Fluorescence collecting device and fluorescence microscope

Abstract

The invention discloses a fluorescence collecting device, which comprises an objective lens for receiving a fluorescence beam and enabling the fluorescence beam to pass through; a fluorescence collection channel extending in a first direction; the fluorescence transmission and collection assembly is arranged in the fluorescence collection channel, transmits and collects the fluorescence beams from the objective lens, and comprises a lens assembly, a first lens assembly and a second lens assembly which are sequentially arranged in the fluorescence collection channel in the first direction; the first end face and the second end face are oppositely arranged in the first direction, the side face is located between the first end face and the second end face in the first direction, the diameter of the first end face is larger than that of the second end face, and fluorescent beams transmitted from the lens assembly enter the circular table mirror from the first end face and penetrate out of the second end face. The side surface reflects the fluorescent beam reaching the side surface to the second end surface; and the photon detector receives the fluorescence beam transmitted from the second end face and converts the fluorescence beam into an electric signal. The fluorescence collecting device is high in collecting efficiency. The invention also discloses a fluorescence microscope.

Description

Technical Field

[0001] The invention relates to the technical field of optical imaging, and in particular to a fluorescence collection device and a fluorescence microscope. Background Art

[0002] Fluorescence microscopy uses lasers of a certain wavelength to excite the fluorescent substances that mark samples under the microscope, causing them to emit fluorescence, and judges the state of the observed sample based on the color and brightness of the collected fluorescence. Fluorescence microscopy has high sensitivity and high resolution, and can achieve real-time observation of the internal structure, function, and activity process of cells and tissues by marking specific molecules or microstructures in biological samples. It has a wide range of applications in cell biology, neuroscience, biomedicine, and other fields, providing an important technical support for life science research.

[0003] Improving the stimulated fluorescence collection efficiency is crucial to improving the performance of fluorescence microscopes. Improving the fluorescence collection efficiency can increase the intensity of the fluorescence signal collected from the sample under the same excitation light intensity and microscopic observation conditions, which helps to improve the signal-to-noise ratio of imaging and make imaging clearer and more accurate. In addition, the improvement of fluorescence collection efficiency can reduce the required excitation light power while maintaining imaging quality, thereby reducing photodamage to the sample. The improvement of fluorescence collection efficiency also means that weaker fluorescence signals can be collected, thereby increasing the imaging depth of the system and the detection limit of fluorescence signals. In practical applications, brain scientists and neuroscientists can use multi-photon fluorescence microscopes with high fluorescence collection efficiency to reduce photodamage to the brains of living animals being observed, extend the in vivo imaging time, and observe deeper neural activity in the brains of living animals.

[0004] In the fluorescence collection system of a fluorescence microscope, the window and sensor / detector are the key components of the photodetector used. The main function of the window is to protect the detector from external contamination while allowing the fluorescence signal to pass through; the sensor / detector is an electronic device used to detect and record fluorescence signals, such as photomultiplier tubes (PMT) or cameras and other photon detection and photoelectric conversion devices. The PMT has a photocathode photosensitive surface. When photons hit the photocathode, electrons are generated and pass through the multiplier, collector, etc. to form a current pulse signal, which is finally converted into a digital signal output.

[0005] Existing fluorescence microscopy systems usually converge fluorescence to the incident window of the photon detector (sensor), and believe that the fluorescence reaching the incident window means that it has reached the photosensitive surface of the detector. However, in fact, there is often a distance between the incident window and the detector. The space at this distance can be called a "corridor", that is, the fluorescence will reach the photocathode photosensitive surface only after passing through a section of the corridor after the incident window. In some systems, the length of the corridor may even reach 8mm. In this case, since the fluorescence beam entering the fluorescence collection system may have both parallel incident and non-parallel incident fluorescence, the existence of the corridor will inevitably reduce the fluorescence intensity detected by some non-parallel incident fluorescence, thereby affecting the overall fluorescence collection efficiency. In addition, the larger the incident angle, the more obvious the influence of the corridor. Especially in the case of a long corridor, the fluorescence propagation distance is longer, more fluorescence is affected, and the fluorescence collection efficiency is lower. Therefore, we cannot simply equate the fluorescence reaching the incident window with the fluorescence reaching the photocathode, especially when the channel is long and there is non-parallel incident fluorescence. We need to further consider the impact of the channel on the fluorescence collection efficiency. Summary of the invention

[0006] In order to solve the above technical problems, an embodiment of the present invention discloses a fluorescence collection device, comprising:

[0007] an objective lens, for receiving the fluorescent beam and allowing the fluorescent beam to pass through;

[0008] A fluorescence collection channel extending along a first direction;

[0009] The fluorescence transmission and collection component is arranged in the fluorescence collection channel and is used to transmit and collect the fluorescence beam from the objective lens, including:

[0010] A lens assembly comprising at least two lenses;

[0011] A truncated cone mirror, comprising a first end face and a second end face arranged opposite to each other along a first direction, and a side face located between the first end face and the second end face in the first direction, wherein a diameter of the first end face is greater than a diameter of the second end face, wherein a fluorescent beam transmitted from the lens assembly enters the truncated cone mirror from the first end face and passes through the second end face, and the side face reflects the fluorescent beam reaching the side face to the second end face;

[0012] A photon detector, used for receiving the fluorescent beam transmitted from the second end surface and converting the fluorescent beam into an electrical signal;

[0013] The first direction is parallel to the optical axis of the fluorescence transmission collection component.

[0014] By adopting the above technical solution, the influence of the channel on the non-parallel incident fluorescence can be reduced, the efficiency of the non-parallel incident fluorescence reaching the photosensitive surface of the photon detector can be ensured, and the collection efficiency of the fluorescence collection device can be improved.

[0015] Optionally, the first end surface is located at a rear focal plane of the lens assembly, and a diameter of the first end surface is greater than or equal to a diameter of the fluorescent beam passing through the lens assembly.

[0016] Optionally, the photon detector includes a photosensitive surface, which senses the fluorescent beam transmitted from the second end surface, and the photosensitive surface is rectangular or circular, and the width of the rectangle or the diameter of the circle is greater than or equal to the diameter of the second end surface.

[0017] Optionally, along the first direction, a detector protection window is provided between the truncated cone mirror and the photosensitive surface, and the fluorescent beam passes through the detector protection window from the second end face to reach the photosensitive surface. The distance between the rear surface of the detector protection window and the photosensitive surface is not greater than 2 mm and not less than 0.1 mm, and the distance between the front surface of the detector protection window and the second end face is not greater than 1 mm and not less than 0.1 mm.

