponding infertility treatment scheme is determined. Clinically, the existing sperm analysis device requires a professional to operate a biological microscope, and has large equipment volume, high cost and complex operation method. The large volume and complexity of the equipment are not suitable for resource-limited areas.
Most of sperm detection devices used in the market at present use bright field imaging, and the imaging mode can not improve contrast between sperms and the background, so that problems of wrong sperm and background identification or unrecognizable sperms and the like can possibly occur during subsequent sperm track tracking. In addition, the collection of more than 200 sperms is ensured at 5 points required in the world health organization semen processing laboratory manual, the existing portable sperm analysis device can not meet the condition of multi-point collection, and the obtained sperm parameter error is larger.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a dark field microscope and an analysis method for sperm motility analysis, which have the advantages of low cost, portability, clear imaging and high picture contrast, so as to solve the problems that the existing portable sperm analysis device cannot meet the requirement of multipoint acquisition, and the sperm and background identification is wrong or the sperm cannot be identified in bright field imaging.
Therefore, the invention provides a dark field microscope for sperm motility analysis, which comprises a shell, an illumination light source, a two-dimensional moving object stage, an objective lens component, an imaging lens, an axial electric control platform, a reflecting mirror component, a CCD camera and a control module; the objective lens component is positioned below the two-dimensional moving object stage and comprises an objective lens and a lens cone, and the central axis directions of the objective lens and the lens cone are parallel to the vertical direction; simultaneously, the imaging lens is positioned right below the lens barrel; and the axial electric control platform is connected with the lens cone to realize the focusing of the objective lens.
The reflecting mirror assembly is arranged below the objective lens assembly and comprises a vertical reflecting mirror bracket, a transverse reflecting mirror bracket, a first reflecting mirror and a second reflecting mirror; the first reflecting mirror is matched with the second reflecting mirror to reflect an image formed in the imaging lens and then transmit the image to the CCD camera, and then sperm motility analysis is realized through a sperm detection module; the illumination light source comprises annular array color LED lamp beads, and the control module is used for controlling the color and brightness of illumination light to realize dark field imaging; the two-dimensional moving object stage is provided with a counting plate; and realizing multipoint acquisition.
As a preferable technical scheme of the invention, the two-dimensional moving object stage comprises a connecting bottom plate, a lower sliding plate, an upper sliding plate, a first screw motor unit and a second screw motor unit; the lower sliding plate is longitudinally matched with the connecting bottom plate in a sliding mode and driven by the second screw motor unit, the upper sliding plate is transversely matched with the lower sliding plate in a sliding mode and driven by the first screw motor unit, and the counting plate is placed on the upper sliding plate.
As a preferable technical scheme of the invention, the side edge of the lens cone is fixedly connected with an objective axial adjusting frame, and the objective axial adjusting frame and the axial electric control platform.
As a preferable technical scheme of the invention, the vertical mirror bracket and the transverse mirror bracket are in a right-angle triangular prism shape, and the bottoms of the vertical mirror bracket and the transverse mirror bracket are fixedly connected with the shell respectively.
As a preferable technical scheme of the invention, the plane of the reflecting mirror I forms an included angle with the central axis direction of the objective lens, so that the vertical direction light rays transmitted from the objective lens are reflected to horizontal and transverse direction light rays; the plane of the second reflecting mirror forms an included angle with the axial direction of the CCD camera lens, so that horizontal transverse light reflected from the reflecting mirror is reflected vertically to form horizontal longitudinal light which is then injected into the CCD camera.
As a preferable technical scheme of the invention, the shell is also provided with a triaxial sliding platform, the illumination light source is connected with the triaxial sliding platform through a supporting rod, and the triaxial sliding platform comprises a Z-axis displacement table and an X-Y axis displacement table and is used for adjusting the position and the height of the light source.
As a preferable embodiment of the present invention, the objective lens is an air lens, na=0.25, and the magnification is 10×, and imaging of each series of objective lenses of 4× to 100× can be supported.
