Multi-mode fluorescence microscope and use method thereof

Abstract

The invention provides a multi-mode fluorescence microscope and a use method thereof. The microscope comprises a microscopic assembly, a near-infrared two-region excitation light path, a visible light confocal excitation light path, a wide-field imaging light path, a near-infrared two-region confocal imaging assembly, a multi-photon intensity / lifetime and harmonic imaging assembly and a visible light confocal and wide-field imaging assembly. The microscopic assembly comprises an objective lens, an optical filter switching assembly, a sleeve lens and a transflective device which are sequentially arranged from the object side to the image side, and the transflective device can be switched to a dichroscope or a reflector; the near-infrared second-area excitation light path, the visible light confocal excitation light path and the wide-field imaging light path are connected with the sleeve lens; the near-infrared two-region confocal imaging assembly, the multi-photon intensity / lifetime and harmonic imaging assembly and the visible light confocal and wide-field imaging assembly are all connected to the sleeve lens. According to the scheme, confocal and wide-field imaging of visible light, confocal and wide-field imaging of a near-infrared second region, and multi-photon intensity / lifetime and harmonic imaging based on near-infrared excitation are realized.

Description

Technical Field

[0001] The present invention belongs to the technical field of optical systems, and in particular relates to a multimodal fluorescence microscope and a use method thereof. Background Art

[0002] Commonly used fluorescence microscope modalities that have been developed include wide-field fluorescence microscope, confocal fluorescence microscope, two-photon / three-photon fluorescence microscope and fluorescence lifetime microscope. These microscopes have their own characteristics, such as fast imaging speed of wide-field fluorescence microscope, high resolution of confocal fluorescence microscope and optical layer cutting ability - able to achieve depth resolution, etc. Therefore, some multimodal microscopes have appeared on the market, which include several of the above microscopic imaging modalities in order to achieve a combination of advantages and meet the needs of different imaging scenarios.

[0003] In addition, the development of near-infrared II microscopic imaging has been in full swing in the past decade or so. Because light in the near-infrared II region has lower scattering and absorption in biological tissues and can penetrate to a greater depth, fluorescence microscopy in the near-infrared II region has also been increasingly developed, and the imaging modality has gradually become consistent with that in the visible light region.

[0004] In the related technology, the imaging modalities of multimodal fluorescence microscopes are relatively limited, and only include a limited number of commonly used imaging modalities. For example, Japan's Nikon's A1R MP has single / two-photon and second harmonic scanning imaging, but no lifetime imaging function, nor near-infrared zone II imaging modality; South Korea's IVI M is basically the same as Nikon's; Germany's Leica's TCSSP8 has two-photon, second harmonic and lifetime imaging modules, but also does not integrate near-infrared zone II imaging, and the system is an inverted microscope and cannot be used for in situ microscopic imaging of living bodies; in the field of near-infrared zone II imaging, existing technologies include near-infrared zone II confocal and wide-field imaging, but their imaging bands are also limited to near-infrared zone II. In other words, there is currently no microscope that can expand the imaging band to a wide range from visible light to near-infrared zone II, and is fully compatible with commonly used imaging modalities, so that users can complete almost all required imaging experiments on one microscope. Summary of the invention

[0005] The technical purpose of the present invention is to provide a multimodal fluorescence microscope and a method of use, which can realize confocal and wide-field imaging of visible light, confocal and wide-field imaging of near-infrared regions, and multi-photon intensity / lifetime and harmonic wave imaging based on near-infrared excitation.

[0006] In order to solve the above technical problems, the present invention is implemented in this way, providing a multimodal fluorescence microscope, including a microscope component, a near-infrared second-zone excitation light path, a visible light confocal excitation light path, a wide-field imaging light path, a near-infrared second-zone confocal imaging component, a multiphoton intensity / lifetime and harmonic wave imaging component, and a visible light confocal and wide-field imaging component; the microscope component includes an objective lens OL, a filter switching component DM4, a sleeve lens TL1 and a transflection device arranged in sequence from the object side to the image side, and the transflection device can be switched to a dichroic mirror DM3 or a reflector M1; the near-infrared second-zone excitation light path, the visible light confocal excitation light path and the wide-field imaging light path are connected to the sleeve lens TL1, and ... The outer second zone excitation light path is used to provide femtosecond laser with a wavelength of 680nm to 1300nm to the microscope component, the visible light confocal excitation light path is used to provide lasers of four confocal bands to the microscope component, and the wide-field imaging light path is used to provide visible light with a continuously distributed band of 400-700nm or near-infrared light with a wavelength of 900-1450nm to the microscope component; the near-infrared second zone confocal imaging component, the multi-photon intensity / lifetime and harmonic wave imaging component, and the visible light confocal and wide-field imaging component are all connected to the tube lens TL1 to receive the returned signal light, and the filter switching component DM4 and the transflective device are used to switch the light transmission state adapted to each of the imaging components.

