Near-infrared two-zone turntable confocal microscopic system

Abstract

The invention relates to a near-infrared two-zone turntable confocal microscopy system, which is characterized in that a laser emitter emits laser, the laser is reflected to a third lens through a dichroscope, the third lens focuses the laser on a turntable, the laser penetrates through a fourth lens, a sample to be measured is placed on an objective table, and the sample is dyed with a fluorescent dye positioned in a near-infrared two-zone window; a sample receives laser and excites fluorescent light, the fourth lens refocuses the fluorescent light on the rotating disc, the dichroscope allows light beams located in a near-infrared two-area window to penetrate through, the fluorescent light penetrates through the third lens and the dichroscope, the camera receives the fluorescent light of the sample and carries out imaging, and at least one ring-area through hole forming an Archimedes spiral pattern is etched in the rotating disc. And the light beam is imaged through the Archimedes spiral pattern in the turntable. Compared with the prior art, the near-infrared turntable confocal microscopic imaging device can provide a high-quality near-infrared turntable confocal microscopic imaging effect under the condition of low cost.

Description

Technical Field

[0001] The invention belongs to the technical field of optical imaging and relates to a near-infrared two-zone rotating disk confocal microscopy system. Background Art

[0002] Spinning disk confocal microscopy is currently the most commonly used biological imaging technology. Compared with the traditional single-point laser scanning confocal microscope (LSCM), the spinning disk confocal microscope has the following advantages: (1) The quantum efficiency of the charge-coupled device (CCD) camera used in the spinning disk confocal microscope is significantly higher than the photomultiplier tube used in the single-point laser scanning confocal microscope, so the spinning disk confocal microscope can better utilize the energy of photons; (2) The higher sensitivity of the spinning disk confocal microscope and the high imaging speed achieved by parallel acquisition make it possible to reduce the exposure time in the experiment compared to the single-point laser scanning confocal microscope, thereby achieving a higher frame rate under the premise of the same signal-to-noise ratio; (3) Compared with the single-point laser scanning confocal microscope, the spinning disk confocal microscope causes less photobleaching, so it can image living cells for a longer time. Existing spinning disk confocal microscopes often require the use of complex optical components and high-power laser light sources. The cost of these optical components and lasers is high, resulting in an expensive overall system. In addition, the requirements for precise mechanical control structures and imaging sensors further increase the cost of equipment manufacturing.

[0003] Most of the current spinning disk confocal microscopes use visible light excitation, which is easily affected by tissue absorption and scattering during imaging. The imaging depth is limited and it is difficult to meet the observation needs of thick samples or deep structures.

[0004] Patent CN118795654A discloses a three-dimensional single-turntable confocal microscope, which is provided in sequence along the excitation light path: a light source, a biconvex lens No. 1, a corner cube prism reflector, a short-pass dichroic mirror, a biconvex lens No. 2, a concave hole plate, a focusing turntable, a biconvex lens No. 3 and a loading plate; and is provided in sequence along the imaging light path: a loading plate, a biconvex lens No. 3, a focusing turntable, a concave hole plate, a biconvex lens No. 2, a short-pass dichroic mirror, a biconvex lens No. 4 and an imaging module; the focal length of the biconvex lens No. 1 is f3, the focal length of the biconvex lens No. 2 is f1, and the vertex of the corner cube prism reflector is located at the focus of the biconvex lens No. 1 and the biconvex lens No. 2; the concave hole plate includes an annular plate, on which a plurality of circular holes are provided, and a concave surface coaxial with the circular hole is provided at a position of the circular hole close to the opening of the biconvex lens No. 2. However, this patent achieves three-dimensional imaging by making a focusing turntable with focusing glass groups of different thicknesses, which mainly solves the problem of focal depth of imaging, but does not solve the problem of achieving rapid cell and living body imaging under low background noise.

[0005] Patent CN114838673A discloses an Archimedean fringe rotating disk for a rotating disk confocal system and a partitioning method thereof, comprising: a rotating disk, the rotating disk using fused quartz as a substrate, the substrate coated with an anti-reflection coating, and the rotating disk stripes are engraved on the anti-reflection coating by electron beam lithography; the rotating disk stripes use a curved pattern structure of Archimedean spirals, the spirals on the rotating disk are used as the aperture, and the other parts block the light from passing through, thereby achieving a confocal effect. However, the purpose of this patent is to design an Archimedean fringe rotating disk for a rotating disk confocal system, and it is not designed for the entire microscope system, and the problem of achieving rapid cell and living body imaging under low background noise has not been solved.

