A three-dimensional single-rotor confocal microscope

Abstract

This invention discloses a three-dimensional single-disc confocal microscope, belonging to the field of focusing microscope technology. Along the excitation optical path, the following components are arranged sequentially: a light source, a first biconvex lens, a cornerstone prism retroreflector, a short-pass dichroic mirror, a second biconvex lens, a concave hole disk, a focusing turntable, a third biconvex lens, and a sample tray. Along the imaging optical path, the following components are arranged sequentially: a sample tray, a third biconvex lens, a focusing turntable, a concave hole disk, a second biconvex lens, a short-pass dichroic mirror, a fourth biconvex lens, and an imaging module. The first biconvex lens has a focal length of f3, and the second biconvex lens has a focal length of f1. The apex of the cornerstone prism retroreflector is located at the focal point of the first and second biconvex lenses. The concave hole disk includes an annular disk with multiple circular holes. A concave surface coaxial with the holes is formed near the opening of the second biconvex lens. This invention enables rapid three-dimensional imaging of living cell tissues.

Description

Technical Field

[0001] This invention belongs to the field of focusing microscope technology, specifically relating to a three-dimensional single-rotor confocal microscope. Background Technology

[0002] As is well known, fluorescence refers to the phenomenon where, when light of a specific wavelength shines on a fluorescent molecule, electrons transition from a lower energy level to a higher energy level. However, because excited-state electrons are unstable, they return to the ground state, releasing energy as light during this return process, thus producing fluorescence. In fluorescence microscopy, fluorescent molecules are used as probes to label specific biomolecules in biological tissues. By detecting the fluorescence signals emitted by excited fluorescent molecules, we can obtain the three-dimensional spatial location information of the sample.

[0003] However, traditional fluorescence microscopy, when illuminating the entire sample and detecting the resulting fluorescence, collects defocused light, including light above and below the focal plane. This causes image blurring and reduces overall image quality. This effect is particularly pronounced when imaging three-dimensional samples, such as cells containing liquid-filled regions that scatter light, leading to information loss.

[0004] To overcome this problem, confocal microscopy employs a pinhole to suppress defocus light, thereby achieving higher resolution and slice capability. In this type of microscope, the focal plane of the sample and the pinhole are in a confocal position. The pinhole only captures fluorescence from the sample's focal plane, thus eliminating defocus signals, resulting in clearer imaging, improved resolution and contrast, and reduced background signal. However, the pinhole can only image a small area of ​​the sample, requiring scanning the entire sample, which is time-consuming and may cause light damage, affecting cell viability.

[0005] Rotating disk confocal microscopy is an improvement on confocal microscopy. It features a spiral array of pinholes on a high-speed rotating, opaque disk. As the disk rotates, the pinholes scan the sample in rows, forming an image. Due to the parallel acquisition method, using a rotating disk significantly increases image acquisition speed and reduces optical damage. However, to overcome crosstalk between pinholes, a large spacing is required, resulting in a laser utilization rate of only 1% to 4%. Dual-rotating disk confocal microscopy adds a microlens disk to the rotating disk confocal microscope. The microlens array focuses the light onto the pinhole array, increasing the laser utilization rate by tens of times. However, the introduction of the microlens disk necessitates placing a dichroic mirror, used to separate the excitation light from the fluorescence, between the pinhole disk and the microlens disk. Due to the short focal length of the lenses, space is limited at this location, requiring a very thin, custom-made dichroic mirror. However, a thin dichroic mirror introduces surface stress, leading to poor separation of excitation light and fluorescence.

[0006] In practical applications, observing complex curved surfaces often requires three-dimensional imaging, and there are several different ways to achieve this.

[0007] One approach is to use objectives with different focal lengths. For example, a published patent document on confocal microscopy (patent publication number CN219370113U) controls the focal length by using different objectives, allowing the laser to scan along the Z-axis of the sample and thus observe different information. However, this method has the disadvantage of slow objective changing speed, and the process may cause further light damage, affecting cell activity. A second approach is to move the objective. For example, another published patent document on confocal microscopy (patent publication number CN111638596A) uses a motorized three-axis turntable to drive the objective, allowing the focusing unit to focus and move the focal plane up and down, thereby imaging information from different layers. This method improves imaging speed compared to the first approach, but still has shortcomings. A third approach is light field microscopy imaging. For example, another published patent document (patent publication number CN109615651A) discloses a three-dimensional microscopic imaging method and system device based on a light field microscopy system. In light field microscopy, information from different angles is integrated into the same image when acquiring light field images. This sacrifices the effective numerical aperture of light field imaging, resulting in poor resolution. Summary of the Invention

[0008] The purpose of this invention is to meet practical needs by providing a three-dimensional single-rotor confocal microscope for achieving rapid three-dimensional imaging of living cell tissues.

[0009] The purpose of this invention is to provide a three-dimensional single-disc confocal microscope, including an excitation optical path and an imaging optical path; wherein:

[0010] Along the excitation optical path are arranged the following components in sequence: light source, first biconvex lens, corner cube prism retroreflector, short-pass dichroic mirror, second biconvex lens, concave aperture plate, focusing turntable, third biconvex lens, and object tray.

[0011] Along the imaging optical path are arranged the following components in sequence: a sample tray, a third biconvex lens, a focusing dial, a concave aperture tray, a second biconvex lens, a short-pass dichroic mirror, a fourth biconvex lens, and an imaging module.

