A fluorescence collection device and fluorescence microscope

Abstract

The application discloses a fluorescence collection device, comprising: an objective lens, receiving a fluorescence beam and making it pass through; a fluorescence collection channel, extending along a first direction; a fluorescence transmission and collection assembly, arranged in the fluorescence collection channel, transmitting and collecting the fluorescence beam from the objective lens, comprising, in sequence along the first direction in the fluorescence collection channel: a lens assembly, comprising at least two lenses; a circular truncated cone mirror, comprising a first end face and a second end face oppositely arranged along the first direction, and a side face between the first end face and the second end face in the first direction, the first end face having a larger diameter than the second end face, the fluorescence beam from the lens assembly entering the circular truncated cone mirror from the first end face and passing out from the second end face, and the side face reflecting the fluorescence beam reaching the side face to the second end face; and a photon detector, receiving the fluorescence beam from the second end face and converting the fluorescence beam into an electrical signal. The fluorescence collection device has high collection efficiency. The application also discloses a fluorescence microscope.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of optical imaging technology, in particular to a fluorescence collection device and a fluorescence microscope. BACKGROUND

[0002] A fluorescence microscope uses laser of certain wavelength to excite fluorescent substance of sample under microscope, so that the fluorescent substance emits fluorescence, and the state of the observed sample is determined according to the color and brightness of the collected fluorescence. The fluorescence microscope has high sensitivity and high resolution, and can realize real-time observation of the internal structure, function and activity process of cells and tissues by marking specific molecules or microstructures in biological samples, and has a wide application in the fields of cell biology, neuroscience, biomedical science, etc., and provides an important technical support for life science research.

[0003] Improvement of stimulated fluorescence collection efficiency is crucial for the performance improvement of fluorescence microscope. Improving fluorescence collection efficiency can increase the intensity of fluorescence signal collected from the sample under the same excitation light intensity and microscopic observation state, which helps to improve the signal-to-noise ratio of imaging, making the imaging clearer and more accurate. In addition, improvement of fluorescence collection efficiency can reduce the required excitation light power while maintaining the imaging quality, thereby reducing the light damage to the sample. Improvement of fluorescence collection efficiency also means that weaker fluorescence signals can be collected, so the imaging depth and fluorescence signal detection limit of the system can be improved. In practical applications, brain scientists and neuroscientists can use multi-photon fluorescence microscopes with high fluorescence collection efficiency to reduce the light damage to the observed brain of living animals, prolong the imaging time of living animals, and at the same time, observe the deeper neural activity of the brain of living animals.

[0004] In the fluorescence collection system of the fluorescence microscope, the window and the sensor / detector are the key components of the used photoelectric detector. The main function of the window is to protect the detector from external pollution while allowing the fluorescence signal to pass through. The sensor / detector is an electronic device for detecting and recording fluorescence signals, such as photomultiplier tube (PMT) or camera, etc. photonic detection and photoelectric conversion device. The PMT has a photocathode photosurface, which generates electrons through the dynode, collector, etc. to form a current pulse signal and finally converts it into a digital signal output when the photon hits the photocathode.

[0005] The existing fluorescence microscopy system usually converges fluorescence to the incident window of a photon sensor, and considers that the fluorescence reaching the incident window means reaching the photosensitive surface of the sensor. However, in fact, there is usually a distance between the incident window and the sensor, and the space on the distance can be called a "channel", that is, the fluorescence passes through the incident window and then passes through the channel to reach the photosensitive surface of the photocathode. In some systems, the length of the channel can even reach 8 mm. In this case, since the fluorescence beam entering the fluorescence collection system can include parallel incident and non-parallel incident fluorescence, for some non-parallel incident fluorescence, the existence of the channel will inevitably reduce the fluorescence intensity detected by the fluorescence, thereby affecting the overall collection efficiency of the fluorescence. The greater the incident angle, the more obvious the influence of the channel. Especially in the case of a long channel, the fluorescence propagates a long distance, and more fluorescence is affected, and the fluorescence collection efficiency is lower. Therefore, the fluorescence reaching the incident window cannot be directly equated with the fluorescence reaching the photocathode, especially in the case of a long channel and non-parallel incident fluorescence, the influence of the channel on the fluorescence collection efficiency needs to be further considered. SUMMARY

[0006] To solve the above technical problems, the embodiments of the present application disclose a fluorescence collection device, comprising:

[0007] an objective lens for receiving the fluorescence beam and passing the fluorescence beam;

[0008] a fluorescence collection channel extending in a first direction;

[0009] a fluorescence transmission and collection assembly arranged in the fluorescence collection channel and configured to transmit and collect the fluorescence beam from the objective lens, comprising, in the fluorescence collection channel, at least two lenses arranged in the first direction in sequence;

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

[0011] a circular truncated cone mirror comprising a first end face and a second end face arranged oppositely in 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, the fluorescence beam from the lens assembly enters the circular truncated cone mirror from the first end face and passes out from the second end face, and the side face reflects the fluorescence beam reaching the side face to the second end face;

[0012] a photon sensor configured to receive the fluorescence beam from the second end face and convert the fluorescence beam into an electrical signal;

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

[0014] By adopting the technical scheme, the influence of the channel on the non-parallel incident fluorescence can be reduced, the efficiency of the non-parallel incident fluorescence reaching the light-sensing 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 back focal plane position of the lens assembly, and a diameter of the first end surface is greater than or equal to a diameter of the fluorescence beam passing out of the lens assembly.

[0016] Optionally, the photon detector comprises a light-sensing surface that senses the fluorescence beam from the second end surface, and the light-sensing surface is rectangular or circular, and a width of the rectangular or a diameter of the circular is greater than or equal to a diameter of the second end surface.