[0006] Patent CN115343835A discloses a three-dimensional isotropic super-resolution imaging method and device based on nonlinear fluorescence excited by mirror interference field, the device includes an excitation light generation module, a multiphoton microscopy scanning module, a reflector sample module and a photoelectric detection module; wherein the excitation light generation module is used to generate a continuous near-infrared laser beam / femtosecond near-infrared laser beam as excitation light, the laser beam is focused to the reflector sample module marked with a fluorescent probe through the multiphoton microscopy scanning module, and different areas of the sample are scanned, and the photoelectric detection module is used to detect the fluorescence signal of the above sample excited by the scanning. However, the point-by-point scanning imaging of this patent leads to a slow speed, which is difficult to meet the real-time imaging requirements of dynamic samples.

[0007] Patent CN111307772A discloses a single objective light sheet fluorescence microscopy imaging device and method based on a micromirror array. The micromirror array is used to combine light sheet excitation and fluorescence collection into one with the same objective lens, so that sample freedom is achieved at the sample without complicated sample processing. Conventional sample slides and cover glass can be used to achieve this, and optical path adjustment is avoided. The micromirror array is used to reflect the fluorescence information of light sheet excitation at different depths of the sample from different positions of the micromirror array system and image at different positions of the signal collection system, so that single objective light sheet fluorescence microscopy imaging can be achieved, and the use of large numerical aperture objective lenses can be realized, thereby improving the limitation that the resolution cannot be further improved in traditional light sheet fluorescence microscopy imaging. However, this patent requires galvanometer scanning, the imaging speed is slow, and the imaging effect of deep samples is poor. Summary of the invention

[0008] The purpose of the present invention is to provide a near-infrared two-zone spinning disk confocal microscopy system in order to overcome at least one of the defects of the above-mentioned prior art, such as the high cost of confocal microscopes and the low resolution of traditional visible light confocal microscopes. The present invention can provide high-quality near-infrared spinning disk confocal microscopy imaging effects at a low cost.

[0009] The purpose of the present invention can be achieved by the following technical solutions:

[0010] One of the technical solutions of the present invention is to provide a near-infrared two-zone rotating disk confocal microscopy system, which includes a laser transmitter, a dichroic mirror, a third lens, a rotating disk, a fourth lens, a stage and a camera. The laser transmitter transmits a laser with a wavelength of 600-900nm required for the experiment. The dichroic mirror allows the reflection of the laser with a wavelength of 600-900nm. The laser is reflected to the third lens through the dichroic mirror. The third lens focuses the laser on a specific position of the rotating disk to ensure that the laser forms a clear excitation spot on the sample, thereby improving the excitation efficiency of fluorescence. The laser passes through the third lens. Four lenses, the stage is used to place the sample to be tested, the sample is dyed with a fluorescent dye located in the 900-1700nm area of ​​the near-infrared zone II window, the sample receives laser light and excites fluorescence, the fourth lens refocuses the fluorescence onto the turntable, thereby effectively filtering the light beams from different focal planes, retaining only the fluorescence signal from the target plane, and achieving a high-resolution confocal imaging effect, the dichroic mirror allows the light beam located in the 900-1700nm area of ​​the near-infrared zone II window to pass through, the fluorescence passes through the third lens and the dichroic mirror, the camera receives the sample fluorescence and performs imaging,

[0011] The dichroic mirror separates the incident laser and the fluorescence emitted by the sample, and accurately focuses the light beams onto the same focal plane of the turntable through the third lens and the fourth lens to achieve confocal imaging of the light beams. The turntable is etched with at least one circle of through holes forming an Archimedean spiral pattern, and the light beam is imaged through the Archimedean spiral pattern in the turntable.

[0012] As a preferred technical solution, the laser power of the laser transmitter is 100-1000mW. Using a laser source with higher light intensity can make the filling factor of the single-disk microscope system much smaller than that of the double-disk microscope system, while obtaining lasers of equal intensity reaching the sample.

[0013] As a preferred technical solution, the laser emitter is connected to a beam shaper, and the beam shaper shapes the laser source.

[0014] Furthermore, the system also includes a first lens and a second lens. After the laser transmitter emits laser, the first lens converges the incident laser, and the second lens converts the laser emitted by the first lens into parallel laser again, while expanding the diameter of the light beam. The first lens and the second lens play the role of beam expansion.

[0015] Furthermore, the system also includes a reflector group, and after the first lens and the second lens expand the laser, the reflector group collimates the optical path of the laser.