[0018] Optionally, the lens assembly includes a first lens, a second lens and a third lens arranged in sequence along a first direction, the diameter of the first lens is 38-50.8 mm, the focal length of the first lens is 75 mm, the diameter of the second lens is 30-50.8 mm, the focal length of the second lens is 39-40 mm, the diameter of the third lens is 25-30 mm, and the focal length of the third lens is 15-16.5 mm.

[0019] Optionally, the fluorescence transmission and collection assembly also includes a truncated cone mirror fixed window located between the truncated cone mirror and the third lens along the first direction, the first end face is fixedly mounted on the truncated cone mirror fixed window, and the distance between the third lens and the truncated cone mirror fixed window is 0.9-1mm.

[0020] Optionally, along the first direction, the distance between the frustum mirror fixed window piece and the photosensitive surface is 7-10 mm, the length of the frustum mirror along the first direction is 7-9 mm, the radius of the first end face is 2.5-3 mm, and the radius of the second end face is 2-2.5 mm.

[0021] Optionally, a dichroic mirror assembly is further provided in the fluorescence transmission and collection assembly, and along the first direction, the dichroic mirror assembly is located before the first lens or between the first lens and the second lens.

[0022] Optionally, the objective lens and the fluorescence collection channel are arranged coaxially.

[0023] Optionally, the fluorescence collection channel further includes:

[0024] Optics holder, including:

[0025] A large fixing frame, used for embedding the objective lens, the first lens and the dichroic mirror assembly, having two openings opposite to each other along the first direction;

[0026] A small fixing frame, used for embedding the second lens, the third lens and the frustum mirror fixing window sheet, having two openings opposite to each other along the first direction;

[0027] A transfer fixing frame, wherein an opening at one end of the large fixing frame and an opening at one end of the small fixing frame are respectively inserted into the transfer fixing frame, so that the large fixing frame and the small fixing frame are coaxially installed;

[0028] The detector fixing frame has an opening and an end surface opposite to each other along the first direction. The edge of the opening at one end of the detector fixing frame abuts against the frustum mirror fixing window sheet, and the detector protection window sheet is embedded in the detector fixing frame.

[0029] Optionally, the distance between one end opening of the detector fixing frame and the detector protection window piece is the corridor length, the length of the truncated cone mirror is less than or equal to the corridor length, and one end opening of the detector fixing frame contacts and abuts against the truncated cone mirror fixing window piece.

[0030] Optionally, the distance between the opening at one end of the detector fixing frame and the detector protection window is the corridor length, the length of the truncated cone mirror is greater than the corridor length, and a gasket is provided between the opening at one end of the detector fixing frame and the truncated cone mirror fixing window.

[0031] Optionally, the fluorescence collection channel is a first fluorescence collection channel, the dichroic mirror assembly is located before the first lens, the dichroic mirror assembly includes a first dichroic mirror and a second dichroic mirror, the fluorescence collection device also includes a second fluorescence collection channel extending along a second direction, the fluorescence beam transmitted through the second dichroic mirror is collected by the first fluorescence collection channel, and the fluorescence beam reflected through the second dichroic mirror is collected by the second fluorescence collection channel, and the second direction is perpendicular to the first direction.

[0032] Optionally, when the angle between the fluorescence beam and the optical axis of the fluorescence collection channel is 2.0°-8.0°, the collection efficiency of the fluorescence collection device is 69.78%-98.64%.

[0033] According to another specific embodiment of the present invention, an embodiment of the present invention discloses a fluorescence microscope, including the above-mentioned fluorescence collection device.

[0034] By adopting the above technical solution, the fluorescence microscope has higher fluorescence collection efficiency and improved imaging performance. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 A schematic diagram showing the structure of a fluorescence collection device according to an embodiment of the present invention is shown;

[0036] Figure 2 A schematic structural diagram of a fluorescence collection device according to another embodiment of the present invention is shown;

[0037] Figure 3 A schematic diagram showing the structure of a fluorescence transmission and collection component according to an embodiment of the present invention;

[0038] Figure 4 A schematic diagram showing the structure of a fluorescence collection device with dual fluorescence collection channels according to an embodiment of the present invention;

[0039] Figure 5 A schematic diagram showing simulation results of fluorescent beams at various incident angles and their collection efficiency received by Example 1 of the present invention;

[0040] Figure 6 A schematic diagram showing simulation results of fluorescent beams at various incident angles and their collection efficiency received by Comparative Example 1 of the present invention;

[0041] Figure 7 The scatter plots of the collection efficiency of Example 1 of the present invention and Comparative Example 1 for fluorescence incident at different angles are shown.

[0042] 1. Fluorescence collection device,

[0043] 10. Objective lens,

[0044] 20. Fluorescence collection channel, 21. Optical element holder, 211. Large holder, 2111. Optical element holder, 2112.

[0045] Fixing frame coaxial rod and fasteners, 212. Small fixing frame, 2121. Optical element fixing frame, 2122. Fixing frame coaxial rod and fasteners, 22. Adapter fixing frame, 23. Detector fixing frame, 24. Shading component, 25. Corridor, 26 Gasket,

[0046] 30. Fluorescence transmission and collection assembly, 31. Lens assembly, 311. First lens, 312. Second lens, 313. Third lens, 32. Frustum mirror, 321. First end surface, 322. Second end surface, 323. Side surface, 33. Photon detector, 331. Photosensitive surface, 34.

[0047] Detector protection window, 35. Frustum mirror fixed window, 36. Dichroic mirror assembly, 361. First dichroic mirror, 362.

[0048] Second dichroic mirror, 37. Filter assembly. DETAILED DESCRIPTION

[0049] The following specific embodiments illustrate the implementation of the present invention, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. Although the description of the present invention will be introduced in conjunction with the preferred embodiment, this does not mean that the features of this invention are limited to this implementation. On the contrary, the purpose of introducing the invention in conjunction with the implementation is to cover other options or modifications that may extend based on the claims of the present invention. In order to provide a deep understanding of the present invention, the following description will include many specific details. The present invention can also be implemented without using these details. In addition, in order to avoid confusion or blurring the focus of the present invention, some specific details will be omitted in the description. It should be noted that the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

[0050] It should be noted that in this specification, similar reference numerals and letters denote similar items in the following drawings, and therefore, once an item is defined in one drawing, it does not need to be further defined and explained in the subsequent drawings.

[0051] The terms “first”, “second”, etc. are only used for distinguishing descriptions and should not be understood as indicating or implying relative importance.

[0052] In the description of this embodiment, it is also necessary to explain that, unless otherwise clearly specified and limited, the terms "set", "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 this embodiment can be understood according to specific circumstances.

[0053] In the embodiments of the present application, the "photon detector" is an integral device consisting of many components such as photosensitizing and photoelectric conversion elements, a housing, a built-in cooling device, a window, etc. In the present application, only a partial structure of the photon detector is shown by diagram.