[0017] Optionally, along the first direction, a detector protection window piece is arranged between the circular truncated cone mirror and the light-sensing surface, the fluorescence beam passes through the detector protection window piece from the second end surface to the light-sensing surface, a distance between a back surface of the detector protection window piece and the light-sensing surface is not greater than 2 mm and not less than 0.1 mm, and a distance between a front surface of the detector protection window piece and the second end surface is not greater than 1 mm and not less than 0.1 mm.

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

[0019] Optionally, the fluorescence transmission and collection assembly further comprises a circular truncated cone mirror fixing window piece located between the circular truncated cone mirror and the third lens along the first direction, and the first end surface is fixedly installed on the circular truncated cone mirror fixing window piece, and a distance between the third lens and the circular truncated cone mirror fixing window piece is 0.9-1 mm.

[0020] Optionally, along the first direction, a distance between the circular truncated cone mirror fixing window piece and the light-sensing surface is 7-10 mm, a length of the circular truncated cone mirror along the first direction is 7-9 mm, a radius of the first end surface is 2.5-3 mm, and a radius of the second end surface is 2-2.5 mm.

[0021] Optionally, a dichroic mirror assembly is further arranged 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 is arranged on the same optical axis as the fluorescence collection channel.

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

[0024] The optical element fixing frame comprises:

[0025] The large fixing frame is used for embedding the objective lens, the first lens and the dichroic mirror assembly, and has two end openings opposite along the first direction.

[0026] The small fixing frame is used for embedding the second lens, the third lens and the fixed window piece of the circular mirror, and has two end openings opposite along the first direction.

[0027] The adapter fixing frame is inserted into the one end opening of the large fixing frame and the one end opening of the small fixing frame respectively, so that the large fixing frame and the small fixing frame are coaxially arranged.

[0028] The detector fixing frame has one end opening and an end surface opposite along the first direction, and the one end opening of the detector fixing frame abuts against the fixed window piece of the circular mirror, and the detector protection window piece is embedded in the detector fixing frame.

[0029] Optionally, the distance between the one end opening of the detector fixing frame and the detector protection window piece is a length of a channel, the length of the circular mirror is less than or equal to the length of the channel, and the one end opening of the detector fixing frame is in contact with the fixed window piece of the circular mirror and abuts against each other.

[0030] Optionally, the distance between the one end opening of the detector fixing frame and the detector protection window piece is a length of a channel, the length of the circular mirror is greater than the length of the channel, and a gasket is arranged between the one end opening of the detector fixing frame and the fixed window piece of the circular mirror.

[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 comprises a first dichroic mirror and a second dichroic mirror, the fluorescence collection device further comprises 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 by 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 included 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 application, the embodiment of the present application discloses a fluorescence microscope comprising the fluorescence collection device.

[0034] By using the above technical scheme, the fluorescence microscope has higher fluorescence collection efficiency, and the imaging performance is improved. BRIEF DESCRIPTION OF DRAWINGS

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

[0036] Figure 2 A structure schematic diagram of a fluorescence collection device according to another embodiment of the present application is shown.

[0037] Figure 3 A structure schematic diagram of a fluorescence transmission collection assembly according to an embodiment of the present application is shown.

[0038] Figure 4 A structure schematic diagram of a fluorescence collection device with double fluorescence collection channels according to an embodiment of the present application is shown.

[0039] Figure 5 A schematic diagram showing the simulation results of the fluorescence beams received at each incident angle and the collection efficiency according to Example 1 of the present application is shown.

[0040] Figure 6 A schematic diagram showing the simulation results of the fluorescence beams received at each incident angle and the collection efficiency according to Comparative Example 1 of the present application is shown.

[0041] Figure 7 A scatter plot showing the collection efficiency of the fluorescence beams with different incident angles according to Example 1 and Comparative Example 1 of the present application is shown.

[0042] 1. A fluorescence collection device,

[0043] 10. An objective lens,

[0044] 20. A fluorescence collection channel, 21. An optical element fixing frame, 211. A large fixing frame, 2111. An optical element fixing frame, 2112.

[0045] A fixing frame coaxial connecting rod and a fastener, 212. A small fixing frame, 2121. An optical element fixing frame, 2122. A fixing frame coaxial connecting rod and a fastener, 22. An adapter fixing frame, 23. A detector fixing frame, 24. A light shielding assembly, 25. A duct, 26. A gasket,

[0046] 30. A fluorescence transmission collection assembly, 31. A lens assembly, 311. A first lens, 312. A second lens, 313. A third lens, 32. A circular table mirror, 321. A first end surface, 322. A second end surface, 323. A side surface, 33. A photon detector, 331. A photosensitive surface, 34.

[0047] a detector guard window 35, a round table mirror fixed window 36, a dichroic mirror assembly 36, a first dichroic mirror 361, a second dichroic mirror 362.

[0048] a second dichroic mirror 37, a filter assembly. DETAILED DESCRIPTION

[0049] The specific embodiments of the present application will be described in the following detailed description with reference to the drawings, but those skilled in the art will readily appreciate that the specific embodiments of the present application are only illustrative of the present application as claimed. The intention is that the present application covers all alternatives, modifications and equivalents falling within the scope of the claims based on the present application. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present application. The present application can be practiced without the specific details. In other instances, well-known methods, procedures, components, and circuits have not been described in detail since not pertinent to the knowledge of the present application. Also, the present application has not been described with the best mode for carrying out the application. It is intended to include all alternatives, modifications and equivalents falling within the scope of the claims based on the present application. It is also noted that like reference numerals refer to like elements throughout the description.