[0016] Furthermore, the system also includes an objective lens. After the laser passes through the fourth lens, the laser passes through the objective lens again. The sample receives the laser and excites fluorescence. The fluorescence is imaged through the objective lens. The fourth lens refocuses the fluorescence onto the turntable, and the objective lens focuses the fluorescence onto the plane of the turntable to perform microscopic imaging of the sample.

[0017] Furthermore, the system also includes a reflector. After the laser passes through the fourth lens, the laser is reflected by the reflector to change the light path to a vertical direction. The laser passes through the objective lens, the sample receives the laser and excites fluorescence, the fluorescence is imaged through the objective lens and reflected by the reflector, and the fourth lens re-focuses the fluorescence onto the turntable. The reflector is used to guide the light path to a vertical direction when an upright microscope is required in situations such as in vivo imaging.

[0018] As a preferred technical solution, the spiral radius, spiral pitch and through-hole diameter of the Archimedean spiral pattern can be adjusted. The spiral radius determines the distribution range of the through-hole relative to the center of the turntable. The through-hole diameter is usually selected from the first minimum diameter of the Airy disk. The calculation formula is as follows:

[0019]

[0020] Among them, ω represents the first minimum diameter of the Airy disk, λ represents the wavelength of the incident light, M represents the magnification of the objective lens, and NA represents the numerical aperture of the objective lens;

[0021] The spiral pitch determines the distance between adjacent through-holes and is usually selected to be 3-10 times the through-hole diameter so that the filling factor can be greater than 1%. The adjustment of the spiral pitch balances the rationality of the filling factor and the through-hole spacing, so that the sample can effectively resist crosstalk between through-holes.

[0022] As a preferred technical solution, the use of multiple concentric spirals will greatly reduce the sensitivity of illumination uniformity to the deviation of the turntable from its true center position. The calculation equation for a set of n concentric Archimedean spirals in polar coordinates is as follows:

[0023]

[0024] Among them, r i represents the radius of the i-th spiral, s represents the spiral pitch, r start represents the position of the innermost through hole, n represents the number of Archimedean spirals, and θ represents the angle in the polar coordinate system;

[0025] In principle, a large number of concentric spirals can be used while keeping the spiral pitch s, but in order to make the lateral spacing of the through holes meet the Nyquist sampling criterion, the radial increment of two adjacent spiral through holes should not exceed the Airy disk diameter of the half-maximum full width.

[0026] Furthermore, the turntable is etched with two concentric circles of through holes forming an Archimedean spiral pattern, so that different through hole arrays can be selected by radially translating the turntable without replacing the turntable, wherein the size and arrangement of one circle are designed for visible light to facilitate debugging of the optical path under visible light, and the size and arrangement of the other circle are designed for the near-infrared zone II window 900-1700nm to facilitate microscopic imaging of the sample.

[0027] As a preferred technical solution, there are at least two circles etched in the near-infrared zone II window of 900-1700nm, corresponding to objective lenses with different parameters. By using Archimedean spiral patterns corresponding to different incident light and microscope parameters, experiments can be conducted conveniently in different situations to provide the most suitable imaging requirements.

[0028] As a preferred technical solution, in order to adjust the confocal position of the turntable, first fix the position of the turntable in the visible light circle, then use a visible light source to replace the laser source of the laser transmitter, use a dichroic mirror that allows visible light to pass through to replace the dichroic mirror that allows light beams located in the 900-1700nm region of the near-infrared second zone window to pass through, turn on the visible light source, and adjust the positions of the third lens and the fourth lens until the through hole in the turntable can be clearly focused on the camera, which means that the turntable is in the confocal position.

[0029] Furthermore, the turntable is coated with an anti-reflection film, which increases the transmittance of light beams in the near-infrared zone II window region of 900-1700nm. The anti-reflection film reduces the reflection of laser on the turntable surface to increase the transmittance of the turntable to incident light.

[0030] As a preferred technical solution, the side of the turntable coated with the anti-reflection film faces the laser emitter and the camera to reduce the background light source generated by reflection.

[0031] As a preferred technical solution, the stage fixes and moves the sample, providing a precise positioning function. By fine-tuning the stage, the sample can be moved in three dimensions, XYZ, making it easier for the objective lens to focus on different positions and depths of the sample, thereby obtaining more comprehensive imaging information.