[0054] In order to make the objectives, technical solutions and advantages of the present invention more clear, the embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0055] The first aspect of the present invention discloses a fluorescence collection device, such as Figure 1-Figure 3 As shown, the fluorescence collection device 1 includes an objective lens 10 , a fluorescence collection channel 20 , and a fluorescence transmission and collection component 30 disposed in the fluorescence collection channel 20 .

[0056] The objective lens 10 is used to receive the fluorescent beam and allow the fluorescent beam to pass through, that is, the objective lens 10 collects light from a sample (such as a biological sample), including reflected light, transmitted light and scattered light, to facilitate subsequent collection and processing.

[0057] The fluorescence collecting channel 20 extends along the first direction, and the fluorescence beam from the objective lens 10 can propagate in the fluorescence collecting channel 20 .

[0058] A fluorescence transmission and collection component 30 is provided in the fluorescence collection channel 20, and the fluorescence transmission and collection component 30 is used to transmit and collect the fluorescence beam from the objective lens 10. The fluorescence transmission and collection component 30 includes the following components which are sequentially arranged in the fluorescence collection channel 20 along the first direction:

[0059] A lens assembly 31, wherein the lens assembly 31 includes at least two lenses;

[0060] The truncated cone mirror 32 includes a first end face 321 and a second end face 322 that are arranged opposite to each other along a first direction, and a side face 323 that is located between the first end face 321 and the second end face 322 in the first direction. The diameter of the first end face 321 is greater than the diameter of the second end face 322. The truncated cone mirror 32 is in a contracted state along the first direction. The fluorescent beam transmitted from the lens assembly 31 enters the truncated cone mirror 32 from the first end face 321 and passes through the second end face 322. The side face 323 reflects the fluorescent beam that reaches the side face 323 to the second end face 322.

[0061] The photon detector 33 is used to receive the fluorescent beam transmitted from the second end surface 322 and convert the fluorescent beam into an electrical signal.

[0062] Specifically, the first direction is as follows: Figure 1-Figure 3 As shown in direction A in the figure, the first direction is parallel to the optical axis of the fluorescence transmission and collection component 30. More specifically, the first direction is parallel to the propagation direction of the incident fluorescence in the fluorescence collection channel 20.

[0063] In the fluorescence collection device 1, according to the propagation path of the fluorescence beam, it passes through the objective lens 10, the lens assembly 31, the truncated cone mirror 32 in sequence, and finally reaches the photosensitive surface 331 of the photon detector 33. In the truncated cone mirror 32, since the first end face 321 and the second end face 322 are arranged in sequence along the first direction, the fluorescence beam enters the truncated cone mirror 32 from the first end face 321, and then leaves the truncated cone mirror 32 from the second end face 322.

[0064] In the arrangement of the present invention, firstly, after the fluorescence parallel to the first direction is converged by the lens assembly 31, it directly reaches the second end surface 322 from the first end surface 321, and then reaches the photosensitive surface 331 of the photon detector 33. Secondly, there may be fluorescence not parallel to the first direction in the fluorescence beam collected by the objective lens 10, that is, when there is fluorescence incident at an angle, especially when the back aperture of the objective lens 10 is large, more large-angle fluorescence enters the fluorescence transmission and collection assembly 30. These non-parallel fluorescences are first converged by the lens assembly 31. Since the fluorescence itself has an angle, they are still in a non-parallel state after convergence, and it is difficult to ensure that they will pass through the second end surface 322. However, due to the existence of the side surface 323, these non-parallel fluorescences can be changed in propagation direction by the side surface 323, that is, by being reflected by the converging truncated mirror 32, these fluorescences can pass through the second end surface 322 and then be captured by the photosensitive surface 331 of the photon detector 33.

[0065] According to the existing method of transmitting directly from a window through a tunnel to a detector (sensor), non-parallel fluorescence may undergo various changes in the tunnel after passing through the window. For example, some fluorescence incident at a large angle may hit the tunnel wall and be consumed. Even if it can eventually reach the photon detector, the fluorescence intensity has been greatly reduced, resulting in the fluorescence intensity detected by the final photosensitive surface 331 being much lower than the fluorescence intensity of the incident fluorescence transmission and collection component 30. In addition, the longer the tunnel is, the lower the intensity of non-parallel incident fluorescence collected by the photosensitive surface 331. Therefore, in fact, the tunnel cannot be ignored. The present invention utilizes the existing tunnel structure and adds a truncated cone mirror 32 in the tunnel. Through the reflection effect of the side 323, the propagation direction of non-parallel incident fluorescence is changed. On the one hand, it can reduce the loss of fluorescence, and on the other hand, it can increase the possibility of fluorescence reaching the photon detector 33, thereby improving the collection efficiency of fluorescence, especially for large-angle incident fluorescence. In addition, the truncated cone structure of the truncated cone mirror 32 is more suitable for the structure of a long and narrow corridor, which is more conducive to its stable installation in the corridor. At the same time, it is precisely because of the influence of the corridor structure itself that in the limited space of the corridor, if a lens group is added, on the one hand, after many simulation experiments, it is found that the effect of a lens on fluorescence is limited, and multiple lenses are needed to improve the collection efficiency, which will make the installation cumbersome and complicated; on the other hand, according to the simulation calculation results, even if it is theoretically feasible to add a lens group, it is actually impossible to find a corresponding usable lens group. Even if there is a usable lens group, the structure of the corridor will make it impossible to install the lens; thirdly, even if the lens assembly 31 is adjusted accordingly to install a feasible lens in the corridor, it may also cause multiple problems such as premature loss of the fluorescent beam. Therefore, in general, the truncated cone mirror 32 not only has a significant ability to improve the collection efficiency, but also its preparation and installation are relatively simple, and it can maximize the overall collection efficiency in the simplest way. In particular, the size of the truncated cone mirror 32 can be changed according to the length of the corridor, and further adapted to the lens assembly 31, so as to adapt to a variety of collection devices. More particularly, the truncated cone mirror 32 is also particularly suitable for longer corridors. Further, by improving the fluorescence collection efficiency of microscopic imaging, the imaging depth and signal-to-noise ratio of the fluorescence microscopic imaging system can be improved at the same imaging laser power, and the sample can be imaged at a lower power on the fluorescence microscopic imaging system to reduce photobleaching and phototoxicity.

[0066] Specifically, the side surface 323 is coated with a high-reflection film, which can reflect the fluorescence incident thereon, thereby allowing the fluorescence to reach the second end surface 322. Preferably, the high-reflection band of the high-reflection film is 300nm-750nm, so that the fluorescence collection device 1 can be adapted to different application scenarios.