As a preferable technical scheme of the invention, the shell comprises a base and a left shell and a right shell, the two-dimensional moving object stage is fixedly arranged at the left upper end of the shell, and the axial electric control platform is fixedly connected with the right shell.
As a preferable technical scheme of the invention, the control module is provided with an Arduino UNO singlechip and is used for controlling an illumination light source, a two-dimensional moving object stage, an axial displacement platform, camera parameters and image acquisition.
The invention provides an analysis method of a dark field microscope for sperm motility analysis, which comprises the following steps:
S1, diluting semen, recording dilution factors, fully mixing, and adding the diluted semen into a sperm counting plate;
S2, loading an objective lens, wherein a 10X infinite objective lens is selected, and the NA value is 0.25;
s3, loading a sperm counting plate filled with a sample on a microscope;
s4, turning on a control program, turning on LED illumination, and adjusting illumination NA to enable the microscope to be in a dark field imaging working mode;
s5, opening a camera, checking whether the image is clear, adjusting the axial position of the objective lens, searching a focal plane, and observing a clear sperm image;
S6, clicking a program, adjusting imaging parameters of a CCD camera, ensuring proper exposure, and shooting more than 30 frames of images per second;
s7, switching functions, previewing the effect of the binarized image, and obtaining an ideal binarized image by adjusting parameters such as brightness, contrast and the like;
s8, selecting a sperm analysis function, calculating sperm movement parameters, sperm number and the like, and switching and controlling a screw motor group to switch the field of view;
S9, integrating analysis results of the multiple fields, and calculating average concentration and activity and motion information.
The light path of the invention adopts the design of a polarizing shaft, the volume of the device is greatly reduced and is only 164 multiplied by 139 multiplied by 250mm 3, thus achieving the miniaturization aim and reducing the manufacturing cost; dark field imaging is adopted, so that the contrast of sperms is improved, the problem that sperms cannot be identified or are identified by mistake during movement tracking is solved, the operation difficulty of a microscope is reduced through integrated control of an upper computer, and an integrated CASA algorithm is written to directly obtain sperm movement parameters. The system has the advantages of clear acquired images, high automation degree, short detection time, small volume and the like.
In addition to the objects, features and advantages described above, the present application has other objects, features and advantages. The present application will be described in further detail with reference to the drawings.
Drawings
The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification, illustrate embodiments of the application and together with the description serve to explain the application. In the drawings:
FIG. 1 is a schematic diagram of a dark field microscope for sperm motility analysis according to the present invention;
FIG. 2 is a schematic diagram of a dark field microscope for sperm motility analysis according to the present invention;
FIG. 3 is a schematic diagram III of the dark field microscope for sperm motility analysis of the present invention;
FIG. 4 is a graph showing the effect of dark field microscopy on sperm detection for use in sperm motility assays in accordance with the present invention;
FIG. 5 is a flow chart of the steps of the dark field microscopy method for sperm motility analysis of the present invention;
Description of the reference numerals
1. An illumination light source; 2. a support rod; 3. a Z-axis displacement table; 4. an X-Y axis displacement table; 5. a first screw motor unit; 6. an upper sliding plate; 7. a screw motor group II; 8. a lower slide plate; 9. a connecting bottom plate; 10. an objective lens; 11. a lens barrel; 12. an objective axial adjusting frame; 13. an imaging lens mount; 14. a vertical mirror mount; 15. a lateral mirror mount; 16. an axial electric control platform; 17. a CCD camera; 18. a housing; 19. and (5) a base.
Detailed Description
It should be noted that, without conflict, the embodiments of the present application and features of the embodiments may be combined with each other. The application will be described in detail below with reference to the drawings in connection with embodiments.