[0012] The focal length of the first biconvex lens is f3, and the focal length of the second biconvex lens is f1; the vertex of the cornerstone prism retroreflector is located at the focal point of the first and second biconvex lenses.

[0013] The concave-hole disk includes an annular disk made of opaque material, with an outer diameter of a and an inner diameter of b; M circular holes are formed on the annular disk, where M is a natural number greater than 0; a concave surface coaxial with the circular holes is formed near the opening of the second biconvex lens; the focal length of the concave surface is f2; the distance between the second biconvex lens and the concave surface is x1.

[0014] h is the depth of the concave surface.

[0015] Preferably: the M circular holes are divided into T groups; the value of T is in the range of 20-30; each group of circular holes is distributed on L Archimedean spirals, where L is a natural number greater than 0, and the starting point of the Archimedean spiral is d and the ending point is c.

[0016] Preferably: d is 20mm, c is 40mm

[0017] Preferably, the diameter of the concave surface is 250 μm, the diameter of the circular holes is 50 μm, and the distance between the circular holes is 300 μm.

[0018] Preferably: L=6, T=20.

[0019] Preferably, the focusing turntable includes several focusing glass groups of different thicknesses; each focusing glass group has an arc-shaped structure, and several focusing glass groups are spliced ​​together to form a fan-shaped ring.

[0020] Preferably, the focusing turntable includes three focusing glass groups with thicknesses of 0.16mm, 0.82mm, and 1.8mm; each focusing glass group has an arc-shaped structure, and the three focusing glass groups are spliced ​​together to form a fan-shaped ring.

[0021] Preferably: when the thickness of the focusing glass group is d1, the refractive index of the focusing glass group is n2, the distance between the focusing glass group and the concave hole disk is x8, the distance between the focusing glass group and the third biconvex lens is x9, and the imaging depth is x. 10 The focal length of the third biconvex lens is f4.

[0022]

[0023] Where: I represents the incident angle, that is, the angle between the outgoing light and the normal of the focusing glass group.

[0024] Preferably, the focusing turntable and the concave hole disc are mounted on the same motor shaft.

[0025] Preferably, it also includes a controller for controlling the motor speed and the imaging module's shooting frequency.

[0026] Preferably, the corner cube prism retroreflector is a solid structure, and includes a planar triangular area at the top of the corner cube prism retroreflector;

[0027] Alternatively, the corner prism reflector may be a hollow corner prism, with a circular or non-circular hole at the top of the hollow corner prism.

[0028] Compared with the prior art, the advantages and positive effects of this application are:

[0029] This invention designs a concave aperture disk with a specific structure. The main function of this concave aperture disk is to reflect excitation light and then collect it a second time before transmitting it to a pinhole. In this process, the front side of the concave aperture disk, that is, the side with the concave surface, plays the role of reflecting the excitation light, while the pinhole is responsible for transmitting the second-collected excitation light. In addition, the pinhole on the back side of the concave aperture disk also functions as an optical filter, which can allow only fluorescence from the focal plane to pass through, thereby effectively improving the imaging resolution.

[0030] Furthermore, this invention employs a focusing turntable with a specific structure. This focusing turntable consists of several focusing glass groups of different thicknesses, each corresponding to a different focal plane. Therefore, through this focusing turntable, this invention can achieve focusing functionality for images with different focal planes.

[0031] In this invention, a motor first drives the focusing turntable and the concave aperture plate to rotate synchronously. Then, a controller controls the motor's rotation speed and the imaging module's image capture frequency to achieve signal synchronization. Specifically, the controller determines the timing relationship between the camera's acquisition cycle and the motion cycles of different focusing glass groups on the focusing turntable. Since the concave aperture plate and the focusing turntable rotate coaxially, the imaging module can acquire signals within the working cycle of each focusing glass group to obtain image information of that focal plane. In this process, the acquisition timing is crucial; it ensures that the imaging module can combine the focal plane signals from all planes to obtain a complete three-dimensional image. This enables three-dimensional microscopic imaging of the target object. Attached Figure Description

[0032] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0033] Figure 1 This is a structural diagram of a fluorescence microscope imaging system in the prior art;

[0034] Figure 2 This is an optical path diagram of a preferred embodiment of the present invention;

[0035] Figure 3 This is a front view of the concave hole disk in a preferred embodiment of the present invention;

[0036] Figure 4 for Figure 3 Enlarged view of section A;

[0037] Figure 5 This is a partially enlarged rear view of the concave hole plate in a preferred embodiment of the present invention;

[0038] Figure 6 This is a schematic diagram of the distribution of 6 sets of spirals in the concave hole disk and 20 sets of spiral array a in a preferred embodiment of the present invention;

[0039] Figure 7 for Figure 6 Enlarged view of section C;

[0040] Figure 8 This is a schematic diagram of the grouped and spaced distribution of concave hole positions in a preferred embodiment of the present invention;

[0041] Figure 9 This is a schematic diagram illustrating the suppression of pinhole crosstalk by a concave hole disk in a preferred embodiment of the present invention;

[0042] Figure 10 This is a schematic diagram illustrating the light convergence between the concave hole plate and the cornerstone prism retroreflector in a preferred embodiment of the present invention;

[0043] Figure 11 This is a schematic diagram of the focusing disk in a preferred embodiment of the present invention;

[0044] Figure 12 This is a schematic diagram of the focusing glass assembly in a preferred embodiment of the present invention;

[0045] Figure 13 This is a schematic diagram of the focusing principle in a preferred embodiment of the present invention;