[0067] In a specific embodiment of the present invention, the first end face 321 is located at the back focal plane of the lens assembly 31, and the diameter of the first end face 321 is greater than or equal to the diameter of the fluorescent beam passing through the lens assembly 31. Through this arrangement, the first end face can collect as much fluorescent beam as possible from the lens assembly 31. Specifically, the back focal plane of the lens assembly 31 refers to the virtual plane in the lens system where the light emitted from the lens converges or appears to converge at a point after passing through the lens. Further, there may be 2 lenses, 3 lenses, or more lenses in the lens assembly 31. More specifically, when there are 3 lenses in the lens assembly 31, the back focal plane of the lens assembly 31 refers to the back focal plane of the equivalent lens of the two lenses arranged at the back along the propagation direction of the fluorescent beam.

[0068] In a specific embodiment of the present invention, Figure 1 and Figure 2 As shown, the photon detector 33 includes a photosensitive surface 331, which senses the fluorescent beam transmitted from the second end surface 322. The photosensitive surface 331 is rectangular or circular, and the width of the rectangle or the diameter of the circle is greater than or equal to the diameter of the second end surface 322. That is, when the photosensitive surface 331 is rectangular, the shorter side of the rectangle is greater than or equal to the diameter of the second end surface 322; when the photosensitive surface 331 is circular, the diameter of the circle is greater than or equal to the diameter of the second end surface 322. Through this arrangement, when the photosensitive surface 331 has different shapes, it can also ensure that the photon detector 33 can detect as many fluorescent beams as possible.

[0069] In a specific embodiment of the present invention, Figure 1 and Figure 2 As shown, along the first direction, a detector protection window 34 is provided between the truncated cone mirror 32 and the photosensitive surface 331. The fluorescent beam passes through the detector protection window 34 from the second end surface 322 to the photosensitive surface 331. The distance between the rear surface of the detector protection window 34 and the photosensitive surface 331 is not greater than 2 mm. More specifically, the distance between the rear surface of the detector protection window 34 and the photosensitive surface 331 is not greater than 2 mm and not less than 0.1 mm. The distance between the front surface of the detector protection window 34 and the second end surface 322 is not greater than 1 mm. More specifically, the distance between the front surface of the detector protection window 34 and the second end surface 322 is not greater than 1 mm and not less than 0.1 mm. The detector protection window 34 can protect the photosensitive surface 331, and under the setting of the distance between the detector protection window 34 and the second end surface 322 and the photosensitive surface 331, the influence of the detector protection window 34 on the collection of the fluorescent beam can be minimized. Specifically, the detector protection window 34 is a light-transmitting medium, including quartz glass and organic glass.

[0070] In a specific embodiment of the present invention, along the first direction, the length of the truncated cone mirror 32 along the first direction is 7-9 mm, the radius of the first end face 321 is 2.5-3 mm, and the radius of the second end face 322 is 2-2.5 mm. In the case of a long corridor, the length of the truncated cone mirror 32 along the first direction is 7-9 mm, which can further reduce the influence of the long corridor on the intensity of the fluorescence beam and reduce the loss of fluorescence. Combined with the setting of the diameters of the first end face 321 and the second end face 322, the photon detector 33 is more efficient in capturing the fluorescence signal of the fluorescence beam.

[0071] In a specific embodiment of the present invention, Figure 1-Figure 3 As shown, the lens assembly 31 includes a first lens 311, a second lens 312 and a third lens 313 arranged in sequence along a first direction, wherein the diameter of the first lens 311 is 38-50.8 mm, the focal length of the first lens 311 is 75 mm, the diameter of the second lens 312 is 30-50.8 mm, the focal length of the second lens 312 is 39-40 mm, the diameter of the third lens 313 is 25-30 mm, and the focal length of the third lens 313 is 15-16.5 mm. The arrangement of the lens assembly 31 can collect fluorescence more efficiently in a limited space of the fluorescence collection device 1. Furthermore, the second lens 312 and the third lens 313 are spaced 1 mm apart.

[0072] In a specific embodiment of the present invention, Figure 1 and Figure 2 As shown, the fluorescence transmission and collection component 30 also includes a truncated cone mirror fixed window piece 35 located between the truncated cone mirror 32 and the third lens 313 along the first direction, the first end face 321 is fixedly mounted on the truncated cone mirror fixed window piece 35, and the spacing between the third lens 313 and the truncated cone mirror fixed window piece 35 is 0.9-1mm. The truncated cone mirror fixed window piece 35 can protect the truncated cone mirror 32, and the truncated cone mirror 32 can be installed and fixed in the corridor more conveniently. At the same time, the spacing between the third lens 313 and the truncated cone mirror fixed window piece 35 is ensured to be 0.9-1mm, so that the influence of the truncated cone mirror fixed window piece 35 on the collection of the fluorescence beam can be minimized. Specifically, the truncated cone mirror fixed window piece 35 is coaxially bonded to the truncated cone mirror 32. More specifically, the truncated cone mirror fixed window piece 35 is a light-transmitting medium, including quartz glass and organic glass. The truncated cone mirror fixed window piece 35 and the truncated cone mirror 32 can be composite components. When installing, for example, the original window in the device can be removed, and the truncated cone mirror fixed window piece 35 and the truncated cone mirror 32 can be placed in the device. Specifically, the installation position of the truncated cone mirror fixed window piece 35 is the original incident window position. When installing, the original incident window can be removed, and then the truncated cone mirror fixed window piece 35 and the truncated cone mirror 32 can be installed. Further, if Figure 1As shown, a passage 25 is provided between the truncated cone mirror fixed window piece 35 and the photosensitive surface 331, a truncated cone mirror 32 is installed in the passage 25, and the installation position P of the truncated cone mirror fixed window piece 35 is the original incident window position.

[0073] Through the coordination of the optical component objective lens 10, lens assembly 31, truncated cone mirror 32, and photon detector 33, specifically, through the adaptation of the objective lens 10, the first lens 311, the second lens 312, the third lens 313, the truncated cone mirror fixed window 35, the truncated cone mirror 32, the detector protection window 34, and the photosensitive surface 331, the characteristics of the optical components are adjusted, and the coordination and coordination between the components are taken into account to build a stable optical system, which effectively improves the fluorescence collection efficiency of the microscopic imaging system. In particular, in the case of a long corridor and large-angle incident fluorescence, the improvement of fluorescence collection efficiency is more obvious.