[0032] As a preferred technical solution, the method for preparing the turntable comprises the following steps:

[0033] S1. Coat an anti-reflection film on a glass ring of appropriate size;

[0034] S2, designing the Archimedean spiral pattern in the turntable and making the corresponding photomask;

[0035] S3. Use a photomask to photolithography the glass ring to obtain a turntable.

[0036] As a preferred technical solution, the material of the glass ring is generally required to have good light transmittance from visible light to near-infrared bands, chemical corrosion resistance, high temperature resistance and the like, and the material is selected from one of silicon dioxide, calcium fluoride, and magnesium fluoride.

[0037] Furthermore, the system also includes a filter. After the fluorescence passes through the dichroic mirror, it passes through the filter to reach the camera. The filter allows the light beam in the 900-1700nm region of the near-infrared second zone window to pass through. The filter blocks the excitation light and filters out other light beams except the sample fluorescence.

[0038] Furthermore, the system also includes a fifth lens. After the fluorescence passes through the dichroic mirror, the fluorescence passes through the filter and the fifth lens to reach the camera. The lens adjusts the size of the light spot entering the camera.

[0039] Furthermore, the rotation speed of the turntable is precisely controlled by a controller, a motor and a motor driver. The motor drives the turntable to rotate. The rotation speed of the turntable directly determines the scanning speed. A higher rotation speed can achieve faster scanning and higher time resolution, while a lower rotation speed will increase the sampling time of each point. The rotation speed can be adjusted simply and quickly according to different imaging requirements. Generally, the exposure of the camera is synchronized with the rotation of the turntable to ensure that the exposure time is equal to an integer multiple of the scanning time.

[0040] The frequency output port F of the motor driver is connected to the digital reading port D1 of the controller, the frequency output port outputs the real-time speed of the motor, the digital reading port receives the frequency signal, and the motor driver transmits the rotation frequency signal to the controller;

[0041] The output voltage port Vout of the motor driver is connected to the power supply voltage port VCC of the motor. The motor driver supplies power to the motor and can control the speed of the motor through the output of the motor driver.

[0042] As a preferred technical solution, the motor driver is connected to an LED screen, and the rotation speed of the turntable is displayed in real time on the LED screen to facilitate real-time adjustment of the rotation speed.

[0043] As a preferred technical solution, the power supply voltage port VCC of the controller and the motor driver is connected to a power supply VCC, and the power supply VCC supplies power to the controller and the motor driver.

[0044] As a preferred technical solution, the ground ports GND of the controller, the motor and the motor driver are connected to the ground point GND, and the ground point GND provides grounding protection for the controller, the motor and the motor driver.

[0045] Compared with the prior art, the present invention has the following beneficial effects:

[0046] (1) The present invention adopts a single turntable structure to achieve continuous dynamic scanning. With the development of laser technology, incident light sources with higher light intensity have become more economical and practical. Compared with the complex double turntable design, the single turntable structure has a simpler design and lower cost by appropriately reducing the fill factor. With a higher light intensity laser source, the reduction in the fill factor has little effect on the imaging quality.

[0047] (2) The present invention uses a laser emitter with a wavelength of 600-900nm, combined with the fluorescent dye of the sample whose emission wavelength is located in the near-infrared zone II window (900-1700nm region), the through hole of the turntable and the anti-reflection film, and the filter. The near-infrared light in this band has significant advantages in non-invasive in vivo imaging, including stronger penetration ability and deeper imaging effect, while significantly reducing the influence of scattering and absorption in biological tissues. Therefore, the design of the present invention is more suitable for observing the internal structure of living tissues and thick samples, and significantly improves the imaging depth and tissue perspective effect;

[0048] (3) The present invention innovatively combines single-disk technology with near-infrared two-zone imaging technology, overcoming the depth and speed limitations of traditional imaging technology. While maintaining high resolution, it achieves rapid three-dimensional imaging and significantly reduces background noise, thereby enabling efficient completion of cell and living body imaging tasks. This fast, low-noise imaging capability provides a more efficient solution for real-time dynamic imaging in biomedical research. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 Schematic diagram of the optical path structure of the near-infrared two-zone spinning disk confocal microscopy system in an embodiment of the present invention;

[0050] Figure 2 It is a schematic diagram of the structure of the turntable in the embodiment of the present invention;

[0051] Figure 3 Schematic diagram of the circuit structure of the controller, motor and motor driver of the turntable in the embodiment of the present invention.