[0046] Figure 14 This is a schematic diagram of the focusing glass group and camera sampling period in a preferred embodiment of the present invention;

[0047] Figure 15 This is an enlarged view of the top triangular area of ​​the first embodiment of the cornerstone prism retroreflector in a preferred embodiment of the present invention;

[0048] Figure 16 This is an enlarged view of the top triangular area of ​​the second embodiment of the cornerstone prism retroreflector in a preferred embodiment of the present invention;

[0049] Figure 17This is a structural diagram of the second embodiment of the cornerstone prism retroreflector in a preferred embodiment of the present invention;

[0050] Figure 18 This is an enlarged view of the top triangular area of ​​the third embodiment of the cornerstone prism retroreflector in a preferred embodiment of the present invention;

[0051] Figure 19 This is a structural diagram of the third embodiment of the cornerstone prism retroreflector in a preferred embodiment of the present invention;

[0052] Figure 20 This is a schematic diagram of the concave surface parameters in a preferred embodiment of the present invention;

[0053] Figure 21 This is a schematic diagram illustrating the calculation of the circular aperture at the second excitation light focusing center in a preferred embodiment of the present invention;

[0054] Figure 22 This is a schematic diagram of imaging depth calculation in a preferred embodiment of the present invention;

[0055] Figure 23 This is a schematic diagram of the confocal turntable assembly in a preferred embodiment of the present invention;

[0056] Figure 24 This is a front view of the integrated base in a preferred embodiment of the present invention;

[0057] Figure 25 This is a schematic diagram of the integrated base in a preferred embodiment of the present invention;

[0058] Figure 26 This is a front view of the outer casing in a preferred embodiment of the present invention;

[0059] Figure 27 This is a schematic diagram of the outer shell in a preferred embodiment of the present invention;

[0060] Figure 28 This is an exploded view of the motor drive module in a preferred embodiment of the present invention;

[0061] Figure 29 This is a schematic diagram of the first structure of the fixing kit in a preferred embodiment of the present invention;

[0062] Figure 30 for Figure 29 Cross-sectional view of DD;

[0063] Figure 31 for Figure 29 Cross-sectional view of EE;

[0064] Figure 32 This is a schematic diagram of the structure of the first bolt in a preferred embodiment of the present invention;

[0065] Figure 33This is a schematic diagram of the structure of the first O-ring in a preferred embodiment of the present invention;

[0066] Figure 34 This is a schematic diagram of the structure of the second O-ring in a preferred embodiment of the present invention;

[0067] Figure 35 This is a schematic diagram of the second structure of the fixing kit in a preferred embodiment of the present invention;

[0068] Figure 36 for Figure 35 Cross-sectional view of GG;

[0069] Figure 37 This is a schematic diagram of the structure of the two O-grooves in a preferred embodiment of the present invention;

[0070] Figure 38 This is a schematic diagram of the structure of the third fixed card holder in a preferred embodiment of the present invention;

[0071] Figure 39 This is a schematic diagram of the structure of two other O-grooves in a preferred embodiment of the present invention;

[0072] Figure 40 This is a schematic diagram of the first connection method in a preferred embodiment of the present invention;

[0073] Figure 41 This is a schematic diagram of the second connection method in a preferred embodiment of the present invention;

[0074] Figure 42 for Figure 41 Enlarged view of section B;

[0075] Figure 43 This is a schematic diagram of the third connection method in a preferred embodiment of the present invention;

[0076] Figure 44 This is a schematic diagram of the overall assembly of a preferred embodiment of the present invention. Detailed Implementation

[0077] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. The components of the embodiments of the present invention described and shown in the accompanying drawings can generally be arranged and designed in various different configurations. Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0078] Please see Figures 1 to 44:

[0079] like Figure 1 As shown, the fluorescence microscope imaging system is used in a variable-focus three-dimensional confocal imaging system to illuminate and excite the imaging area where the sample is located, and the reflected fluorescence reaches the image processing module. In the fluorescence microscope imaging system: light source 1 is used to provide excitation light 2, and the wavelength of excitation light 2 is set to 488nm. Excitation light 2 passes through excitation filter 3, and the bandpass of excitation filter 3 is 480nm~500nm. Then excitation light 2 passes through short-pass dichroic mirror 4 placed at 45°, and the transmission band of short-pass dichroic mirror 4 is 400nm~635nm. Excitation light 2 continues to pass through first biconvex lens 5 and reaches the focal plane corresponding to sample 6 to excite fluorescent molecules in biological tissue. The excited fluorescence 8, with a wavelength of 800nm ​​(represented by a dashed line), passes through the first biconvex lens 5 to the short-pass dichroic mirror 4. The reflection band of the short-pass dichroic mirror 4 is 680nm to 1200nm. The fluorescence 8 passes through the emission filter 7, which has a bandpass band of 600nm to 1000nm and filters out other stray light. After passing through the third biconvex lens 10, the fluorescence 8 is processed on the imaging module 9.