As shown in fig. 1 to 3, the dark field microscope for sperm motility analysis adopts an inverted microscope imaging mode, and comprises a shell, an illumination light source 1, a triaxial sliding platform, a two-dimensional moving object stage, an objective lens assembly, an axial electric control platform, a lens assembly, a reflecting mirror assembly, a CCD camera and a control module; the counting plate with sperms is arranged on the two-dimensional moving object stage; the axial electric control platform controls the objective lens component to move so as to adjust the focal length of the objective lens component; the CCD camera receives the image transmitted by the reflecting mirror assembly, the two-dimensional moving object stage drives the counting plate to move to realize multi-point acquisition, and meanwhile, the three-axis sliding platform drives the illumination light source to adjust the position and the height of the light source, so that the illumination condition is always consistent in the observation process.
Specifically, the shell comprises a base 19, a left shell 18 and a right shell 18, and the two-dimensional moving object stage is fixedly arranged at the left upper end of the shell 18 and comprises a connecting bottom plate 9, a lower sliding plate 8, an upper sliding plate 6, a first screw motor unit 5 and a second screw motor unit 7; the connecting bottom plate 9 is fixedly connected with the left shell 18, the lower sliding plate 8 is longitudinally matched with the connecting bottom plate 9 in a sliding way and driven by the screw motor group II 7, and the upper sliding plate 6 is transversely matched with the lower sliding plate 8 in a sliding way and driven by the screw motor group I5; and the counting plate is placed on the upper sliding plate 6 and moves along with the two-dimensional moving object stage, so that multipoint acquisition is realized.
The two-dimensional moving object stage is connected with the object carrying platform through two lead screw motors, so that the switching of imaging view fields is realized. The movement of the screw motor is controlled by the upper computer, the visual field can be smoothly and stably switched, the two-dimensional moving objective table supplements a manual driving module, and the visual field can be manually adjusted and switched when the power is off.
As shown in fig. 2, the objective lens assembly is located below the two-dimensional moving stage, and includes an objective lens 10 and a lens barrel 11, and the lens assembly includes an imaging lens and an imaging lens mount 13; the central axis direction of the objective lens 10 and the lens cone 11 is parallel to the vertical direction, and the objective lens 10 and the lens cone 11 are fixedly connected, so that the objective lens can be manually switched; the imaging lens is mounted on the imaging lens mount 13 and located directly below the lens barrel 11; the lens barrel 11 is connected with an axial electric control platform 16, the side edge of the lens barrel 11 is fixedly connected with an objective axial adjusting frame 12, the objective axial adjusting frame 12 is fixedly connected with the axial electric control platform 16, and the axial electric control platform 16 is fixedly connected with a right side shell 18; the axial electric control platform 16 drives the lens barrel 11 to move up and down, so that the distance between the objective lens 10 and the counting plate is adjusted, the observation focal length is changed, and the objective lens focusing is realized. Likewise, the axial control platform 16 also has a manual drive module that can switch the field of view by manual adjustment when the power is off.
The microscope is an infinite objective lens, a plurality of objective lenses are selectable, the magnification factor is changed in a manual switching mode, the magnification factor of the objective lenses is 10 times, 20 times, 40 times, 60 times, 100 times and the like, and the numerical aperture is between 0.25 and 1.35, and the microscope comprises water immersion, oil immersion and air system objective lenses. The axial displacement of the objective lens is regulated by an axial electric control platform, the position of the objective lens can be regulated in a program control or manual regulation mode, and an imaging focal plane is searched.
Specifically, the microscope objective 10 used is an air mirror, na=0.25, magnification is 10×, imaging of each series of objective of 4× 100× can be supported, and manual replacement can be performed as required. The lens adopts double-cemented achromatic lens, and can correct chromatic dispersion effect to a certain extent. The stepper motor used by the microscope can reach micron-scale precision, and can realize focusing and accurate control of an imaging view field.
As shown in fig. 2, the mirror assembly is disposed below the objective lens assembly and includes a vertical mirror frame 14, a lateral mirror frame 15, and two mirrors; the vertical mirror bracket 14 and the transverse mirror bracket 15 are in a right-angle triangular prism shape as a whole, and the bottoms of the vertical mirror bracket 14 and the transverse mirror bracket 15 are respectively fixedly connected with the base 19; the vertical mirror frame 14 is provided with a first mirror, and the plane of the first mirror forms an included angle of 45 degrees with the central axis direction of the objective lens 10, so that the vertical direction light rays transmitted from the objective lens 10 are reflected into horizontal transverse direction light rays.