[0074] In a specific embodiment of the present invention, Figure 1 and Figure 2 As shown, the fluorescence transmission and collection assembly 30 is further provided with a dichroic mirror assembly 36. Along the first direction, the dichroic mirror assembly 36 is located before the first lens 311 or between the first lens 311 and the second lens 312. Figure 1 , along the propagation direction of the fluorescent beam, the dichroic mirror assembly 36 is located before the first lens 311, and the fluorescent beam first reaches the dichroic mirror assembly 36 and then reaches the first lens 311. Or, Figure 2 , along the propagation direction of the fluorescent beam, the dichroic mirror assembly 36 is located between the first lens 311 and the second lens 312, and the fluorescent beam first reaches the first lens 311, then reaches the dichroic mirror assembly 36, and then reaches the second lens 312. Specifically, the dichroic mirror assembly 36 is composed of one, two, three or more dichroic mirrors, which can be adjusted according to actual conditions.

[0075] In a specific embodiment of the present invention, Figure 1-Figure 3 As shown, the objective lens 10 is coaxially arranged with the fluorescence collection channel 20, and the back aperture of the objective lens 10 is greater than or equal to 20 mm. That is, the objective lens 10, the lens assembly 31, the truncated cone mirror 32, the photon detector 33, the detector protection window 34, and the truncated cone mirror fixed window 35 are all coaxially arranged.

[0076] Preferably, the dichroic mirror assembly 36 is a dichroic mirror placed between the objective lens 10 and the first lens 311, and is used to separate the fluorescence excitation wavelength and the stimulated fluorescence wavelength. It can transmit the stimulated fluorescence wavelength and reflect the incident laser wavelength of the microscope system used to excite the fluorescence, or it can reflect the stimulated fluorescence wavelength and transmit the incident laser wavelength of the microscope system used to excite the fluorescence. Alternatively, in another specific embodiment of the present invention, the dichroic mirror close to the objective lens 10 in the dichroic mirror assembly 36 composed of multiple dichroic mirrors is used to separate the fluorescence excitation wavelength and the stimulated fluorescence wavelength, and the other dichroic mirrors can be placed between the objective lens 10 and the first lens 311, or can be placed between the lenses in the lens assembly 31, and are used to separate stimulated fluorescence of different wavelengths, so that the stimulated fluorescence of the target wavelength is collected by the target channel in the multi-channel fluorescence collection system.

[0077] In a specific embodiment of the present invention, Figure 1 and Figure 2 As shown, the fluorescence transmission and collection component 30 is also provided with a filter component 37. Along the first direction, that is, along the propagation path of the fluorescence beam, the filter component 37 is located after the dichroic mirror component 36, and is used to further filter out stray light other than the stimulated fluorescence of the target wavelength, so that only the stimulated fluorescence of the target wavelength enters the photon detector 33. Specifically, the filter component 37 includes a filter, and can also be composed of two, three or even more filters. More specifically, the filter component 37 including two or more filters also includes a filter rotation fixing frame (not shown in the figure), and the filter rotation fixing frame is composed of a plurality of rotatable filter fixing slots. The selected filter can be rotated into the fluorescence collection light path and fixed coaxially with other optical elements in the fluorescence collection light path according to the needs, and the unnecessary filter can be rotated out of the fluorescence collection light path by rotating the fixing slot.

[0078] Specifically, the filter in the filter assembly 37 is placed between the lens assembly 31 and the truncated cone mirror 32, or between the lenses in the lens assembly 31. Specifically, in a filter assembly comprising two or more filters, the filters can be placed adjacent to each other or interspersed non-adjacently. Preferably, the filter in the filter assembly 37 is a bandpass filter, and narrow bandpass filters such as 438±10nm, 532±10nm that only transmit the stimulated fluorescence wavelength of a specific fluorescent indicator can be selected, or bandpass filters that can transmit the stimulated fluorescence wavelengths of multiple fluorescent indicators can be selected, such as a bandpass filter with a transmission light band of 300nm-750nm.

[0079] Specifically, the lens assembly 31 and the filter assembly 37 may include a reflector to meet the design requirements of different optical paths, and the lens assembly 31 and the filter assembly 37 are placed on the same optical axis.

[0080] In a specific embodiment of the present invention, Figure 1 and Figure 2 As shown, the fluorescence collection channel 20 includes:

[0081] The optical element fixing frame 21 includes a large fixing frame 211 and a small fixing frame 212. The large fixing frame 211 is used to embed the objective lens 10, the first lens 311 and the dichroic mirror assembly 36, and has two openings opposite to each other along the first direction; the small fixing frame 212 is used to embed the second lens 312, the third lens 313 and the frustum mirror fixing window piece 35, and has two openings opposite to each other along the first direction. Since the frustum mirror fixing window piece 35 is fixedly connected to the frustum mirror 32, the frustum mirror 32 is also fixedly installed in the channel;

[0082] The adapter fixing frame 22, one end opening of the large fixing frame 211 and one end opening of the small fixing frame 212 are respectively inserted into the adapter fixing frame 22, so that the large fixing frame 211 and the small fixing frame 212 are coaxially installed. That is to say, due to the difference in size of the fixed optical elements, the size of the large fixing frame 211 and the small fixing frame 212, such as the size of the caliber, are different. In order to stably connect them, the larger large fixing frame 211 and the smaller small fixing frame 212 can be connected through the adapter fixing frame 22. Furthermore, the filter assembly 37 can be installed on the adapter fixing frame 22;

[0083] The detector fixing frame 23 has an opening at one end and an end surface opposite to each other along the first direction. The opening at one end of the detector fixing frame 23 abuts against the frustum mirror fixing window piece 35 , and the detector protection window piece 34 is embedded in the detector fixing frame 23 .

[0084] More specifically, if Figure 1 As shown, the truncated cone mirror fixed window piece 35 abuts against and is fixedly connected to an opening at one end of the detector fixing frame 23. Alternatively, in another embodiment of the present invention, as Figure 2 As shown, the frustum mirror fixing window piece 35 can be directly embedded in the detector fixing frame 23 .

[0085] In each embodiment of the present invention, Figure 2 As shown, the distance between the opening at one end of the detector fixing frame 23 and the detector protection window piece 34 is the length of the corridor, the length of the truncated cone mirror 32 is less than or equal to the length of the corridor, and the opening at one end of the detector fixing frame 23 is in contact with the truncated cone mirror fixing window piece 35. That is, the opening at one end of the detector fixing frame 23 is in direct contact with the truncated cone mirror fixing window piece 35.