[0080] Please see Figure 2 A three-dimensional single-rotor confocal microscope, comprising:

[0081] The excitation optical path guides the laser emitted by the light source to the sample, causing the sample to fluoresce under the influence of the laser. Along the excitation optical path are arranged the following components in sequence: light source 1, first biconvex lens 5, corner cube prism retroreflector, short-pass dichroic mirror 4, second biconvex lens 25, concave aperture plate 11, focusing turntable 22, third biconvex lens 10, and sample tray. In this embodiment, the main function of light source 1 is to generate laser light of the required wavelength or intensity; its core component is the laser. The sample tray is used to hold the sample. The first biconvex lens 5 focuses the laser light emitted to the corner cube prism. At the top corner of the retroreflector, the laser passes through the corner prism retroreflector and is filtered by the short-pass dichroic mirror 4. Then, the laser passes through the second biconvex lens 25 to reach the concave hole disk 11. After reflection, the laser passes through the second biconvex lens 25 and the short-pass dichroic mirror 4 to reach the corner prism retroreflector and completes a 180-degree turn. After turning, the laser passes through the short-pass dichroic mirror 4 and the second biconvex lens 25 in sequence and enters the circular hole on the concave hole disk 11. The laser passing through the circular hole passes through the focusing turntable 22 to complete the optical path adjustment and first passes through the third biconvex lens 10, and then reaches the sample on the sample tray.

[0082] The imaging optical path guides the fluorescence generated by the sample to the imaging module. Along the imaging optical path are arranged the following components in sequence: a sample tray, a third biconvex lens 10, a focusing turntable 22, a concave hole plate 11, a second biconvex lens 25, a short-pass dichroic mirror 4, a fourth biconvex lens 26, and the imaging module 9. In this embodiment, the fluorescence generated by the sample on the sample tray passes through the third biconvex lens 10, the focusing turntable 22, the concave hole plate 11, the second biconvex lens 25, the short-pass dichroic mirror 4, and the fourth biconvex lens 26 in sequence before entering the imaging module 9.

[0083] The focal length of the first biconvex lens 5 is f3, and the focal length of the second biconvex lens 25 is f1. The relationship between the two can be: f1 = 2f3; the vertex of the corner cube prism retroreflector is located at the focal point of the first biconvex lens and the second biconvex lens 25.

[0084] The concave hole disk 11 includes an annular disk made of opaque material, with an outer diameter of a and an inner diameter of b. M circular holes are formed on the annular disk, where M is a natural number greater than 0. A concave surface coaxial with the circular holes is formed near the opening of the second biconvex lens 25. The focal length of the concave surface is f2. The distance between the second biconvex lens 25 and the concave surface is x1.

[0085] h is the depth of the concave surface.

[0086] The concave plate serves to reflect the excitation light and then converge the laser light to the pinhole, such as... Figure 3 , Figure 4 , Figure 5 As shown, the concave surface on the front reflects the excitation light, while the pinhole transmits the secondary collected excitation light. The pinhole on the back also acts as an optical filter, allowing only fluorescence from the focal plane to pass through, thus improving imaging resolution.

[0087] The focusing turntable uses the distribution of glass of different thicknesses, such as... Figure 11 , Figure 12 , Figure 13 As shown, each thickness corresponds to a different focal plane, achieving the shift of different focal planes.

[0088] like Figure 15 As shown, the corner pyramid prism retroreflector can be a Sorebo corner retroreflector, which has a top plane triangular area 27 at its top.

[0089] like Figure 16 and Figure 17 As shown, since the top of the corner cube prism retroreflector should include a top planar triangular area, but some domestic corner cube prism retroreflectors do not include this area, it is necessary to cut and process a planar triangular area at the top.

[0090] like Figure 18 and Figure 19As shown, the corner cube prism retroreflector can be a hollow corner cube prism, which already contains a top triangular area, and the top of the prism is perforated (the hole can be circular or non-circular).

[0091] The signal synchronization module is used to determine the timing relationship between the camera's acquisition cycle and the movement cycles of different focusing glass surfaces on the focusing dial, such as... Figure 14 As shown, since the concave aperture plate and the focusing turntable rotate coaxially, the camera acquires signals in each focusing glass working cycle, obtains the corresponding focal plane image information, and synchronously acquires timing information.

[0092] The image processing module, after the acquisition sequence is completed, combines the focal plane signals from all planes to obtain three-dimensional image information, thereby achieving three-dimensional microscopic imaging of the target. The focusing turntable and the concave aperture plate are mounted on the same motor shaft. The controller is used to control the motor speed and the imaging module's image capture frequency.

[0093] like Figures 3 to 9 As shown, the concave disk 11 includes an annular disk made of opaque material, with M circular holes on the annular disk, where M is a natural number greater than 0. A concave surface coaxial with the circular holes is formed near the opening of the second biconvex lens 25. The inner diameter of the annular disk is 12.7 mm, the outer diameter is 95.6 mm, and the thickness is 0.2 mm. The function of the concave surface is to reflect the first excitation light back to the corner prism retroreflector along the optical path through the focal point. After passing through the corner prism retroreflector, the direction of the first excitation light is flipped by 180° to form the second excitation light, which converges at the central circular hole of the concave surface.

[0094] The concave-faced disk has a starting radius of 20 mm and a stopping radius of 40 mm. 315 concave faces are distributed along an Archimedean spiral, with a concave diameter of 250 μm, an aperture of 50 μm, and a spacing of 300 μm. These 315 concave faces illuminate and excite fluorescent molecules within a 20 mm linear region. Rotation of the concave-faced disk allows for a complete scan of the 20 mm linear region. One revolution of one spiral scans the region once. To accelerate the scanning speed, 120 spirals are arrayed on the disk, achieving a scanning speed 120 times faster than a single spiral. To ensure a more uniform field of view, the 120 spirals are divided into 20 groups of 6 spirals each: spiral a, spiral b, spiral c, spiral d, spiral e, and spiral f. The initial concave hole position on spiral b is offset by 50µm compared to the initial concave hole position on spiral a, and so on. Within each group of spirals, the initial concave hole position on spiral c is offset by 50µm compared to spiral b, and so on. The initial concave hole position on spiral f is offset by 50µm compared to the initial concave hole position on spiral e. Compared to 120 identical spirals scanning the same area for each hole, the 6-group interval distribution design can fill a larger area of ​​the linear region with a hole spacing of 300µm. When the pinhole array disk rotates, it provides more uniform illumination for the entire field of view.