The second reflecting mirror is arranged on the transverse reflecting mirror bracket 15, the second reflecting mirror extends along the vertical direction, and meanwhile, an included angle of 45 degrees is formed between the plane of the second reflecting mirror and the axial direction of the lens of the CCD camera 17, so that horizontal transverse light reflected by the reflecting mirror is vertical to form horizontal longitudinal light which is then injected into the CCD camera 17, and the CCD camera 17 shoots a picture and analyzes sperm activity through analysis software.
The central axis of the objective lens 10 is perpendicular to the different plane of the lens axis of the CCD camera 17, the vertical mirror frame 14 and the transverse mirror frame 15 are provided with mirrors for reflecting the light path, the imaging light path is folded, the device volume is compressed to 164 multiplied by 139 multiplied by 250mm 3, and the microscope volume is reduced.
As shown in fig. 1, an illumination light source 1 is positioned right above a two-dimensional moving object stage, the illumination light source 1 is connected with a three-axis sliding platform arranged on a right end shell through a support rod 2, and the three-axis sliding platform comprises a Z-axis displacement platform 3 and an X-Y axis displacement platform 4; the Z-axis displacement table 3 adjusts the height of the light source, and the X-Y axis displacement table 4 adjusts the horizontal position of the light source, so that the position of the illumination light source is three-dimensionally adjustable; the light conditions are always consistent in the observation process by matching the light conditions with the movement of the two-dimensional moving object stage.
The illumination light source used for the dark field microscope for sperm motility analysis is an annular array formed by intelligent external control LED lamp beads packaged by 5050, so that 0-255 chromaticity accurate regulation and brightness control of three RGB channels can be realized, and the typical central wavelengths of RGB are 630, 523 and 470nm respectively; the illumination distance D can be controlled to be 30-90 cm.
The annular LED array light source is annular illumination light composed of color LED lamp beads, the color and the brightness of the illumination light can be controlled through a program, and the position of the light source is regulated through a three-dimensional regulating device composed of a Z-axis displacement platform and a X, Y-axis displacement platform, so that a better imaging effect is obtained.
In addition, the control module of the microscope is used for controlling the LED light source, controlling the two-dimensional carrying platform, controlling the objective axial displacement platform, controlling the camera parameters and collecting images. An Arduino UNO singlechip is used as a controller to control an illumination light source, an objective table and an axial electric control platform, communication is carried out between the Arduino UNO singlechip and the LabVIEW, illumination light source control, objective table displacement control, axial platform control and CCD camera control are integrated. The computer is used for carrying out centralized control on the dark field microscope, so that focusing, field switching, illumination light adjustment, exposure and video setting are realized, a sample can be controlled by the computer only by placing on the microscope, and the sperm detection is carried out without too much complex operation.
The function control module also comprises a sperm detection module, wherein the sperm detection module comprises reading of a camera, tracking of sperm tracks and measurement of sperm movement parameters. Through function switching, a video sequence binarization preview page is opened, parameters such as contrast, brightness and the like are adjusted, a binarization video sequence with proper size is obtained, then motion tracking is clicked, calculation of the motion trail of sperms can be achieved, and through a written algorithm, the motion parameters of sperms are obtained, and sperm motility is analyzed. The microscope is equipped with LabCASA sperm detection program, which can detect parameters including sperm concentration, motility, total number, average curve speed, average linear speed and the like.