[0052] Description of the markings in the figure:

[0053] 1—laser transmitter, 2—first lens, 3—second lens, 4—reflector group, 5—dichroic mirror, 6—third lens, 7—turntable, 8—fourth lens, 9—reflector, 10—objective lens, 11—stage, 12—filter, 13—fifth lens, 14—camera, 15—controller, 16—motor, 17—motor driver. DETAILED DESCRIPTION

[0054] The present invention is described in detail below in conjunction with specific embodiments. This embodiment is implemented based on the technical solution of the present invention, and provides a detailed implementation method and a specific operation process, but the protection scope of the present invention is not limited to the following embodiments.

[0055] In the description of the present invention, it should be noted that the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inside", "outside", etc. indicate positions or positional relationships based on the positions or positional relationships shown in the accompanying drawings, which are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as a limitation on the present invention. In addition, the terms "first", "second", "third", etc. are used to describe common objects, which only refer to different instances of the same object, and are not intended to imply that the objects described in this way must adopt a given order, whether in time, space, order or any other way.

[0056] In the description of the present invention, it should be noted that, unless otherwise clearly specified and limited, the terms "installed", "connected", and "connected" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection, or it can be indirectly connected through an intermediate medium, or it can be the internal communication of two components. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0057] Example:

[0058] A near-infrared two-zone spinning disk confocal microscopy system, such as Figure 1As shown, it includes a laser emitter 1, a dichroic mirror 5, a third lens 6, a turntable 7, a fourth lens 8, a stage 11 and a camera 14. The laser emitter 1 emits a laser with a wavelength of 600-900nm required for the experiment, preferably 785nm in this embodiment. The dichroic mirror 5 allows the reflection of the laser with a wavelength of 600-900nm. The laser is reflected to the third lens 6 through the dichroic mirror 5. The third lens 6 focuses the laser to a specific position on the turntable 7 to ensure that the laser forms a clear excitation spot on the sample, thereby improving the excitation efficiency of the fluorescence. The laser passes through the fourth lens 8. The stage 11 is placed on the sample to be measured. The sample is dyed with a fluorescent dye located in the 900-1700nm region of the near-infrared zone II window, preferably 1000-1300nm in this embodiment. The sample receives laser light and excites fluorescence. The fourth lens 8 refocuses the fluorescence onto the turntable 7, thereby effectively filtering the light beams from different focal planes and retaining only the fluorescence signal from the target plane to achieve a high-resolution confocal imaging effect. The dichroic mirror 5 allows the light beam located in the 900-1700nm region of the near-infrared zone II window to pass through. The fluorescence passes through the third lens 6 and the dichroic mirror 5. The camera 14 receives the sample fluorescence and performs imaging.

[0059] The dichroic mirror 5 separates the incident laser light and the fluorescence emitted by the sample, and the light beams are precisely focused on the same focal plane of the rotating disk 7 through the third lens 6 and the fourth lens 8 to achieve confocal imaging of the light beams. The rotating disk 7 is etched with at least one through hole forming an Archimedean spiral pattern, and the light beam is imaged through the Archimedean spiral pattern in the rotating disk 7.

[0060] The laser power of the laser emitter 1 is 100-1000 mW, preferably 200 mW in this embodiment. Using a laser source with higher light intensity can make the filling factor of the single-disk microscope system much smaller than that of the double-disk microscope system, and obtain the same intensity of laser reaching the sample;

[0061] The laser transmitter 1 is connected to a beam shaper, and the beam shaper shapes the laser source;

[0062] The system also includes a first lens 2 and a second lens 3. After the laser emitter 1 emits laser light, the first lens 2 converges the incident laser light, and the second lens 3 converts the laser light emitted by the first lens 2 into parallel laser light again, while expanding the diameter of the light beam. The first lens 2 and the second lens 3 play the role of beam expansion.

[0063] The system further comprises a reflector group 4, after the first lens 2 and the second lens 3 expand the laser beam, the reflector group 4 collimates the optical path of the laser beam;

[0064] The system also includes an objective lens 10. After the laser passes through the fourth lens 8, the laser passes through the objective lens 10 again. The sample receives the laser and excites fluorescence. The fluorescence is imaged through the objective lens 10. The fourth lens 8 refocuses the fluorescence onto the turntable 7. The objective lens 10 focuses the fluorescence onto the plane of the turntable 7 to perform microscopic imaging of the sample. The system also includes a reflector 9. After the laser passes through the fourth lens 8, the laser is reflected by the reflector 9 to change the optical path to a vertical direction. The laser passes through the objective lens 10. The sample receives the laser and excites fluorescence. The fluorescence is imaged through the objective lens 10 and reflected by the reflector 9. The fourth lens 8 refocuses the fluorescence onto the turntable 7. The reflector 9 is used to guide the optical path to a vertical direction when an upright microscope is required in situations such as in vivo imaging.