[0086] In a specific embodiment of the present invention, Figure 1As shown, the distance between the opening at one end of the detector fixing frame 23 and the detector protection window sheet 34 is the length of the corridor, the length of the truncated cone mirror 32 is greater than the length of the corridor, and a gasket 26 is provided between the opening at one end of the detector fixing frame 23 and the truncated cone mirror fixing window sheet 35. That is, the opening at one end of the detector fixing frame 23 is not directly in contact with the truncated cone mirror fixing window sheet 35. Since the truncated cone mirror 32 is longer, the gasket 26 is used for installation, and the opening at one end of the detector fixing frame 23 and the truncated cone mirror fixing window sheet 35 are indirectly connected through the gasket. Specifically, the gasket 26 has a hollow circle, the diameter of which is smaller than the diameter of the truncated cone mirror fixing window sheet 35 and larger than the diameter of the first end face 321, the truncated cone mirror 32 passes through the hollow circle of the gasket 26 and does not directly contact the gasket 26, and the thickness of the gasket 26 is greater than or equal to the difference between the length of the truncated cone mirror 32 and the length of the corridor.

[0087] Furthermore, if Figure 1 and Figure 2 As shown, the large fixing frame 211 includes an optical element fixing frame 2111 and a fixing frame coaxial connecting rod and a fastener 2112, and multiple optical element fixing frames 2111 are connected to the fastener 2112 through the fixing frame coaxial connecting rod, and the optical elements are coaxially installed; the small fixing frame 212 includes an optical element fixing frame 2121 and a fixing frame coaxial connecting rod and a fastener 2122, and multiple optical element fixing frames 2121 are connected to the fastener 2122 through the fixing frame coaxial connecting rod, and the optical elements are coaxially installed. Each fixing frame does not block the propagation of the fluorescent beam.

[0088] In a specific embodiment of the present invention, Figure 1 and Figure 2 As shown, the fluorescence collection channel 20 is also provided with a shading component 24, which is located outside the objective lens 10 and the fluorescence transmission and collection component 30, and is fixed to the outside of each fixing frame and completely covers it, so as to block external stray light. Preferably, the shading component 24 is composed of a non-transparent metal medium such as aluminum and stainless steel, and the surface is oxidized and blackened. The oxidation treatment is used to reduce the conductivity of the shading component 24 and improve the safety of the system. The blackening treatment is used to reduce the reflection and scattering of light by the shading component 24. More preferably, the shading component 24 is composed of a black high-strength non-metallic medium, which is not conductive, can withstand high temperatures of 350°C, and is not easily deformed by heat.

[0089] In a specific embodiment of the present invention, Figure 1 and Figure 2 The fluorescence collecting device 1 includes a single fluorescence collecting channel 20 , and the objective lens 10 is coaxially arranged with the fluorescence collecting channel 20 .

[0090] In a specific embodiment of the present invention, the fluorescence collection device 1 includes a plurality of fluorescence collection channels, such as Figure 4As shown in FIG. 1 , the fluorescence collection device 1 includes a first fluorescence collection channel 20 and a second fluorescence collection channel 20′. Specifically, a fluorescence transmission collection component 30 is provided in the first fluorescence collection channel 20, and the fluorescence transmission collection component 30 includes a first direction (eg, Figure 4 The dichroic mirror assembly 36, the filter assembly 37, the lens assembly 31, the truncated cone fixed window 35, the truncated cone 32, the detector protection window 34, and the photon detector 33 are sequentially arranged in the direction A shown in FIG. More specifically, the dichroic mirror assembly 36 is located before the first lens 311, and the dichroic mirror assembly 36 includes a first dichroic mirror 361 and a second dichroic mirror 362. The second fluorescence collection channel 20' is provided with a fluorescence transmission collection assembly 30', and the fluorescence transmission collection assembly 30' includes a fluorescence transmission collection assembly 30' along the second direction (as shown in FIG. Figure 4 In the direction B) shown in FIG. 1 , a dichroic mirror assembly 36', a filter assembly 37', a lens assembly 31', a truncated mirror fixed window 35', a truncated mirror 32', a detector protection window 34', and a photon detector 33' are sequentially arranged. More specifically, the dichroic mirror assembly 36' is located before the first lens 311', and the dichroic mirror assembly 36' includes a second dichroic mirror 362. That is, the first fluorescence collection channel 20 and the second fluorescence collection channel 20' share the second dichroic mirror 362, and the fluorescence beam reflected by the first dichroic mirror 361 is divided into two fluorescence collection channels of different wavelength bands by the second dichroic mirror 362, that is, after passing through the second dichroic mirror 362, the transmitted fluorescence beam is collected by the first fluorescence collection channel 20, and the fluorescence beam reflected by the second dichroic mirror 362 is collected by the second fluorescence collection channel 20', and the second direction is perpendicular to the first direction. Through the multi-channel setting, fluorescence signal collection can be performed more efficiently and conveniently.

[0091] In a specific embodiment of the present invention, when the angle between the fluorescent beam and the optical axis of the fluorescent collection channel is 2.0°-8.0°, the collection efficiency of the fluorescent collection device is 69.78%-98.64%. The collection efficiency of non-parallel incident fluorescent beams, especially large-angle fluorescent beams, is greatly improved.

[0092] A second aspect of the present invention discloses a fluorescence microscope, comprising the above-mentioned fluorescence collecting device.

[0093] The following will describe based on more specific implementation methods.

[0094] Embodiment 1:

[0095] Embodiment 1 is a fluorescence collection device with a single fluorescence collection channel.

[0096] Structural reference of Example 1 Figure 1It includes an objective lens 10 and a fluorescence collecting channel 20 coaxially placed along a first direction. A fluorescence transmission and collection component 30 is provided in the fluorescence collection channel 20. The fluorescence transmission and collection component 30 is composed of a dichroic mirror component 36, a first lens 311, a filter component 37, a second lens 312, a third lens 313, a truncated cone mirror fixed window 35, a truncated cone mirror 32, a detector protection window 34, and a photon detector 33, which are sequentially placed along the first direction.

[0097] The first lens 311, the second lens 312, and the third lens 313 are respectively a LA1386-A lens with a focal length of 75 mm, a LA1274-A lens with a focal length of 40 mm, and an ACL25416U-A aspherical lens with a focal length of 16 mm from the Thorlabs brand. The second lens 312 and the third lens 313 are spaced 1 mm apart and can be combined to be equivalent to a lens with a smaller focal length, which serves to further reduce the incident light spot.

[0098] The back aperture of the objective lens 10 coincides with the front focal plane of the first lens 311 , the back focal plane of the first lens 311 coincides with the front focal plane of the second lens 312 , and the equivalent lens back focal planes of the second lens 312 and the third lens 313 coincide with the first end face 321 of the frustum mirror 32 .

[0099] The dichroic mirror assembly 36 uses a Semrock FF725-SDi01-25x36x3.5 dichroic mirror, which transmits 430nm to 700nm and reflects 750nm to 1140nm. Its function is to separate two-photon excitation light and stimulated fluorescence. The fluorescence will pass through the dichroic mirror and enter the lens assembly 31.