As shown in fig. 5, according to the above-mentioned dark field microscope for sperm motility analysis, the present invention further provides an analysis method of dark field microscope for sperm motility analysis, comprising the steps of:
S1, diluting and fully mixing semen, recording dilution times, and adding the dilution times into a 10 mu m deep sperm counting plate;
S2, loading an objective lens, wherein a 10X infinite objective lens is selected, and the NA value is 0.25;
s3, loading a sperm counting plate filled with a sample on a microscope;
s4, turning on a control program, turning on LED illumination, and adjusting illumination NA to enable the microscope to be in a dark field imaging working mode;
s5, opening a camera, checking whether the image is clear, adjusting the axial position of the objective lens, searching a focal plane, and observing a clear sperm image;
S6, clicking a program, adjusting imaging parameters of a CCD camera, ensuring proper exposure, and shooting more than 30 frames of images per second;
s7, switching functions, previewing the effect of the binarized image, and obtaining an ideal binarized image by adjusting parameters such as brightness, contrast and the like;
s8, selecting a sperm analysis function, calculating sperm movement parameters, sperm number and the like, and switching a control motor to switch a field of view;
S9, integrating analysis results of a plurality of fields of view, and calculating average concentration and activity and motion information;
In an embodiment, dark field imaging is selected by calculating the matching relationship between the illumination aperture NA illu and the objective aperture NA obj, wherein:
R is the radius of the LED array, D is the illumination distance, and α is the illumination aperture angle. When NA illu≤NAobj, the bright field illumination condition is satisfied; when NA illu>NAobj, dark field lighting conditions are satisfied, dark field imaging can be achieved. According to the calculation, the illumination distance D is adjusted to enable the microscope to be in a working state of dark field imaging for sperm analysis.
Sperm motility analysis is processed according to the sixth WHO manual of human sperm examination laboratory, and sperm motility parameters are classified into a curvilinear movement Velocity (VCL), a linear movement Velocity (VSL), a mean path Velocity (VAP), etc., and sperm motility, and sperm quality can be evaluated by analyzing sperm motility status. The specific calculation method is as follows:
1) The CCD camera is used for collecting an image sequence of about 1s, the frame rate is 25-40fps, the microscope matched sperm identification and tracking program LabCASA is used for analyzing the video sequence to obtain the number of sperms and the movement track, the movement distance is calculated to obtain the speed parameter of the sperms, and the concentration and the activity of the sperms are evaluated (as shown in figure 4).
2) VSL is determined by determining the linear distance between the first and last points of the trajectory and correcting according to sperm movement time, and the VSL calculation formula is shown in formula (2)
3) VCL is the real time average velocity of the sperm head along its actual curve, i.e. the time average velocity of the two-dimensional motion track seen under the microscope, reflecting the sperm motility, the calculation method is to find the sum of the distances along the track, then correct according to time, the curve path of the sperm refers to the total distance covered by the sperm head during the observation period. VCL calculation formula is shown in the following formula (3)
4) The VAP is the average path rate of the sperm head, i.e. the time-average rate of the movement of the sperm head along its average path, and the average movement path and average path rate of the sperm are obtained by averaging the actual movement path of the sperm, and the VAP calculation formula is shown in formula (4).
In the formulas (2), (3) and (4), n is the number of sperm, x and y are the path coordinates, and M and Δt are the number of frames and the inter-frame time consumption of video, respectively. In the formula (4), the amino acid sequence of the compound,And/>The average route point calculation method is represented by taking an average value calculation once using five points. (x, y) is coordinates representing specified points of five of the paths. Where (xj, yj) is the coordinates representing the j-th specified point in the path.
5) Sperm motility is obtained based on sperm motility parameters if the VAP of sperm is greater than 25 μm/s; the fast forward characteristic is marked that the sperm velocity is more than 5 mu m/s and less than 25 mu m/s; slow forward, sperm velocity less than 5 μm/s, greater than 0, and non-forward; wherein, the average activity g=e/F of sperm; e is the total number of motile sperm in all fields of view, e=e1+e2+ & gt en, en representing the number of all motile sperm in the nth field of view; f is the total number of sperm in all fields, f=f1+f2+ & fn, fn indicating the number of all sperm in the nth field.
The working principle of the dark field microscope and method for sperm motility analysis of the present invention is briefly described below with reference to the accompanying drawings.