[0095] The principle of the Archimedean spiral design is to transform the points to be scanned on a straight line into pinholes on the Archimedean spiral. For example, if the starting point is 20mm from the origin and the ending point is 40mm from the origin, the required scanning length of the straight line is 40-20=20mm. When the concave holes are distributed on the straight line, approximately 20 / (0.3)≈66 concave holes can be arranged. When the concave holes are distributed using the Archimedean spiral r=a+bθ, where r is the distance to the origin, a is the radius at the starting point of the spiral, and b affects the distance between each spiral, a=20mm. Here, we illustrate this with three different numbers of Archimedean spirals. When selecting method one with 0.5 turns (θ1=0, θ2=π), the ending radius is 40mm. Calculations show:

[0096]

[0097] The formula for the arc length of a curve in polar coordinates:

[0098]

[0099] Substituting the above into the arc length formula, we get...

[0100]

[0101] The number of holes that can be placed is approximately 315 (94.594 / (0.3)).

[0102] When selecting option two and completing one lap, the calculation...

[0103]

[0104] Substituting into the arc length formula

[0105]

[0106] The number of holes that can be placed is approximately 188.84 / (0.3)≈629.

[0107] When the selected method is 3 laps and the number of laps is 2, the calculation is as follows:

[0108]

[0109] Substituting into the arc length formula

[0110]

[0111] The number of holes that can be placed is approximately 1257 (377.34 / (0.3)).

[0112] That is, by using different numbers of solenoid coils, the 20mm straight line is extended to 94.594mm, 188.84mm, and 377.34mm. The number of concave holes is increased from 66 to 315, 629, and 1257. Correspondingly, the entire 20mm area is scanned by rotating 0.5, 1, and 2 revolutions. Therefore, rapid scanning of the line is achieved by the rapid rotation of the concave hole disk 11, thus scanning the entire plane. At the same time, the scanning speed can be further accelerated by using a circular array of solenoid coils, and uniform illumination of the field of view can be achieved by increasing the number of holes. However, increasing the number of Archimedean solenoid coils further compresses the distance between each group of solenoids. When the distance between two concave holes between adjacent solenoids decreases, it will lead to increased pinhole crosstalk. Furthermore, the more concave holes there are, the more difficult the processing becomes, and the higher the quantity, the higher the cost and the greater the difficulty in quality control.

[0113] Therefore, based on this Archimedean spiral, this patent selects the corresponding number of spiral groups according to the ratio of hole spacing S to hole diameter D, where N is an integer;

[0114]

[0115] The aperture spacing S is used to reduce pinhole crosstalk from fluorescence signals from other focal planes. In confocal microscopy, a typical aperture spacing S is 250 μm, and the aperture diameter D is 50 μm, which effectively suppresses pinhole crosstalk. Because this patent uses a concave hole design, selecting a concave diameter of 250 μm, consistent with the typical aperture spacing S, causes adjacent concave holes to be tangent. During the processing of the concave surface, deformation easily occurs at the tangent points. Therefore, considering the processing technology of the concave surface, this patent changes the typical aperture spacing S to 300 μm, still achieving the effect of suppressing pinhole crosstalk. At this point, there is still a region between two aperture spacings in the Archimedean spiral that cannot be illuminated by six times the aperture diameter. Reflected in sample measurement, this means that fluorescence signals within six times the aperture spacing on the sample cannot pass through the circular aperture for imaging. Taking N=2, a six-group spiral interval design is used, with spiral b offset from spiral a by one aperture diameter, spiral c offset from spiral b by one aperture diameter, and so on, compensating for the six-times aperture spacing through six groups of spirals. With N=2, 12 sets of spirals can compensate for a 6-fold aperture spacing. The intermittent spiral design allows for complete scanning of the entire area; fluorescence signals within a 6-fold aperture spacing of adjacent samples can pass through the intermittently distributed circular holes at corresponding spirals b, c, etc., for imaging. Another advantage of the intermittent spiral design is that it reduces the number of holes by selecting Archimedean spirals with fewer turns. For example, an Archimedean spiral with 0.5 turns requires 315 holes per spiral. An Archimedean spiral with 2 turns requires 1257 holes, reducing the hole processing cost by four times. For the same 2-turn rotation scanning of a complete 20mm linear area, an Archimedean spiral with 0.5 turns requires a total of 315*4=1260 holes, while an Archimedean spiral with 2 turns requires 1257 holes. Within the same period, the number of holes for both types of scanning is approximately the same, and the intermittent spiral design compensates for the aperture spacing, resulting in a brighter scanning field of view.