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

[0051] The terms "first", "second", and the like, are used only to distinguish descriptions, and cannot be understood as indicating or implying relative importance.

[0052] In the description of the present embodiments, it should also be noted that unless specifically defined and limited, the terms "set", "connected", "connected", should be understood broadly, for example, it can be fixedly connected, or it can be detachably connected, or integrally connected; it can be mechanically connected, or it can be electrically connected; it can be directly connected, or it can be indirectly connected through an intermediate medium; it can be the communication inside two elements. For those skilled in the art, the specific meaning of the above terms in the present embodiments can be understood according to the specific circumstances.

[0053] In the embodiments of the present application, the "photon detector" is a whole device composed of many components such as a light-sensing and photoelectric conversion element, a shell, a built-in cooling device, a window, etc. In the present application, only part of the structure of the photon detector is shown by the drawing.

[0054] To make the purpose, technical solutions and advantages of the present application clearer, the embodiments of the present application will be further described in detail below with reference to the drawings.

[0055] A first aspect of the present application discloses a fluorescence collection device, such as Figures 1-3 As shown in the drawings, the fluorescence collection device 1 comprises an objective lens 10, a fluorescence collection channel 20, and a fluorescence transmission and collection assembly 30 arranged in the fluorescence collection channel 20.

[0056] The objective lens 10 is used to receive the fluorescence beam and make the fluorescence beam 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, for subsequent collection and processing.

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

[0058] The fluorescence transmission and collection assembly 30 is arranged in the fluorescence collection channel 20, and the fluorescence transmission and collection assembly 30 is used to transmit and collect the fluorescence beam from the objective lens 10. The fluorescence transmission and collection assembly 30 comprises, arranged in the fluorescence collection channel 20 along the first direction in turn:

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

[0060] A circular truncated cone mirror 32, comprising a first end face 321 and a second end face 322 arranged oppositely along the first direction, and a side face 323 located between the first end face 321 and the second end face 322 along the first direction, the diameter of the first end face 321 being greater than the diameter of the second end face 322, the circular truncated cone mirror 32 being in a state of contraction along the first direction, the fluorescence beam from the lens assembly 31 enters the circular truncated cone mirror 32 from the first end face 321 and exits from the second end face 322, and the side face 323 reflects the fluorescence beam reaching the side face 323 to the second end face 322;

[0061] A photon detector 33 for receiving the fluorescence beam from the second end face 322 and converting the fluorescence beam into an electrical signal.

[0062] Specifically, the first direction is as shown by direction A in Figures 1-3 The first direction is parallel to the optical axis of the fluorescence transmission and collection assembly 30, and 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 sequentially passes through the objective lens 10, the lens assembly 31, the circular truncated cone mirror 32, and finally reaches the photosensitive surface 331 of the photon detector 33, and in the circular truncated cone mirror 32, the first end face 321 and the second end face 322 are arranged in turn along the first direction, so that the fluorescence beam enters the circular truncated cone mirror 32 from the first end face 321 and exits the circular truncated cone mirror 32 from the second end face 322.

[0064] In the arrangement of the present application, firstly, the fluorescent light parallel to the first direction is directly from the first end surface 321 to the second end surface 322 after converging via the lens assembly 31, and then to the light sensing surface 331 of the photon detector 33. Secondly, when there is fluorescent light not parallel to the first direction in the fluorescent light beam collected by the objective 10, i.e. there is fluorescent light incident at an angle, especially when the back aperture of the objective 10 is large, more large-angle fluorescent light enters the fluorescent light transfer and collection assembly 30. These non-parallel fluorescent light is first converging by the lens assembly 31, and due to the angle of the fluorescent light itself, they are still in a non-parallel state after converging, and it is difficult to ensure that they will pass out from the second end surface 322, but due to the existence of the side surface 323, the non-parallel fluorescent light can change the propagation direction by the side surface 323, i.e. reflected by the converging circular truncated cone mirror 32, and these fluorescent light can pass out from the second end surface 322, and then can be captured by the light sensing surface 331 of the photon detector 33.

[0065] According to the existing mode of passing through the window and directly transmitting to the sensor through the channel, after the non-parallel fluorescence passes through the window, various changes may occur in the channel, for example, some large-angle incident fluorescence will be shot on the channel wall and consumed, even if it can finally reach the photon detector, the fluorescence intensity has been greatly reduced, resulting in that the fluorescence intensity detected by the final photosensitive surface 331 is much lower than the fluorescence intensity of the incident fluorescence transmission and collection assembly 30, and the longer the channel, the lower the intensity of the non-parallel incident fluorescence collected by the photosensitive surface 331, therefore, in fact, the channel cannot be ignored. The application utilizes the existing channel structure, and adds a circular truncated cone mirror 32 in the channel. Through the reflection of the side surface 323, the propagation direction of the non-parallel incident fluorescence is changed, which can reduce the loss of fluorescence on one hand, and increase the possibility of fluorescence reaching the photon detector 33 on the other hand, thereby improving the collection efficiency of fluorescence, especially the collection efficiency of large-angle incident fluorescence. In addition, the circular truncated cone structure of the circular truncated cone mirror 32 is more conducive to its stable installation in the channel, which is suitable for the structure of a longer and narrower channel. At the same time, due to the influence of the structure of the channel itself, if a lens group is added in the limited space of the channel, on one hand, through multiple simulation experiments, it is found that the influence of a lens on fluorescence is limited, and multiple lenses are needed to improve the collection efficiency, which will lead to complicated installation; on the other hand, according to the simulation calculation result, even if the addition of the lens group is theoretically feasible, it is actually difficult to find a corresponding available lens group, and even if there is an available lens group, the structure of the channel will make it impossible to install the lens; on the third hand, even if the lens assembly 31 and the like are adjusted accordingly to make the installation of the available lens in the channel feasible, it may also bring multiple problems such as early loss of fluorescence beam. Therefore, in summary, the circular truncated cone mirror 32 not only has obvious ability to improve the collection efficiency, but also is simple to prepare and install, and can maximize the overall collection efficiency in the simplest way. In particular, according to the length of the channel, the size of the circular truncated cone mirror 32 can be changed, and further adapted to the lens assembly 31, which can adapt to various collection devices. More particularly, the circular truncated cone mirror 32 is particularly suitable for longer channels. 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 under the same imaging laser power, and the sample can be imaged with 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 shot thereon, so that the fluorescence reaches the second end surface 322. Preferably, the high-reflection film has a high-reflection band of 300-750 nm, which can make the fluorescence collection device 1 adapt to different application scenarios.