[0065] The stage 11 fixes and moves the sample, providing a precise positioning function. By fine-tuning the stage 11, the sample can be moved in three dimensions, XYZ, so that the objective lens 10 can focus on different positions and depths of the sample, thereby obtaining more comprehensive imaging information.

[0066] The system also includes a filter 12. After the fluorescence passes through the dichroic mirror 5, the fluorescence passes through the filter 12 and reaches the camera 14. The filter 12 allows the light beam located in the 900-1700nm area of ​​the near-infrared second zone window to pass through. The filter 12 blocks the excitation light and filters out other light beams except the sample fluorescence.

[0067] The system also includes a fifth lens 13. After the fluorescence passes through the dichroic mirror 5, the fluorescence passes through the filter 12 and the fifth lens 13 to reach the camera 14. The lens 13 adjusts the size of the light spot entering the camera 14.

[0068] The spiral radius, spiral pitch and through-hole diameter of the Archimedean spiral pattern can be adjusted. The spiral radius determines the distribution range of the through-holes relative to the center of the turntable 7. The through-hole diameter is usually selected from the first minimum diameter of the Airy disk. The calculation formula is as follows:

[0069]

[0070] Wherein, ω represents the first minimum diameter of the Airy disk, λ represents the wavelength of the incident light, M represents the magnification of the objective lens 10, and NA represents the numerical aperture of the objective lens 10;

[0071] The spiral pitch determines the distance between adjacent through-holes, and is usually selected to be 3-10 times the through-hole diameter, so that the filling factor can be greater than 1%. In this embodiment, it is preferably 5 times. The adjustment of the spiral pitch balances the rationality of the filling factor and the through-hole spacing, so that the sample can effectively resist the crosstalk between through-holes.

[0072] At the same time, the use of multiple concentric spirals results in a much reduced sensitivity of the illumination uniformity to the deviation of the turntable 7 from its true center position. The calculation equation for a set of n concentric Archimedean spirals in polar coordinates is as follows:

[0073]

[0074] Among them, r i represents the radius of the i-th spiral, s represents the spiral pitch, r start represents the position of the innermost through hole, n represents the number of Archimedean spirals, and θ represents the angle in the polar coordinate system;

[0075] In principle, a large number of concentric spirals can be used while keeping the spiral pitch s, but in order to make the lateral spacing of the through holes meet the Nyquist sampling criterion, the radial increment of two adjacent spiral through holes should not exceed the Airy disk diameter of the half-maximum full width;

[0076] like Figure 2 As shown, two concentric circles of through holes forming an Archimedean spiral pattern are etched in the turntable 7, so that different through hole arrays can be selected by radially translating the turntable 7 without replacing the turntable 7, wherein the size and arrangement of one circle are designed for visible light to facilitate debugging of the optical path under visible light, and the size and arrangement of the other circle are designed for the near-infrared second zone window 900-1700nm to facilitate microscopic imaging of the sample;

[0077] In this embodiment, three concentric circles of through holes forming an Archimedean spiral pattern are etched in the turntable 7. The size and arrangement of the outermost circle are designed for visible light, and the size and arrangement of the inner two circles are designed for the near-infrared second window 1000-1300nm. At the same time, two objective lenses 10 with different parameters are corresponding. Through the Archimedean spiral patterns corresponding to different incident light and microscope parameters, experiments can be carried out conveniently in different situations to provide the most suitable imaging requirements.

[0078] To adjust the confocal position of the turntable 7, first fix the position of the turntable 7 in the visible light circle area, then replace the laser source of the laser emitter 1 with a visible light source, which is a white light source in this embodiment, and replace the dichroic mirror 5 that allows the light beam located in the 900-1700nm area of ​​the near-infrared second zone window to pass through with a dichroic mirror that allows visible light to pass through, which is a dichroic mirror that allows white light to pass through in this embodiment, turn on the visible light source, and adjust the positions of the third lens 6 and the fourth lens 8 until the through hole in the turntable 7 can be clearly focused on the camera 14, which means that the turntable 7 is in the confocal position;

[0079] The turntable 7 is coated with an anti-reflection film, which increases the transmittance of the light beam in the 900-1700nm region of the near-infrared zone II window. The anti-reflection film reduces the reflection of the laser on the surface of the turntable 7 to increase the transmittance of the turntable 7 to the incident light;