The device volume is compressed to 164×139×250mm 3 by adopting the optical path off-axis and multipath reflection design. The imaging light route comprises an annular LED array light source, a three-dimensional adjusting platform of the light source, a two-dimensional electric control object carrying platform, a microscope objective, an axial electric control platform, an imaging lens, two silver plating reflectors and a CCD camera. The function control module comprises a control module for controlling the brightness of the LED light source, a control module for controlling the two-dimensional object carrying platform, a control module for controlling the axial displacement platform of the objective lens, a control module for controlling the parameters of the camera and the image acquisition. The sperm detection module comprises reading of a camera, sperm track tracking and sperm movement parameter measurement.
The imaging system of the inverted microscope is adopted, the magnification is manually switched, the microscope objective lenses with 4 multiplied by 100 multiplied by different multiplying power can be used for imaging, including water immersion, oil immersion and air system objective lenses, and the imaging focal plane and microscopic view field are changed in an electric control mode.
The annular LED array light source is annular illumination light composed of color LED lamp beads, the color and the brightness of the illumination light can be controlled through a program, and the position of the light source can be adjusted, so that a better imaging effect can be obtained. The two-dimensional displacement electric control platform and the axial electric control platform are both supplemented with manual displacement platforms, and manual operation can be used when power is off.
The computer performs centralized control on the dark field microscope to realize focusing, field switching, illumination light adjustment, exposure and video setting, and the sperm detection can be performed by computer control only by placing a sample on the microscope without too much complex operation.
Through function switching, a video sequence binarization preview page is opened, parameters such as contrast, brightness and the like are adjusted, a binarization video sequence with proper size is obtained, then motion tracking is clicked, calculation of the motion trail of sperms can be achieved, and through a written algorithm, the motion parameters of sperms are obtained, and sperm motility is analyzed. The microscope is equipped with LabCASA sperm detection program, which can detect parameters including sperm concentration, motility, total number, average curve speed, average linear speed and the like.
The above description is only of the preferred embodiments of the present application and is not intended to limit the present application, but various modifications and variations can be made to the present application by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.
Claims (10)
1. The dark field microscope for sperm motility analysis is characterized by comprising a shell, an illumination light source (1), a two-dimensional moving object stage, an objective lens assembly, an imaging lens, an axial electric control platform, a reflecting mirror assembly, a CCD camera and a control module;
the objective lens assembly is positioned below the two-dimensional moving object stage and comprises an objective lens (10) and a lens barrel (11), and the central axis directions of the objective lens (10) and the lens barrel (11) are parallel to the vertical direction; simultaneously, the imaging lens is positioned right below the lens barrel (11); the axial electric control platform (16) is connected with the lens cone (11) to realize focusing of the objective lens (10);
The reflector assembly is arranged below the objective lens assembly and comprises a vertical reflector bracket (14), a transverse reflector bracket (15), a first reflector and a second reflector; the first reflecting mirror is matched with the second reflecting mirror to reflect an image formed in the imaging lens and then transmit the image to the CCD camera (17), and then sperm motility analysis is realized through a sperm detection module;
The illumination light source (1) comprises annular array color LED lamp beads, and the control module is used for controlling the color and brightness of illumination light to realize dark field imaging; the two-dimensional moving object stage is provided with a counting plate; and realizing multipoint acquisition.
2. Dark-field microscope for sperm motility analysis according to claim 1, characterized in that the two-dimensional mobile stage comprises a connection base plate (9), a lower slide plate (8), an upper slide plate (6), a first screw motor group (5) and a second screw motor group (7); the lower sliding plate (8) is longitudinally matched with the connecting bottom plate (9) in a sliding mode and driven by the screw motor group II (7), the upper sliding plate (6) is transversely matched with the lower sliding plate (8) in a sliding mode and driven by the screw motor group I (5), and the counting plate is placed on the upper sliding plate (6).