[0067] In some embodiments of the present application, the first end surface 321 is located at the back focal plane of the lens assembly 31, and the diameter of the first end surface 321 is greater than or equal to the diameter of the fluorescent light beam passing out of the lens assembly 31. In this way, the first end surface can collect as much fluorescent light beam as possible from the lens assembly 31. Specifically, the back focal plane of the lens assembly 31 refers to a virtual plane in the lens system, where the light rays passing out of the lens converge or appear to converge at a point after passing through the lens. Further, the lens assembly 31 can include two lenses, three lenses, or more lenses. More specifically, when the lens assembly 31 includes three lenses, 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 in the direction of propagation of the fluorescent light beam.

[0068] In some embodiments of the present application, as shown in Figure 1 and Figure 2 , the photon detector 33 includes a light sensing surface 331 that senses the fluorescent light beam from the second end surface 322. The light sensing surface 331 is rectangular or circular, and the width of the rectangular or the diameter of the circular is greater than or equal to the diameter of the second end surface 322. That is, when the light sensing 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 light sensing surface 331 is circular, the diameter of the circle is greater than or equal to the diameter of the second end surface 322. In this way, the photon detector 33 can detect as much fluorescent light beam as possible, regardless of the shape of the light sensing surface 331.

[0069] In some embodiments of the present application, as shown in Figure 1 and Figure 2 , a detector protection window 34 is arranged between the circular mirror 32 and the light sensing surface 331 in the first direction, and the fluorescent light beam passes through the detector protection window 34 from the second end surface 322 to the light sensing surface 331. The distance between the back surface of the detector protection window 34 and the light sensing surface 331 is not greater than 2 mm, and more specifically, the distance between the back surface of the detector protection window 34 and the light sensing 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, and 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 light sensing surface 331, and the arrangement of the distance between the detector protection window 34 and the second end surface 322 and the light sensing surface 331 can minimize the impact of the detector protection window 34 on the collection of the fluorescent light beam. Specifically, the detector protection window 34 is a light-transmitting medium, including quartz glass or organic glass.

[0070] In some specific embodiments of the present application, along the first direction, the length of the circular 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 longer channel, the length of the circular truncated cone mirror 32 along the first direction is 7-9 mm, which can further reduce the influence of the longer channel on the intensity of the fluorescent beam and reduce the loss of fluorescence. In combination with the diameter of the first end face 321 and the second end face 322, the efficiency of the photon detector 33 in capturing the fluorescent signal of the fluorescent beam is higher.

[0071] In some specific embodiments of the present application, as shown in Figures 1-3 the lens assembly 31 comprises a first lens 311, a second lens 312, and a third lens 313 arranged in sequence along the 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 more efficiently collect fluorescence in a limited fluorescent collection device 1 space. Further, the second lens 312 and the third lens 313 are spaced apart by 1 mm.

[0072] In some specific embodiments of the present application, as shown in Figure 1 and Figure 2 the fluorescent transmission collection assembly 30 further comprises a circular truncated cone mirror fixing window piece 35 located between the circular truncated cone mirror 32 and the third lens 313 along the first direction, the first end face 321 is fixedly installed on the circular truncated cone mirror fixing window piece 35, and the spacing between the third lens 313 and the circular truncated cone mirror fixing window piece 35 is 0.9-1 mm. The circular truncated cone mirror fixing window piece 35 can protect the circular truncated cone mirror 32 and can more conveniently install and fix the circular truncated cone mirror 32 in the channel. At the same time, ensuring that the spacing between the third lens 313 and the circular truncated cone mirror fixing window piece 35 is 0.9-1 mm can make the influence of the circular truncated cone mirror fixing window piece 35 on the collection of the fluorescent beam as low as possible. Specifically, the circular truncated cone mirror fixing window piece 35 is coaxially bonded with the circular truncated cone mirror 32. More specifically, the circular truncated cone mirror fixing window piece 35 is a light-transmitting medium, including quartz glass and organic glass. The circular truncated cone mirror fixing window piece 35 and the circular truncated cone mirror 32 can be a composite element. When installing, for example, the original window in the device can be removed, and the circular truncated cone mirror fixing window piece 35 and the circular truncated cone mirror 32 can be placed in the device. Specifically, the installation position of the circular truncated cone mirror fixing window piece 35 is the original incident window position. When installing, the original incident window can be removed, and the circular truncated cone mirror fixing window piece 35 and the circular truncated cone mirror 32 can be installed. Further, as shown in Figure 1As shown, the conical mirror fixed window piece 35 and the photosensitive surface 331 are connected by a channel 25, the conical mirror 32 is installed in the channel 25, and the installation position P of the conical mirror fixed window piece 35 is the original incident window position.