[0080] The side of the turntable 7 coated with an anti-reflection film faces the laser emitter 1 and the camera 14 to reduce the background light source generated by reflection;

[0081] The specific steps of the preparation method of the turntable 7 are as follows:

[0082] S1. Coat an anti-reflection film on a glass ring of appropriate size;

[0083] The material of the glass ring is generally required to have good light transmittance from visible light to near-infrared band, chemical corrosion resistance, high temperature resistance, etc. The material is selected from one of silicon dioxide, calcium fluoride, and magnesium fluoride. In this embodiment, quartz glass is preferably used;

[0084] S2, designing the Archimedean spiral pattern in the turntable 7 and making a corresponding photomask;

[0085] S3, using a photomask to perform photolithography on the glass ring to obtain a turntable 7;

[0086] like Figure 3 As shown, the rotation speed of the turntable 7 is precisely controlled by the controller 15, the motor 16 and the motor driver 17. The motor 16 drives the turntable 7 to rotate. The rotation speed of the turntable 7 directly determines the scanning speed. A higher rotation speed can achieve faster scanning and higher time resolution. A lower rotation speed will increase the sampling time of each point. The rotation speed can be adjusted simply and quickly according to different imaging requirements. Generally, the exposure of the camera 14 is synchronized with the rotation of the turntable 7 to ensure that the exposure time is equal to an integer multiple of the scanning time.

[0087] The motor driver 17 is connected to the LED screen, and the rotation speed of the turntable 7 is displayed in real time on the LED screen to facilitate real-time adjustment of the rotation speed;

[0088] The controller 15 uses the YWRobot Arduino Uno control board. The controller 15 receives the frequency signal output by the motor driver 17, and outputs the speed and then displays it on the LED screen for real-time display of the speed.

[0089] For the sake of simplicity, the motor 16 directly uses the Seagate ST500DM002 hard disk motor, which can achieve a high speed and high precision control speed of 4000-10000 revolutions per minute;

[0090] The motor driver 17 uses JYQD_BLDC V6.7, and the speed of the turntable 7 can be controlled by controlling the output power of the motor driver 17;

[0091] The frequency output port F of the motor driver 17 is connected to the digital reading port D1 of the controller 15. The frequency output port outputs the real-time rotation speed of the motor 16. The digital reading port receives the frequency signal. The motor driver 17 transmits the rotation frequency signal to the controller 15.

[0092] The output voltage port Vout of the motor driver 17 is connected to the power supply voltage port VCC of the motor 16. The motor driver 17 supplies power to the motor 16 and can control the speed of the motor 16 through the output of the motor driver 17.

[0093] The power supply voltage port VCC of the controller 15 and the motor driver 17 is connected to the power supply VCC, and the power supply VCC supplies power to the controller 15 and the motor driver 17;

[0094] The ground ports GND of the controller 15 , the motor 16 and the motor driver 17 are connected to the ground point GND, and the ground point GND provides grounding protection for the controller 15 , the motor 16 and the motor driver 17 .

[0095] The present invention proposes a rotating disk confocal microscopy system based on the near-infrared zone II, aiming to achieve high-quality imaging effects at a low cost. The present invention adopts a laser with a wavelength of 600-900nm as an excitation light source, and combines an optimized optical path with an innovatively designed rotating disk structure to improve imaging performance. Specifically, a laser emitter 1 emits a laser of 600-900nm, and the laser is reflected by a dichroic mirror 5, which allows the reflection of a light beam in the 900-1700nm region of the near-infrared zone II window. The reflected laser is focused by a lens onto the surface of a rotating disk 7, on which a through hole with an Archimedean spiral pattern is etched. The laser passing through the through hole further passes through a lens and an objective lens 10 and irradiates a sample on a stage 11. The sample to be tested is stained with a fluorescent dye located in the near-infrared zone II window, and emits a fluorescent signal under laser excitation. The lens refocuses the fluorescence onto the surface of the rotating disk 7, and the fluorescence is transmitted through the dichroic mirror 5, and is finally received by a camera 14 and imaging is completed. The Archimedean spiral pattern through-hole design in the turntable 7 not only optimizes the optical path efficiency, but also realizes accurate fluorescence collection, significantly improving the imaging quality. The present invention combines the near-infrared second-zone optical technology with the innovative turntable design to solve the limitations of traditional technologies in imaging depth and optical path complexity. Compared with traditional systems, the present invention not only achieves high signal-to-noise ratio and deep-level tissue imaging, but also achieves a good balance between cost and efficiency, and is particularly suitable for the imaging needs of living tissues and thick samples.