[0100] The filter assembly 37 is a Semrock FF01-593 / 46 filter, which allows only red stimulated fluorescence in the target wavelength band to enter the photon detector.

[0101] The diameter of the first end face 321 of the truncated cone mirror 32 is 5.85 mm, and the diameter of the second end face 322 is 4.45 mm. The length of the truncated cone mirror 32 along the first direction is 8.5 mm. When setting the truncated cone mirror 32, the Hamamatsu H7422 series of photomultiplier tubes is used as the basis. The length of the channel between the incident window and the photocathode front window is about 7.4 mm. The 8.5 mm length of the truncated cone mirror is greater than the 7.4 mm long channel of the Hamamatsu H7422 photomultiplier tube. Therefore, a gasket is required when fixing the truncated cone mirror fixing window plate 35 and the truncated cone mirror 32. There is an original glass window (incident window) on the outside of the photomultiplier tube channel. In order to make the truncated cone mirror output end as close to the PMT photocathode front window as possible, it is necessary to first remove the original glass window outside the channel as a whole, and in this process, prevent dust, debris, etc. from entering the channel. The truncated cone mirror fixed window piece 35 is coaxially bonded to the truncated cone mirror 32, and the truncated cone mirror fixed window piece 35 is coaxially bonded to a hollow annular gasket with a thickness of 1.3 mm. After the annular gasket is coaxially bonded to the detector fixing frame 23, the truncated cone mirror is coaxial with the PMT photocathode, and the distance between the front surface of the detector protection window piece 34 and the second end surface 322 of the truncated cone mirror 32 is 0.2 mm. The truncated cone mirror fixed window piece 35 is made of glass. For the Hamamatsu H7422 photomultiplier tube, the position where the annular gasket is bonded is the position where the original glass window outside the PMT channel is located.

[0102] The photon detector photosensitive surface 331 of the Hamamatsu H7422 photomultiplier tube is square in shape, with a side length of 5 mm, which is larger than the diameter of the second end surface 322. The detector protection window 34 is placed in front of the photosensitive surface 331 and fixed on the detector fixing frame 23. The detector protection window 34 is made of glass, and its front surface is about 1.5 mm away from the photosensitive surface 331.

[0103] Comparative Example 1:

[0104] Comparative Example 1 is a traditional fluorescence collection device without a truncated cone mirror. The 8.5 mm long truncated cone mirror is removed, and the equivalent rear focal planes of the second lens 312 and the third lens 313 coincide with the plane where the corridor entrance is located. The Hamamatsu H7422 original incident window piece is tightly attached to the corridor entrance and has a thickness of 1 mm, that is, the equivalent rear focal planes of the second lens 312 and the third lens 313 coincide with the rear surface of the original window piece. Other settings remain unchanged, and the corridor length remains 7.4 mm.

[0105] The configurations of Comparative Example 1 and Example 1 were simulated respectively, fluorescence at different fluorescence angles was collected, and data on collection efficiency were acquired. In the simulation, it was assumed that the thickness of the detector protection window sheet 34 was 1 mm.

[0106] In the simulation, the back aperture of the objective lens with a diameter of 20 mm is used as the fluorescence incident light source. The fluorescence collected by the objective lens enters the fluorescence collection device through the back aperture of the objective lens. The angles of the fluorescence emitted from the back aperture of the objective lens and the optical axis are set to 0°, 1.0°, 2.0°, 3.0°, 4.0°, 4.5°, 5.0°, 5.5°, 6.0°, 6.5°, 7.0°, and 8.0°, respectively. The incident fluorescence intensity at each angle is set to 1W. The Zemax software is used to record the fluorescence intensity at the angle received by the 5 mm square photon detector under ideal conditions, so as to calculate the fluorescence collection efficiency at different angles.

[0107] Among them, the fluorescence collection efficiency at different angles obtained by the configuration simulation of Example 1 is as follows: Figure 5 It can be seen that when the angle between the incident fluorescence and the optical axis is within 5.5°, the fluorescence collection efficiency at all angles is above 86%, and when the angle between the incident fluorescence and the optical axis is 8°, the fluorescence collection efficiency is still 69.78%.

[0108] The fluorescence collection efficiency at different angles obtained by the configuration simulation of Comparative Example 1 is as follows: Figure 6 As shown, it can be seen that when the angle between the incident fluorescence and the optical axis is 3.0°, the fluorescence collection efficiency is as low as 50.01%, the collection efficiency is less than 2% when the angle is 5°, and the fluorescence at an angle above 5.5° can no longer be collected.

[0109] Figure 7 The scatter plots of the collection efficiency of the fluorescence incident at different angles for Example 1 and Comparative Example 1 are shown in FIG. Figure 7 It can be seen that the collection efficiency of the two devices for the fluorescence incident along the optical axis is 100%, but Example 1 has a better collection effect for the fluorescence incident at a large angle.

[0110] Embodiment 2:

[0111] Embodiment 2 is a fluorescence collection device with two fluorescence collection channels, which is used to divide the stimulated fluorescence into a red fluorescence collection channel and a green fluorescence collection channel for dual-channel collection.

[0112] The specific structure of Example 2 is shown in FIG. Figure 4 The fluorescence collection device 1 has a first fluorescence collection channel 20 and a second fluorescence collection channel 20'. The first fluorescence collection channel 20 is provided with a fluorescence transmission collection component 30. The fluorescence transmission collection component 30 is formed along a first direction (such as Figure 4The second fluorescence collection channel 20' is provided with a fluorescence transmission collection component 30', which is composed of a first dichroic mirror 361, a second dichroic mirror 362, a filter assembly 37, a first lens 311, a second lens 312, a third lens 313, a truncated cone mirror fixed window 35, a truncated cone mirror 32, a detector protection window 34, and a photon detector 33. ... Figure 4 The optical filter assembly 37', the first lens 311', the second lens 312', the third lens 313', the truncated cone mirror fixed window 35', the truncated cone mirror 32', the detector protection window 34', and the photon detector 33' are arranged in sequence in the direction B shown in FIG.

[0113] After the stimulated fluorescence passes through the objective lens 10, it is reflected by the first dichroic mirror 361. The first dichroic mirror 361 adopts Semrock FF735-Di02-25x36, transmits 750nm~1600nm, and reflects 350nm~720nm. Its function is to separate the two-photon excitation light and the stimulated fluorescence. The fluorescence beam reflected by the first dichroic mirror 361 is divided into two fluorescence collection channels of different wavelengths (the first fluorescence collection channel 20 and the second fluorescence collection channel 20') by the second dichroic mirror 362. The second dichroic mirror 362 adopts Semrock FF552-Di02-25x36, transmits red fluorescence and reflects green fluorescence, so that the collected red fluorescence and green fluorescence beams are collected by the red fluorescence collection light path (the first fluorescence collection channel 20) and the green fluorescence collection light path (the second fluorescence collection channel 20'). The filter assembly 37 uses Semrock FF01-593 / 46-25, and the filter assembly 37 ′ uses Semrock FF01-520 / 35-25 to filter out other fluorescence wavelengths and stray light outside the target fluorescence band.