[0073] By the cooperation of the optical assembly objective 10, the lens assembly 31, the conical mirror 32, and the photon detector 33, specifically by the cooperation of the objective 10, the first lens 311, the second lens 312, the third lens 313, the conical mirror fixed window piece 35, the conical mirror 32, the detector protection window piece 34, and the photosensitive surface 331, the characteristics of the optical elements are adjusted, and the cooperation and coordination between the elements are considered, a stable optical system is constructed, and the fluorescence collection efficiency of the microscopic imaging system is effectively improved. Especially in the case of long channel and large-angle incident fluorescence, the fluorescence collection efficiency is improved more obviously.

[0074] In a specific embodiment of the present application, as shown in Figure 1 and Figure 2 , the fluorescence transmission and collection assembly 30 further comprises 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. That is, as shown in Figure 1 , along the fluorescence beam propagation direction, the dichroic mirror assembly 36 is located before the first lens 311, and the fluorescence beam first reaches the dichroic mirror assembly 36 and then reaches the first lens 311. Alternatively, as shown in Figure 2 , along the fluorescence beam propagation direction, the dichroic mirror assembly 36 is located between the first lens 311 and the second lens 312, and the fluorescence 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 application, as shown in Figures 1-3 , the objective 10 is coaxially arranged with the fluorescence collection channel 20, and the back aperture of the objective 10 is greater than or equal to 20 mm. That is, the objective 10, the lens assembly 31, the conical mirror 32, the photon detector 33, the detector protection window piece 34, and the conical mirror fixed window piece 35 are coaxially arranged.

[0076] Preferably, the dichroic mirror assembly 36 is a dichroic mirror placed between the objective lens 10 and the first lens 311 for separating the fluorescence excitation wavelength and the excited fluorescence wavelength, which can transmit the excited fluorescence wavelength and reflect the incident laser wavelength of the fluorescence excitation of the microscope system, or can reflect the excited fluorescence wavelength and transmit the incident laser wavelength of the fluorescence excitation of the microscope system. Alternatively, in another specific embodiment of the present application, the dichroic mirror assembly 36 is composed of multiple dichroic mirrors, and the dichroic mirror close to the objective lens 10 is used to separate the fluorescence excitation wavelength and the excited fluorescence wavelength, and the other dichroic mirrors can be placed between the objective lens 10 and the first lens 311 or between the lenses in the lens assembly 31 for separating different wavelengths of the excited fluorescence, so that the excited 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 application, as shown in Figure 1 and Figure 2 The fluorescence transmission and collection assembly 30 further comprises a filter assembly 37, which is placed behind the dichroic mirror assembly 36 along the first direction, i.e., along the propagation path of the fluorescence beam, for further filtering out stray light other than the excited fluorescence of the target wavelength, so that only the excited fluorescence of the target wavelength is incident on the photon detector 33. Specifically, the filter assembly 37 comprises a filter, which can also be composed of two, three or even more filters. More specifically, the filter assembly 37 comprising two or more filters further comprises a filter rotating fixture (not shown in the figure), which is composed of multiple rotatable filter fixing slots, which can rotate the selected filter into the fluorescence collection light path and fix it coaxially with other optical elements in the fluorescence collection light path by rotating the fixing slots, and rotate the unnecessary filters out of the fluorescence collection light path.

[0078] Specifically, the filters in the filter assembly 37 are placed between the lens assembly 31 and the circular mirror 32, or between the lenses in the lens assembly 31. Specifically, in the filter assembly comprising two or more filters, the filters can be placed adjacently or interspersed non-adjacently. Preferably, the filters in the filter assembly 37 are bandpass filters, which can be selected from narrow bandpass filters such as 438±10nm, 532±10nm, etc. that only transmit the excited fluorescence wavelength of a specific fluorescence indicator, or bandpass filters that can transmit multiple excited fluorescence wavelengths of fluorescence indicators, such as a bandpass filter with a transmission wavelength range of 300nm-750nm.

[0079] Specifically, the lens assembly 31 and the filter assembly 37 can comprise mirrors to meet the design requirements of different light paths, and the lens assembly 31 and the filter assembly 37 are placed coaxially.

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

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

[0082] The adapter bracket 22 has one end opening of the large bracket 211 and one end opening of the small bracket 212 respectively inserted into it, so that the large bracket 211 and the small bracket 212 are coaxially installed. That is to say, since there is a difference in the size of the fixed optical element, there is a difference in the size of the large bracket 211 and the small bracket 212, such as the aperture size. In order to connect them stably, the larger large bracket 211 and the smaller small bracket 212 can be connected by the adapter bracket 22. Furthermore, the filter assembly 37 can be installed on the adapter bracket 22.

[0083] The detector mounting bracket 23 has an opening and an end face opposite each other along a first direction. The opening of the detector mounting bracket 23 abuts against the fixed window plate 35 of the frustum mirror, and the detector protective window plate 34 is embedded in the detector mounting bracket 23.

[0084] More specifically, such as Figure 1 As shown, the frustum mirror fixing window 35 abuts against and is fixedly connected to one end opening of the detector fixing bracket 23. Alternatively, in another embodiment of the invention, as... Figure 2 As shown, the fixed window 35 of the frustum mirror can be directly embedded in the detector mounting bracket 23.

[0085] In each specific embodiment of the present invention, such as Figure 2 As shown, the distance between one end opening of the detector mounting bracket 23 and the detector protective window 34 is the length of the passageway. The length of the frustum mirror 32 is less than or equal to the length of the passageway. One end opening of the detector mounting bracket 23 is in contact with the frustum mirror fixing window 35. That is, one end opening of the detector mounting bracket 23 directly abuts against the frustum mirror fixing window 35.