[0116] In a fluorescence microscope imaging system, the addition of a concave aperture disk 11 can suppress the formation of a confocal microscopy imaging system by defocusing light. This patent selects an aperture diameter of 50 μm and an aperture spacing of 300 μm as parameter values, which can both receive high-quality fluorescence signals and suppress pinhole crosstalk. The principle of suppressing pinhole crosstalk is as follows: Figure 9 As shown: When fluorescent molecules in sample 7 are excited and emit fluorescence, focal plane 12, first defocus surface 13, and second defocus surface 14 emit fluorescence 9, first discrete light 15, and second discrete light 16, respectively. These light rays pass through the first biconvex lens 5 and reach the concave aperture plate 11. Only the fluorescence 8 emitted from focal plane 12 can pass through the 50µm circular aperture, and the 300µm aperture spacing blocks the first and second discrete light rays 15 and 16, preventing them from participating in imaging. Fluorescence 9 passes through the concave aperture plate 11 and the third biconvex lens 10 to reach the imaging module 9, achieving point-by-point and line-by-line scanning imaging of the sample at focal plane 12.

[0117] The concave hole disk 11 has concave surfaces with a diameter D1 of 250 μm and circular holes with a diameter D of 50 μm distributed on its front side, with a hole spacing S of 300 μm. Since the laser energy collected by a single pinhole is approximately 3%, very little energy is available for exciting fluorescent substances on the sample surface. For example... Figure 10 As shown, the combination of the concave surface and the cornerstone prism retroreflector serves to concentrate energy, increasing the pinhole energy to approximately 69%. The optical path diagram is as follows: Let the focal length of the second biconvex lens 17 be f1, and the vertex of the cornerstone prism retroreflector 18 be located at its focal point. When the first excitation light 19, collimated by the second biconvex lens 17, enters the concave surface of the concave hole disk 11, the concave surface is coated with a reflective film with a focal length of f2. The reflected light converges at the point and extends to the second biconvex lens 17, then refracts into the cornerstone prism retroreflector 18. Within the cornerstone prism retroreflector, after three reflections (only two reflections are shown in the front view), the light finally rotates 180° in the same direction, emitting the second excitation light 20, which converges through the second biconvex lens 17 at the 50µm diameter circular hole on the front of the concave hole disk 11. After being reflected by the concave surface and flipped by the cornerstone prism retroreflector, the laser beam converges at the pinhole. This means that the laser energy collected by the individual pinhole is replaced by the laser energy collected by the entire concave surface, increasing the laser throughput and resulting in a stronger excitation signal on the sample. This is crucial for many optical and microscopic applications, especially in confocal microscopy, where sufficient excitation light is required to excite the fluorescence signal in the sample.

[0118] The focusing dial is used to adjust the focal plane of the microscope imaging to the corresponding focal plane. The focusing dial 22 includes three sets of focusing glass groups 21 arranged in a ring, with thicknesses of 0.16 mm, 0.82 mm, and 1.8 mm respectively. The working principle of the focusing dial is as follows: Figure 13 As shown: Without the focusing glass group 21, the fluorescence 8 emitted by the focal plane 12 within sample 6 reaches the imaging module through the confocal microscopy imaging system. When the focusing glass group 21 is present, refraction occurs, and the second fluorescence 24 generated at the second focal plane 23 reaches the imaging module through the confocal microscopy imaging system. In other words, by adding focusing glass groups 21 of different thicknesses on the focusing disk 22, different shifts in the focal plane are achieved, resulting in rapid zooming and simultaneous observation of multiple focal plane images.

[0119] The signal synchronization module includes: a mechanism for determining the timing relationship between the acquisition cycles of the concave aperture plate, the focusing plate, and the imaging module (e.g., a CCD camera), and for synchronously acquiring the timing data; such as... Figure 10 As shown, different focal planes corresponding to different thicknesses of focusing glass groups 21 are scanned and imaged at different times when the motor rotates. The signal synchronization module determines the timing information and transmits it to the image processing module.

[0120] The image processing module is used to obtain 3D model information based on the 3D shape distribution of the target and the focal plane signals of all planes after the acquisition sequence ends. By combining the rotation speed of the concave hole disk 11, the timing of image capture for each hole is precisely calculated, thereby segmenting the image into continuous lines or frames. The CCD camera can then combine these lines or frames into a complete image. By stitching together image information from different focal planes, 3D sample image information is obtained.

[0121] The concave-hole disc rotates at 2500 rpm, and the time interval between the rotations of adjacent spiral groups is:

[0122]

[0123] Assuming the CCD camera's maximum imaging speed is 180fps (180 photos per second), with each photo taking a minimum of 5.6ms, and assuming the first image exposure begins 6ms (167fps) after the initial exposure, the next image exposure will commence to capture the next image. Since the camera's dormancy time is measured in microseconds (µs), it can be ignored when it is very short; therefore, the exposure time is set to 6ms.

[0124] Since the interval between rotations of adjacent spiral groups is 0.2ms, a total of 30 spiral groups are rotated within a 6ms exposure time, covering an angle of 90°. The concave lens dial 11 has 4 focusing glass groups, each occupying a 90° angle. The first focusing glass is used within 6ms, including a very short unexposed time. The next focusing glass is used after 6ms. The total exposure time for the concave lens dial 11 is 6 x 4 = 24ms; therefore, the concave lens dial 11 should be rotated at a speed of 1 / 24 * 60 = 2500 rpm.

[0125] The first 6ms imaging focal length is a1, the second is a2, the third is a3, the fourth is a4, the fifth is a1, and so on. That is, the (4n+1)th frame is the image at focal length a1, the (4n+2)th frame is the image at focal length a2, the (4n+3)th frame is the image at focal length a3, and the (4n+4)th frame is the image at focal length a4. The image processing module combines the image information from different focal lengths to obtain the three-dimensional image information of the sample.