[0096] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A near-infrared two-zone spinning disk confocal microscopy system, characterized in that: The system comprises a laser emitter (1), a dichroic mirror (5), a third lens (6), a turntable (7), a fourth lens (8), an object stage (11) and a camera (14). The laser emitter (1) emits laser light with a wavelength of 600-900 nm. The laser light is reflected to the third lens (6) through the dichroic mirror (5). The third lens (6) focuses the laser light onto the turntable (7). The laser light passes through the fourth lens (8). A sample to be measured is placed on the object stage (11). The sample is dyed with a fluorescent dye in the 900-1700 nm region of the near-infrared zone II window. The sample receives the laser light and excites fluorescence. The fourth lens (8) refocuses the fluorescence onto the turntable (7). The dichroic mirror (5) allows the light beam in the 900-1700 nm region of the near-infrared zone II window to pass through. The fluorescence passes through the third lens (6) and the dichroic mirror (5). The camera (14) receives the sample fluorescence and performs imaging. At least one through hole forming an Archimedean spiral pattern is etched in the rotating disk (7), and the light beam is imaged through the Archimedean spiral pattern in the rotating disk (7).

2. A near-infrared two-zone spinning disk confocal microscopy system according to claim 1, characterized in that: The system further comprises a first lens (2) and a second lens (3); after the laser emitter (1) emits laser light, the first lens (2) converges the incident laser light, and the second lens (3) converts the laser light emitted by the first lens (2) back into parallel laser light, while expanding the diameter of the light beam.

3. A near-infrared two-zone spinning disk confocal microscopy system according to claim 2, characterized in that: The system further comprises a reflector group (4), and after the first lens (2) and the second lens (3) expand the laser beam, the reflector group (4) collimates the optical path of the laser beam.

4. A near-infrared two-zone spinning disk confocal microscopy system according to claim 1, characterized in that: The system also includes an objective lens (10). After the laser passes through the fourth lens (8), the laser passes through the objective lens (10) again. The sample receives the laser and excites fluorescence. The fluorescence is imaged through the objective lens (10). The fourth lens (8) refocuses the fluorescence onto the turntable (7).

5. A near-infrared two-zone spinning disk confocal microscopy system according to claim 4, characterized in that: The system further comprises a reflector (9). After the laser passes through the fourth lens (8), the laser is reflected by the reflector (9) to change the optical path to a vertical direction. The laser passes through the objective lens (10). The sample receives the laser and excites fluorescence. The fluorescence is imaged through the objective lens (10) and reflected by the reflector (9). The fourth lens (8) refocuses the fluorescence onto the turntable (7).

6. A near-infrared two-zone spinning disk confocal microscopy system according to claim 1, characterized in that: The rotating disk (7) is etched with two concentric circles of through holes forming an Archimedean spiral pattern, wherein the size and arrangement of one circle are designed for visible light, and the size and arrangement of the other circle are designed for the near-infrared second zone window 900-1700nm.

7. A near-infrared two-zone spinning disk confocal microscopy system according to claim 1, characterized in that: The rotating disk (7) is coated with an anti-reflection film, which increases the transmittance of a light beam in the 900-1700 nm region of the near-infrared zone II window.

8. The near-infrared two-zone spinning disk confocal microscopy system according to claim 1, characterized in that: The system also includes a filter (12). After the fluorescence passes through the dichroic mirror (5), the fluorescence passes through the filter (12) to reach the camera (14). The filter (12) allows the light beam located in the 900-1700nm region of the near-infrared second zone window to pass through.

9. A near-infrared two-zone spinning disk confocal microscopy system according to claim 8, characterized in that: The system also includes a fifth lens (13). After the fluorescence passes through the dichroic mirror (5), the fluorescence passes through the filter (12) and the fifth lens (13) to reach the camera (14). The lens (13) adjusts the size of the light spot entering the camera (14).

10. The near-infrared two-zone spinning disk confocal microscopy system according to claim 1, characterized in that: The rotation speed of the turntable (7) is controlled by a controller (15), a motor (16) and a motor driver (17), and the motor (16) drives the turntable (7) to rotate; The frequency output port of the motor driver (17) is connected to the digital reading port of the controller (15), and the motor driver (17) transmits the rotation frequency signal to the controller (15); The output voltage port of the motor driver (17) is connected to the power supply voltage port of the motor (16); the motor driver (17) supplies power to the motor (16), and controls the rotation speed of the motor (16) through the output of the motor driver (17).

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