[0086] In a specific embodiment of the present invention, such as Figure 1As shown, the distance between the one end opening of the probe fixing frame 23 and the probe protection window sheet 34 is the length of the channel, the length of the circular truncated cone mirror 32 is greater than the length of the channel, and a gasket 26 is arranged between the one end opening of the probe fixing frame 23 and the circular truncated cone mirror fixing window sheet 35. That is, the one end opening of the probe fixing frame 23 is not directly in abutment with the circular truncated cone mirror fixing window sheet 35, and since the circular truncated cone mirror 32 is longer, the gasket 26 is used for installation, and the one end opening of the probe fixing frame 23 indirectly abuts against the circular truncated cone mirror fixing window sheet 35 through the gasket. Specifically, the gasket 26 has a hollow circle, the diameter of the hollow circle is less than the diameter of the circular truncated cone mirror fixing window sheet 35 and greater than the diameter of the first end face 321, the circular 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 circular truncated cone mirror 32 and the length of the channel.

[0087] Further, as shown in Figure 1 and Figure 2 , the large fixing frame 211 includes optical element fixing frames 2111 and fixing frame coaxial connecting rods and fasteners 2112, and the plurality of optical element fixing frames 2111 are connected by the fixing frame coaxial connecting rods and fasteners 2112 and coaxially installed; the small fixing frame 212 includes optical element fixing frames 2121 and fixing frame coaxial connecting rods and fasteners 2122, and the plurality of optical element fixing frames 2121 are connected by the fixing frame coaxial connecting rods and fasteners 2122 and coaxially installed. Each fixing frame does not block the propagation of the fluorescent beam.

[0088] In a specific embodiment of the present application, as shown in Figure 1 and Figure 2 , the fluorescence collection channel 20 is further provided with a light shielding assembly 24, the light shielding assembly 24 is located outside the objective lens 10 and the fluorescence transmission and collection assembly 30, and is fixed outside each fixing frame and completely covers the same, for shielding external stray light. Preferably, the light shielding assembly 24 is composed of non-transparent metal medium such as aluminum and stainless steel, and the surface is treated by oxidation and blackening, the oxidation treatment is used to reduce the conductivity of the light shielding assembly 24 and improve the safety of the system, and the blackening treatment is used to reduce the reflection and scattering of light by the light shielding assembly 24. More preferably, the light shielding assembly 24 is composed of black high-strength non-metal medium, which does not have conductivity and is resistant to 350℃ high temperature and is not easy to be deformed by heat.

[0089] In a specific embodiment of the present application, as shown in Figure 1 and Figure 2 , the fluorescence collection device 1 includes a single fluorescence collection channel 20, and the objective lens 10 is coaxially arranged with the fluorescence collection channel 20.

[0090] In a specific embodiment of the present application, the fluorescence collection device 1 includes a plurality of fluorescence collection channels, for example Figure 4As shown, the fluorescence collection device 1 includes a first fluorescence collection channel 20 and a second fluorescence collection channel 20'. Specifically, the first fluorescence collection channel 20 is provided with a fluorescence transfer collection component 30, which includes components along a first direction (e.g., Figure 4 The dichroic mirror assembly 36, filter assembly 37, lens assembly 31, frustum mirror fixing window 35, frustum mirror 32, detector protective window 34, and photon detector 33 are sequentially arranged in direction A. 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. A fluorescence transfer collection assembly 30' is provided in the second fluorescence collection channel 20', and the fluorescence transfer collection assembly 30' includes components along the second direction (e.g., ...). Figure 4 The dichroic mirror assembly 36', filter assembly 37', lens assembly 31', frustum mirror fixing window 35', frustum mirror 32', detector protective window 34', and photon detector 33' are sequentially arranged in direction B. 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. The fluorescence beam reflected by the first dichroic mirror 361 is split into two fluorescence collection channels of different wavelengths by the second dichroic mirror 362. That is, the fluorescence beam transmitted through the second dichroic mirror 362 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. By setting up multiple channels, fluorescence signal acquisition can be performed more efficiently and conveniently.

[0091] In one specific embodiment of the present invention, 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%. The collection efficiency of non-parallel incident fluorescence beams, especially large-angle fluorescence beams, is significantly improved.

[0092] A second aspect of the present invention discloses a fluorescence microscope including the above-described fluorescence collection device.

[0093] The following will describe a more specific implementation method.

[0094] Example 1:

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

[0096] Structural reference of Example 1 Figure 1The device comprises an objective lens 10 and a fluorescence collection channel 20 coaxially arranged along a first direction, and a fluorescence transmission collection assembly 30 arranged in the fluorescence collection channel 20, wherein the fluorescence transmission collection assembly 30 comprises, in sequence along the first direction, a dichroic mirror assembly 36, a first lens 311, a filter assembly 37, a second lens 312, a third lens 313, a circular truncated cone mirror fixed window piece 35, a circular truncated cone mirror 32, a photodetector protection window piece 34, and a photodetector 33.

[0097] The first lens 311, the second lens 312, and the third lens 313 are respectively LA1386-A lenses with a focal length of 75 mm, LA1274-A lenses with a focal length of 40 mm, and ACL25416U-A aspherical lenses with a focal length of 16 mm of Thorlabs brand.

[0098] The back aperture of the objective lens 10 coincides with the front focal surface of the first lens 311, the rear focal surface of the first lens 311 coincides with the front focal surface of the second lens 312, and the equivalent rear focal surface of the second lens 312 and the third lens 313 coincides with the first end surface 321 of the circular truncated cone mirror 32.