3. Dark-field microscope for sperm motility analysis according to claim 1, characterized in that the lens barrel (11) is fixedly connected with an objective axial adjusting frame (12) at the side, the objective axial adjusting frame (12) and the axial electric control platform (16).
4. Dark field microscope for sperm motility analysis according to claim 1, characterized in that the vertical mirror holder (14) and the lateral mirror holder (15) are in the shape of a right triangular prism as a whole, and the bottoms of the vertical mirror holder (14) and the lateral mirror holder (15) are fixedly connected with the housing, respectively.
5. Dark-field microscope for sperm motility analysis according to claim 1, characterized in that the plane of the mirror-plane is at an angle of 45 ° to the direction of the central axis of the objective lens (10), reflecting the vertical light rays propagating out of the objective lens (10) into horizontal transverse light rays; the plane of the second reflecting mirror forms an included angle of 45 degrees with the axial direction of the lens of the CCD camera (17), so that horizontal and transverse light rays reflected by the reflecting mirror are reflected vertically to form horizontal and longitudinal light rays, and the horizontal and longitudinal light rays are injected into the CCD camera (17).
6. Dark-field microscope for sperm motility analysis according to claim 1, characterized in that the housing is further provided with a three-axis sliding platform, the illumination source (1) being connected to the three-axis sliding platform by means of struts (2), and the three-axis sliding platform comprising a Z-axis displacement stage (3) and an X-Y-axis displacement stage (4) for adjusting the position and the height of the light source.
7. Dark-field microscope for sperm motility analysis according to claim 1, characterized in that the objective lens (10) is an air lens, na=0.25, magnification is 10×, imaging of each series of objective lenses 4× 100× can be supported.
8. Dark-field microscope for sperm motility analysis according to claim 1, characterized in that the housing comprises a base (19) and left and right housings (18), the two-dimensional mobile stage is fixedly mounted at the upper left end of the housing (18), and the axial electric control platform (16) is fixedly connected with the right housing (18).
9. Dark-field microscope for sperm motility analysis according to claim 1, characterized in that the control module has an Arduino UNO single-chip microcomputer for controlling the illumination source (1), the two-dimensional moving stage, the axial displacement stage (16), the camera parameters and the image acquisition.
10. A method of dark-field microscopy for sperm motility analysis as described in any one of claims 1 to 9, comprising the steps of:
S1, diluting semen, recording dilution factors, fully mixing, and adding the diluted semen into a sperm counting plate;
S2, loading an objective lens, wherein a 10X infinite objective lens is selected, and the NA value is 0.25;
s3, loading a sperm counting plate filled with a sample on a microscope;
s4, turning on a control program, turning on LED illumination, and adjusting illumination NA to enable the microscope to be in a dark field imaging working mode;
s5, opening a camera, checking whether the image is clear, adjusting the axial position of the objective lens, searching a focal plane, and observing a clear sperm image;
S6, clicking a program, adjusting imaging parameters of a CCD camera, ensuring proper exposure, and shooting more than 30 frames of images per second;
s7, switching functions, previewing the effect of the binarized image, and obtaining an ideal binarized image by adjusting parameters such as brightness, contrast and the like;
s8, selecting a sperm analysis function, calculating sperm movement parameters, sperm number and the like, and switching and controlling a screw motor group to switch the field of view;
S9, integrating analysis results of the multiple fields, and calculating average concentration and activity and motion information.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202410265353.8A CN118131466A (en) | 2024-03-08 | 2024-03-08 | Dark field microscope for sperm motility analysis and analysis method |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410265353.8A CN118131466A (en) | 2024-03-08 | 2024-03-08 | Dark field microscope for sperm motility analysis and analysis method |
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| Publication Number | Publication Date |
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| CN118131466A true CN118131466A (en) | 2024-06-04 |
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| CN202410265353.8A Pending CN118131466A (en) | 2024-03-08 | 2024-03-08 | Dark field microscope for sperm motility analysis and analysis method |
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| Country | Link |
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| CN (1) | CN118131466A (en) |
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