[0126] A complete optical path diagram is as follows Figure 2 As shown, let the focal length of the first biconvex lens 5 be f3, and the excitation radius be w1, as follows... Figures 15 to 19As shown, the triangular region 27 on the top plane of the corner prism retroreflector 18 is coaxial with the first biconvex lens 5 and located at the focal point. Therefore, the first excitation light 19 converges at the focal point, and without changing direction, it exits through the interior of the corner prism retroreflector 18 and passes through a short-pass dichroic mirror to reach the second biconvex lens 25. The focal length of the second biconvex lens 25 is f1 = 2f3, and its focal point coincides with that of the first biconvex lens 5. Therefore, after collimating the first excitation light 19, the excitation light radius is 2w1, and it exits onto the concave surface of the concave facet disk 11. The concave surface parameters are as follows... Figure 16 As shown: Let the depth of the concave surface be h, Let a be a real number with radius of curvature R, (Rh) 2 +(2w1) 2 =R 2 , From the mirror grinder formula Then its concave focal length f2, The concave surface reflects and converges the excitation light to the focal point, where it propagates to the second biconvex lens 25. After refraction, it reaches the cornerstone prism retroreflector 18, where its direction is flipped 180 degrees to form the second excitation light 20, which is then refracted by the second biconvex lens 25 and reaches the central circular hole of the concave surface of the concave hole disk 11. When the above relationships are satisfied, the parameter relationships are as follows: Figure 21 As shown, let x1 be the distance between the optical center of the concave disk 11 and the optical center of the second biconvex lens 25. From the similarity relationship, we get:

[0127] x4 = x2 - x3 x5 = x4 x6 = x3 - x5

[0128] When the second excitation light converges at the central circular aperture, i.e., x7 = x1 + h, we get:

[0129]

[0130] Solving for:

[0131] The second excitation light 20 propagates through the circular aperture to the focusing glass assembly 21. Given that the thickness of the focusing glass assembly 21 is d1, the refractive index of the glass is n2, the distance from the point where the pinhole beam converges is x8, and the distance from the third biconvex lens 10 is x9, calculate its imaging depth x. 10 By the law of refraction: x 11 =d1 tan I1,x 12 =x 11 cot I,x 13 =d1-x 12 ,x 14 =x8-x 13 +x9, based on similarity relations Solving for:

[0132] That is, the excitation is directed to a distance of 10 x from the third biconvex lens. 10 Fluorescence information at focal plane 12. When fluorescent molecules in biological tissue are excited, they emit fluorescence 8 with a longer wavelength, indicated by a double-headed dashed line. Due to the reversible optical path, fluorescence 8 passes through the third biconvex lens 10, the focusing glass group 21, and the circular hole in the concave hole plate 11, then passes through the second biconvex lens 25 to reach the short-pass dichroic mirror 4. Because the wavelength of fluorescence 8 is too long, it cannot pass through the short-pass dichroic mirror 4 and is reflected through the fourth biconvex lens 26. The focal length of the fourth biconvex lens is f5. The imaging module 9 is placed at the point where fluorescence 8 converges to process the image information at focal plane 12. When the thickness d1 of the focusing glass group 21 is different, image information at different focal planes can be obtained. After time-series processing by the signal synchronization module, the image processing module obtains three-dimensional sample image information.

[0133] In the concave hole disk 11, if the diameter D1 of the concave surface is 250 μm, then 2w1 = 125 μm, and the width of the first excitation light w1 = 62.5 μm. Let a = 64, then substituting the radius of curvature R = 8mm, focal length f2 = 16mm, and assuming the thickness of the second biconvex lens 25 is 4mm and the focal length f1 = 40mm, when the distance from the concave hole disk 11 to the second biconvex lens 25 is x1, the following condition is met. The second excitation light 20 is emitted precisely along the circular aperture. The third biconvex lens 10 is at a distance x from the circular aperture. 14 =20mm, focal length f4=10mm, when the glass thickness d1=0 of focusing glass group 21, x 14 =x8+x9, Substitute into the formula for the depth of the imaging focal plane. The above is the optical path analysis for a single concave aperture. The actual excitation light radius is greater than 62.5 μm. Simultaneously, a beam expansion method can be used to adjust the focal length of the second biconvex lens 25 to increase the diameter of the collimated beam, thereby illuminating multiple microlens apertures and expanding the imaging range at the depth of the focal plane. When the glass thickness d1 of the focusing glass group 21 is not equal to 0, a corresponding shift in the focal plane occurs and is imaged onto the imaging module.

[0134] To further illustrate the technical solution of the present invention, the following description is in conjunction with the appendix. Figure 23 Specific embodiments of the present invention will be described below. The fluorescence confocal microscope body is prior art, and its components, including the laser assembly, dichroic mirror assembly, lens assembly, imaging module assembly, and sample, will not be described again in this schematic diagram.

[0135] Specifically, such as Figures 24 to 27As shown: The integrated base 28 includes a front baffle 33 and a rear baffle 34, with a wire harness inlet 30, a first light inlet slot 31, and a second light inlet slot 32 distributed on the front and rear baffles. The outer shell 35 is connected to the integrated base 28 via the second bolt hole 33 and the first bolt hole 29, thereby achieving a fixed assembly of the outer shell and the bottom.