[0099] The dichroic mirror assembly 36 is a Semrock FF725-SDi01-25x36x3.5 dichroic mirror, which transmits 430 nm to 700 nm and reflects 750 nm to 1140 nm, and separates two-photon excitation light and stimulated fluorescence, and the stimulated fluorescence is transmitted through the dichroic mirror and then incident on the lens assembly 31.

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

[0101] The first end surface 321 of the circular truncated cone mirror 32 has a diameter of 5.85 mm, and the second end surface 322 has a diameter of 4.45 mm. The length of the circular truncated cone mirror 32 along the first direction is 8.5 mm. When the circular truncated cone mirror 32 is arranged, the length of the circular truncated cone mirror 8.5 mm is greater than the length of the Hamamatsu H7422 photomultiplier tube 7.4 mm, so a gasket is needed when the circular truncated cone mirror fixing window piece 35 and the circular truncated cone mirror 32 are fixed. There is an original glass window (an incident window) outside the photomultiplier tube channel. In order to make the exit end of the circular truncated cone mirror as close as possible to the front window of the photomultiplier tube, the original glass window outside the channel is removed as a whole, and dust, debris and the like are prevented from entering the channel during this process. The circular truncated cone mirror fixing window piece 35 is coaxially bonded to the circular truncated cone mirror 32, the circular truncated cone mirror fixing window piece 35 is coaxially bonded to the annular gasket with a hollow thickness of 1.3 mm, and then the annular gasket is coaxially bonded to the detector fixing frame 23. The circular truncated cone mirror and the photomultiplier tube cathode are coaxial, and the distance between the front surface of the detector protection window piece 34 and the second end surface 322 of the circular truncated cone mirror 32 is 0.2 mm. The circular truncated cone mirror fixing window piece 35 is made of glass. For the Hamamatsu H7422 photomultiplier tube, the position of the annular gasket is the position of the original glass window outside the PMT channel.

[0102] The photonic detector photosensitive surface 331 of the Hamamatsu H7422 photomultiplier tube is square-shaped with a side length of 5 mm, which is greater than the diameter of the second end surface 322. The detector protection window piece 34 is placed in front of the photosensitive surface 331 and fixed on the detector fixing frame 23. The detector protection window piece 34 is made of glass, and the distance between the front surface of the detector protection window piece 34 and the photosensitive surface 331 is about 1.5 mm.

[0103] Comparative Example 1:

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

[0105] The configurations of Comparative Example 1 and Example 1 are simulated respectively, different angles of fluorescence are collected, and data of collection efficiency are collected. In the simulation, it is assumed that the thickness of the detector protection window piece 34 is 1 mm.

[0106] In the simulation, an objective back aperture with a diameter of 20 mm is used as the fluorescent light source, the fluorescent light collected by the objective is incident on the fluorescent light collection device through the back aperture of the objective, and the angle between the fluorescent light emitted by the back aperture of the objective and the optical axis is 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, and the incident fluorescent light intensity at each angle is set to 1 W. The fluorescent light intensity received by a 5 mm square photonic detector under the ideal condition at the angle is recorded by using Zemax software, so as to calculate the fluorescent light collection efficiency at different angles.

[0107] The fluorescent light collection efficiency at different angles obtained by simulation according to the configuration of Example 1 is shown in Figure 5 It can be seen that when the incident fluorescent light has an angle of 5.5° or less with the optical axis, the fluorescent light collection efficiency at each angle is more than 86%, and the fluorescent light collection efficiency at an angle of 8° with the optical axis is still 69.78%.

[0108] The fluorescent light collection efficiency at different angles obtained by simulation according to the configuration of Comparative Example 1 is shown in Figure 6 It can be seen that when the incident fluorescent light has an angle of 3.0° with the optical axis, the fluorescent light collection efficiency is as low as 50.01%, the collection efficiency at an angle of 5° is less than 2%, and the fluorescent light at an angle of 5.5° or more cannot be collected.

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

[0110] Example 2:

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

[0112] The specific structure of Example 2 is shown in Figure 4 The fluorescent light collection device 1 has a first fluorescent light collection channel 20 and a second fluorescent light collection channel 20', and the first fluorescent light collection channel 20 is provided with a fluorescent light transfer and collection assembly 30, and the fluorescent light transfer and collection assembly 30 is composed of a first fluorescent light transfer and collection component 31 and a second fluorescent light transfer and collection component 32 arranged along a first direction (for example, the direction of the optical axis). Figure 4The first dichroic mirror 361, the second dichroic mirror 362, the filter assembly 37, the first lens 311, the second lens 312, the third lens 313, the fixed window piece 35 of the conical mirror, the conical mirror 32, the protective window piece 34 of the detector, and the photon detector 33 are sequentially arranged in the direction A shown in FIG. 6. The fluorescence transmission and collection assembly 30' is arranged in the second fluorescence collection channel 20'. The fluorescence transmission and collection assembly 30' is composed of the first lens 311', the second lens 312', the third lens 313', the fixed window piece 35' of the conical mirror, the conical mirror 32', the protective window piece 34' of the detector, and the photon detector 33' arranged in the direction B shown in FIG. 6. Figure 4 The first dichroic mirror 361, the second dichroic mirror 362, the filter assembly 37, the first lens 311, the second lens 312, the third lens 313, the fixed window piece 35 of the conical mirror, the conical mirror 32, the protective window piece 34 of the detector, and the photon detector 33 are sequentially arranged in the direction A shown in FIG. 6. The fluorescence transmission and collection assembly 30' is arranged in the second fluorescence collection channel 20'. The fluorescence transmission and collection assembly 30' is composed of the first lens 311', the second lens 312', the third lens 313', the fixed window piece 35' of the conical mirror, the conical mirror 32', the protective window piece 34' of the detector, and the photon detector 33' arranged in the direction B shown in FIG. 6.