[0007] Furthermore, the near-infrared zone II excitation optical path includes a wavelength tunable femtosecond laser, a reflective beam expander BE, an achromatic lens L1, a pinhole PH1, an achromatic lens L2, a long-pass dichroic mirror DM1, a first scanning module, a scanning lens SL1 and a dichroic mirror DM2 which are sequentially arranged along the optical path. The scanning lens SL1 increases transmittance at 700-1700nm, the output optical path of the dichroic mirror DM2 is toward the transflective device, and the wavelength tunable femtosecond laser is continuously adjustable at a wavelength of 680nm to 1300nm.

[0008] Furthermore, the first scanning module includes scanners Scanner1, 2, an achromatic lens L3, an achromatic lens L4 and a scanner Scanner3.

[0009] Furthermore, the near-infrared two-zone confocal imaging assembly includes a long-pass filter, a reflector M2, an achromatic lens L5, a pinhole PH2, a multimode optical fiber MMF1 and a photomultiplier tube PMT1 arranged in sequence on the optical path away from the long-pass dichroic mirror DM1, and the pinhole PH2 is on the focal plane of the achromatic lens L5.

[0010] Furthermore, the multi-photon intensity / lifetime and harmonic imaging component includes a short-reflecting dichroic mirror DM5, a collimating lens L6, a dichroic mirror DM6, a dichroic mirror DM7, and a filtering Fresnel lens F1 and a photomultiplier tube PMT2 in the transmission direction of the dichroic mirror DM6, a filtering Fresnel lens F2 and a photomultiplier tube PMT3 in the reflection direction of the dichroic mirror DM7, and a filtering Fresnel lens F3 and a photomultiplier tube PMT4 in the transmission direction of the dichroic mirror DM7; wherein, for near-infrared excited multi-photon or harmonic imaging, the transflective device is switched to the dichroic mirror DM3, and the filtering Fresnel lens F1 and the photomultiplier tube PMT2 in the transmission direction of the dichroic mirror DM6 are sequentially arranged. The filtering Fresnel lens F2 is composed of a filter and a Fresnel lens, the photomultiplier tube PMT2 is responsible for second / third harmonic imaging, the photomultiplier tube PMT3 is responsible for two-photon / three-photon imaging, and the detection band of the photomultiplier tube PMT2 and the photomultiplier tube PMT3 is 380-720nm; for near-infrared excited multi-photon fluorescence lifetime imaging, the transflective device is switched to a dichroic mirror DM3, the photomultiplier tube PMT3 is a counting detector, and the time-correlated single photon counting method (TCSPC) is used to achieve precise lifetime measurement. The filtering Fresnel lens F3 is composed of a filter and a Fresnel lens, and the detection band of the photomultiplier tube PMT4 is 280-720nm.

[0011] For the multi-path synchronous imaging mode of near-infrared excitation, a short-pass dichroic mirror is used to split the light at the dichroic mirror DM3, so that the excitation light and longer single-photon signals are reflected, and the two-photon fluorescence and harmonic wave signals are transmitted.

[0012] Furthermore, the visible light confocal excitation optical path includes a four-wavelength laser module, a single-mode optical fiber SMF, a 10x objective lens L7, a multi-bandpass dichroic mirror DM8, a second scanning module, a scanning lens SL2 and a dichroic mirror DM2 which are arranged in sequence along the optical path. The four-wavelength laser module includes four lasers with wavelengths of 405nm, 488nm, 561nm and 638nm. The lasers of each laser are respectively combined and coupled to the single-mode optical fiber SMF through a dichroic mirror. The scanning lens SL2 increases transmittance in the visible light band, and the dichroic mirror DM2 is also used for combining with the near-infrared second zone excitation optical path.

[0013] Furthermore, the second scanning module includes scanners Scanner4, 5, an achromatic lens L8, a 2-inch elliptical reflector M3, an achromatic lens L9 and a scanner Scanner6 which are arranged in sequence.