[0136] Specifically, the motor drive module, such as Figure 28 As shown: The motor drive module includes a motor module 42, driven by a brushless motor drive board with an adjustable speed of 0-7500 rpm. The drive board can be purchased directly; the circuit diagram is not detailed here. The motor module is fixed to the motor mounting slot 38 in the motor base 41, which is bolted to the integrated base 28 via the third bolt hole 36. The motor base 41 includes a third light-entry slot 37, serving as the laser illumination path. The motor module 42 includes a first fixed shaft 39 and a fourth bolt hole 40, acting as a connector to the other modules.

[0137] Specifically, fixed kits such as Figures 29 to 39 As shown: The outer side of the first fixing bracket 43 has three fifth bolt holes 44, which are bolted to the fourth bolt holes 40 using first bolts 47 to fix it to the first fixing shaft 39 of the motor module 42. The inner side of the second fixing bracket 50 has three eighth bolt holes 56 and three seventh bolt holes 55, which are bolted to the sixth bolt holes 45 using first bolts 47 to fix it to the second fixing shaft 46 of the first fixing bracket 43. First O-rings 48 and second O-rings 49 are used to fill the first O-groove 51 and the second O-groove 52. The inner side of the third fixing bracket 58 has three ninth bolt holes 57, which are bolted to the sixth bolt holes 45 using first bolts 47 to fix it to the first fixing bracket 43. The back side also has third O-groove 53 and fourth O-groove 54.

[0138] Specifically, such as Figures 40 to 43 As shown, the connection and assembly are as follows: First, connect the concave hole plate 11 to the motor module 42, then connect the focusing plate 22 to the concave hole plate 11. Use the aforementioned O-rings to fasten the components, and connect the bolts to the corresponding bolt holes. The final assembly diagram is shown below. Figure 44 As shown.

[0139] The above description is only a preferred embodiment of the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A three-dimensional single-rotating-disc confocal microscope, characterized in that, This includes the excitation optical path and the imaging optical path; where: Along the excitation light path are arranged the following components in sequence: light source (1), first biconvex lens (5), corner prism retroreflector, short-pass dichroic mirror (4), second biconvex lens (25), concave hole plate (11), focusing turntable (22), third biconvex lens (10) and loading plate; Along the imaging optical path are arranged the following components in sequence: a sample tray, a third biconvex lens (10), a focusing turntable (22), a concave aperture tray (11), a second biconvex lens (25), a short-pass dichroic mirror (4), a fourth biconvex lens (26), and an imaging module (9). The focal length of the first biconvex lens (5) is f3, and the focal length of the second biconvex lens (25) is f1; the vertex of the corner prism retroreflector is located at the focal point of the first biconvex lens and the second biconvex lens (25); The concave hole disk (11) includes an annular disk made of opaque material, on which M circular holes are opened, where M is a natural number greater than 0. A concave surface coaxial with the circular holes is opened at the position of the circular holes near the opening of the second biconvex lens (25); the focal length of the concave surface is f2; the distance between the second biconvex lens (25) and the concave surface is x1. h is the depth of the concave surface.

2. The three-dimensional single-rotor confocal microscope according to claim 1, characterized in that, The M circular holes are divided into T groups; the value of T ranges from 20 to 30; each group of circular holes is distributed on L Archimedean spirals, where L is a natural number greater than 0, and the starting point of the Archimedean spiral is d, and the ending point is c.

3. The three-dimensional single-rotor confocal microscope according to claim 1, characterized in that, d is 20mm, c is 40mm.

4. The three-dimensional single-rotor confocal microscope according to any one of claims 1-3, characterized in that, The concave surface has a diameter of 250 μm, the circular holes have a diameter of 50 μm, and the distance between the circular holes is 300 μm; L=6, T=20.

5. The three-dimensional single-rotor confocal microscope according to claim 1, characterized in that, The focusing turntable (22) includes several focusing glass groups (21) of different thicknesses; each focusing glass group (21) has an arc-shaped structure, and several focusing glass groups (21) are spliced ​​together to form a fan-shaped ring.

6. The three-dimensional single-rotor confocal microscope according to claim 5, characterized in that, The focusing turntable (22) includes three focusing glass groups (21) with thicknesses of 0.16mm, 0.82mm and 1.8mm; each focusing glass group (21) has an arc-shaped structure, and the three focusing glass groups (21) are spliced ​​together to form a fan-shaped ring.

7. The three-dimensional single-rotor confocal microscope according to claim 5, characterized in that, When the thickness of the focusing glass group (21) is d1, the refractive index of the focusing glass group (21) is n2, the distance between the focusing glass group (21) and the concave hole disk (11) is x8, the distance between the focusing glass group (21) and the third biconvex lens (10) is x9, and the imaging depth is x. 10 The focal length of the third biconvex lens (10) is f4; ； Where: I represents the incident angle, that is, the angle between the outgoing light and the normal of the focusing glass group.

8. The three-dimensional single-rotor confocal microscope according to claim 5 or 6, characterized in that, The focusing turntable (22) and the concave hole turntable (11) are mounted on the same motor shaft.

9. The three-dimensional single-rotor confocal microscope according to claim 8, characterized in that, It also includes a controller for controlling the motor speed and the imaging module (9) taking pictures.

10. The three-dimensional single-rotor confocal microscope according to claim 1, characterized in that: The cornerstone prism retroreflector is a solid structure, and includes a planar triangular region at the top of the cornerstone prism retroreflector. Alternatively, the corner prism reflector may be a hollow corner prism, with a circular or non-circular hole at the top of the hollow corner prism.

Images