[0113] The stimulated fluorescence transmitted through the objective lens 10 is reflected by the first dichroic mirror 361, which is a Semrock FF735-Di02-25x36, transmitting 750nm-1600nm and reflecting 350nm-720nm, and functions 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 (the first fluorescence collection channel 20 and the second fluorescence collection channel 20') of different wavebands by the second dichroic mirror 362, which is a Semrock FF552-Di02-25x36, transmitting red fluorescence and reflecting 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') respectively. The filter assembly 37 is a Semrock FF01-593 / 46-25, and the filter assembly 37' is a Semrock FF01-520 / 35-25, which filter out other fluorescence wavelengths and stray light except the target fluorescence waveband.

[0114] Although the present application has been illustrated and described with reference to certain preferred embodiments thereof, it should be understood that the foregoing is intended to cover all modifications and variations of this application that are within the scope of the present application, which is defined by the appended claims. It is intended to include all changes and modifications that fall within the scope of the present application.

Claims

1. A fluorescence collection device, characterized by, The utility model relates to a fluorescence collection device, comprising: an objective lens for receiving a fluorescence beam and passing the fluorescence beam through; a fluorescence collection channel extending along a first direction; a fluorescence transfer and collection assembly arranged in the fluorescence collection channel for transferring and collecting the fluorescence beam from the objective lens, comprising, in order along the first direction in the fluorescence collection channel: a lens assembly comprising at least two lenses; a circular truncated cone mirror comprising a first end face and a second end face oppositely arranged along the first direction, and a side face between the first end face and the second end face along the first direction, the first end face having a diameter greater than that of the second end face, wherein the fluorescence beam from the lens assembly enters the circular truncated cone mirror from the first end face and passes out from the second end face, and the side face reflects the fluorescence beam reaching the side face to the second end face; a photon detector for receiving the fluorescence beam from the second end face and converting the fluorescence beam into an electrical signal; the first direction is parallel to an optical axis of the fluorescence transfer and collection assembly.

2. A fluorescence collection device as claimed in claim 1, characterized in that the first end face is located at a back focal plane position of the lens assembly, and the diameter of the first end face is greater than or equal to that of the fluorescence beam passing out from the lens assembly.

3. A fluorescence collection device as claimed in claim 1, wherein, the photon detector comprises a photosensitive surface for sensing the fluorescence beam from the second end face, the photosensitive surface being rectangular or circular, the width of the rectangular or the diameter of the circular being greater than or equal to that of the second end face.

4. A fluorescence collection device as claimed in claim 3, wherein, a detector protection window piece is arranged between the circular truncated cone mirror and the photosensitive surface along the first direction, the fluorescence beam from the second end face passes through the detector protection window piece to reach the photosensitive surface, the distance between the back surface of the detector protection window piece 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 piece 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, wherein, the lens assembly comprises, in order along the first direction, a first lens, a second lens and a third lens, 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, wherein, the fluorescence transfer and collection assembly further comprises a circular truncated cone mirror fixing window piece between the circular truncated cone mirror and the lens assembly along the first direction, and the first end face is fixedly installed on the circular truncated cone mirror fixing window piece, and the distance between the third lens and the circular truncated cone mirror fixing window piece is 0.9-1 mm.

7. A fluorescence collection device as claimed in claim 6, wherein along the first direction, the distance between the circular truncated cone mirror fixing window piece and the photosensitive surface is 7-10 mm, the length of the circular truncated cone 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, wherein, The fluorescence transfer and collection assembly further comprises a dichroic mirror assembly, which is located before the first lens or between the first lens and the second lens along the first direction.

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

10. A fluorescence collection device as claimed in claim 9, wherein, The fluorescence collection channel comprises: An optical element fixing frame comprises: A large fixing frame for embedding the objective lens, the first lens and the dichroic mirror assembly, having two end openings opposite along the first direction; A small fixing frame for embedding the second lens, the third lens and the circular mirror fixing window piece, having two end openings opposite along the first direction; An adapter fixing frame, one end opening of the large fixing frame and one end opening of the small fixing frame are respectively inserted into the adapter fixing frame, so that the large fixing frame and the small fixing frame are coaxially installed; A detector fixing frame having one end opening and an end surface opposite along the first direction, the one end opening of the detector fixing frame abuts against the circular mirror fixing window piece, and the detector protection window piece is embedded in the detector fixing frame.

11. A fluorescence collection device as claimed in claim 10, wherein, The distance between the one end opening of the detector fixing frame and the detector protection window piece is a duct length, the length of the circular mirror is less than or equal to the duct length, and the one end opening of the detector fixing frame contacts the circular mirror fixing window piece.

12. A fluorescence collection device as claimed in claim 10, wherein, The distance between the one end opening of the detector fixing frame and the detector protection window piece is a duct length, the length of the circular mirror is greater than the duct length, and a gasket is arranged between the one end opening of the detector fixing frame and the circular mirror fixing window piece.

13. A fluorescence collection device as claimed in claim 8, wherein, The fluorescence collection channel is a first fluorescence collection channel, the dichroic mirror assembly is located before the first lens, the dichroic mirror assembly comprises a first dichroic mirror and a second dichroic mirror, the fluorescence collection device further comprises 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 by 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, wherein, When the included 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 The fluorescence collection device comprises the fluorescence collection device according to any one of claims 1-14. The fluorescence collection device comprises the fluorescence collection device according to any one of claims 1-14.

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