[0114] Although the present invention has been illustrated and described with reference to certain preferred embodiments of the present invention, it should be understood by those skilled in the art that the above is a further detailed description of the present invention in conjunction with specific embodiments, and it cannot be determined that the specific implementation of the present invention is limited to these descriptions. Those skilled in the art may make various changes in form and details, including making several simple deductions or substitutions, without departing from the spirit and scope of the present invention.

Claims

1. A fluorescence collection device, characterized in that: include: an objective lens, for receiving the fluorescent beam and allowing the fluorescent beam to pass therethrough; A fluorescence collection channel extending along a first direction; A fluorescence transmission and collection component is arranged in the fluorescence collection channel, and is used to transmit and collect the fluorescence beam from the objective lens, and includes: A lens assembly comprising at least two lenses; A truncated cone mirror, comprising a first end face and a second end face arranged opposite to each other along the first direction, and a side face located between the first end face and the second end face in the first direction, wherein the diameter of the first end face is greater than the diameter of the second end face, wherein the fluorescent beam transmitted from the lens assembly enters the truncated cone mirror from the first end face and passes through the second end face, and the side face reflects the fluorescent beam reaching the side face to the second end face; a photon detector, configured to receive the fluorescent beam transmitted from the second end surface and convert the fluorescent beam into an electrical signal; The first direction is parallel to the optical axis of the fluorescence transmission and collection component.

2. A fluorescence collection device as claimed in claim 1, characterized in that: The first end surface is located at the rear focal plane of the lens assembly, and the diameter of the first end surface is greater than or equal to the diameter of the fluorescent beam passing through the lens assembly.

3. A fluorescence collection device as claimed in claim 1, characterized in that: The photon detector comprises a photosensitive surface, which senses the fluorescent beam transmitted from the second end surface. The photosensitive surface is rectangular or circular, and the width of the rectangle or the diameter of the circle is greater than or equal to the diameter of the second end surface.

4. A fluorescence collection device as claimed in claim 3, characterized in that: Along the first direction, a detector protection window is provided between the truncated cone mirror and the photosensitive surface, and the fluorescent beam passes through the detector protection window from the second end face to reach the photosensitive surface. The distance between the rear surface of the detector protection window and the photosensitive surface is not greater than 2 mm and not less than 0.1 mm, and the distance between the front surface of the detector protection window and the second end face is not greater than 1 mm and not less than 0.1 mm.

5. A fluorescence collection device as claimed in claim 4, characterized in that: The lens assembly includes a first lens, a second lens and a third lens arranged in sequence along a first direction, the diameter of the first lens is 38-50.8 mm, the focal length of the first lens is 75 mm, the diameter of the second lens is 30-50.8 mm, the focal length of the second lens is 39-40 mm, the diameter of the third lens is 25-30 mm, and the focal length of the third lens is 15-16.5 mm.

6. A fluorescence collection device as claimed in claim 5, characterized in that: The fluorescence transmission and collection assembly also includes a truncated cone mirror fixed window located between the truncated cone mirror and the lens assembly along the first direction, the first end face is fixedly mounted on the truncated cone mirror fixed window, and the spacing between the third lens and the truncated cone mirror fixed window is 0.9-1mm.

7. A fluorescence collection device as claimed in claim 6, characterized in that: Along the first direction, the distance between the frustum mirror fixed window plate and the photosensitive surface is 7-10 mm, the length of the frustum mirror along the first direction is 7-9 mm, the radius of the first end face is 2.5-3 mm, and the radius of the second end face is 2-2.5 mm.

8. A fluorescence collection device as claimed in claim 5, characterized in that: A dichroic mirror assembly is also provided in the fluorescence transmission and collection assembly. Along the first direction, the dichroic mirror assembly is located before the first lens or between the first lens and the second lens.

9. A fluorescence collection device as claimed in claim 8, characterized in that: The objective lens is coaxially arranged with the fluorescence collecting channel.

10. A fluorescence collection device as claimed in claim 9, characterized in that: The fluorescence collection channel comprises: Optics holder, including: A large fixing frame, used for embedding the objective lens, the first lens and the dichroic mirror assembly, having two openings opposite to each other along the first direction; A small fixing frame, used for embedding the second lens, the third lens and the frustum mirror fixing window sheet, having two openings opposite to each other along the first direction; A transfer fixing frame, wherein an opening at one end of the large fixing frame and an opening at one end of the small fixing frame are respectively inserted into the transfer fixing frame, so that the large fixing frame and the small fixing frame are coaxially installed; The detector fixing frame has an opening at one end and an end surface opposite to each other along the first direction. The opening at one end of the detector fixing frame abuts against the frustum mirror fixing window sheet, and the detector protection window sheet is embedded in the detector fixing frame.

11. A fluorescence collection device as claimed in claim 10, characterized in that: The distance between the opening at one end of the detector fixing frame and the detector protection window piece is the corridor length, the length of the frustum mirror is less than or equal to the corridor length, and the opening at one end of the detector fixing frame contacts the frustum mirror fixing window piece.

12. A fluorescence collection device as claimed in claim 10, characterized in that: The distance between the opening at one end of the detector fixing frame and the detector protection window piece is the corridor length, the length of the frustum mirror is greater than the corridor length, and a gasket is provided between the opening at one end of the detector fixing frame and the frustum mirror fixing window piece.

13. A fluorescence collection device as claimed in claim 8, characterized in that: The fluorescence collection channel is a first fluorescence collection channel, the dichroic mirror assembly is located before the first lens, the dichroic mirror assembly includes a first dichroic mirror and a second dichroic mirror, the fluorescence collection device also includes a second fluorescence collection channel extending along a second direction, the fluorescence beam transmitted through the second dichroic mirror is collected by the first fluorescence collection channel, the fluorescence beam reflected through the second dichroic mirror is collected by the second fluorescence collection channel, and the second direction is perpendicular to the first direction.

14. A fluorescence collection device as claimed in claim 1, characterized in that: When the angle between the fluorescence beam and the optical axis of the fluorescence collection channel is 2.0°-8.0°, the collection efficiency of the fluorescence collection device is 69.78%-98.64%.

15. A fluorescence microscope, characterized in that: Comprising a fluorescence collection device as described in any one of claims 1-14.

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