[0014] Furthermore, the visible light confocal and wide-field imaging component includes a reflecting mirror M4, a focusing lens L10, a pinhole PH3, a multimode optical fiber MMF2, a focusing lens L11, a dichroic mirror DM9, a dichroic mirror DM10, a filtering Fresnel lens F4 and a photomultiplier tube PMT5 on the reflected light path of the dichroic mirror DM9, a filtering Fresnel lens F5 and a photomultiplier tube PMT6 on the reflected light path of the dichroic mirror DM10, and a filtering Fresnel lens F6 and a photomultiplier tube PMT7 on the transmitted light path of the dichroic mirror DM10, which are arranged in sequence on the light path of the multi-bandpass dichroic mirror DM8 away from the 10x objective lens L7. The photomultiplier tubes PMT5, PMT6, and PMT7 constitute three imaging channels to respectively detect the blue band, the green band, and the red band.

[0015] Further, the microscope assembly includes a frame fluorescent arm on which the filter switching assembly DM4 is arranged, the frame fluorescent arm is provided with an excitation light source entrance corresponding to the position of the filter switching assembly DM4, the wide-field imaging optical path includes a white light LED or a near-infrared LED switchably installed at the excitation light source entrance, the white light LED excitation band can be continuously distributed in the range of 400-700nm, and the peak wavelength of the near-infrared LED is between 900-1450nm;

[0016] The visible light confocal and wide-field imaging component includes a short-reflecting dichroic mirror DM5 and a switchable camera on the optical path of the transflective device away from the filter switching component DM4. The switchable camera can be switched to a visible light camera and a near-infrared camera. The detection band of the visible light camera is 380-1000nm, and the detection band of the near-infrared camera is 900-1700nm.

[0017] Furthermore, a method for using a multimodal fluorescence microscope is provided, which is applied to the multimodal fluorescence microscope as described in any one of the above items, comprising:

[0018] Visible widefield imaging mode:

[0019] A white light LED is configured at the entrance of the excitation light source, and its output spectrum covers 400nm-700nm. First, turn on the white light LED and rotate the filter switching component DM4 to the filter position corresponding to the fluorescence emission color of the sample; then turn on the visible light camera, and set the dichroic mirror DM3 and the dichroic mirror DM5 to empty magnetic cores to ensure the passage of light. Then, place the sample on the stage and adjust the height of the stage to achieve focus. Focus is achieved by observing the image of the camera;

[0020] Near-infrared wide-field imaging in the second region:

[0021] A near-infrared LED is configured at the entrance of the excitation light source, and its luminous band is adapted to the dye marking situation; the visible light camera is replaced with a near-infrared camera, and then the filter switching component DM4 is rotated to the 4-slot position, and the dichroic mirror DM3 and the dichroic mirror DM5 are set to empty magnetic cores to ensure the passage of light, and then the sample is placed on the stage, and the height of the stage is adjusted to achieve focusing, which is achieved by observing the image of the camera;

[0022] Visible light confocal imaging:

[0023] Turn on the light source of the four-wavelength laser module and turn on the laser output of the corresponding wavelength; turn on the power of scanners Scanner4, 5, 6, and turn on the power of the electric pinhole wheel PH3 and the photomultiplier tubes PMT5, 6, 7; insert a magnetic core with a reflector at the dichroic mirror DM3 to achieve the function of full reflection; rotate the dichroic mirror DM4 to slot No. 5 to allow all light to pass; place the sample on the stage, open the imaging software to start scanning, and adjust the left and right position and height of the sample, as well as the size of the pinhole PH3 according to the image results obtained by the scan; realize the synchronous acquisition of the three-way signals of the photomultiplier tubes PMT5, 6, and 7 through multiple acquisition channels to achieve three-color synchronous imaging;

[0024] Near-infrared zone 2 confocal imaging:

[0025] Turn on the power of the near-infrared second-zone photomultiplier tube PMT1 and wait for 1 hour of cooling time; turn on the wavelength tunable femtosecond laser Femtosecond Turn on the power of the Laser, wait for 15 minutes of warm-up time, then set the required output wavelength and start the laser output; turn on the power of scanners Scanner1, 2, and 3, preset the reverse voltage of the photomultiplier tube PMT1 to -500V, and turn on the power of the electric pinhole wheel PH2; insert the magnetic core with a reflector at the dichroic mirror DM3 to achieve the function of full reflection, and rotate the dichroic mirror DM4 to slot 5 to allow all light to pass through; place the sample on the stage and open the imaging software to start scanning and imaging; if multi-color imaging is required, switch the magnetic core at the dichroic mirror DM1, configure the dichroic mirror / filter required for excitation / imaging of the corresponding band, and then image in batches; when the sample is marked with both visible light fluorescent dyes and near-infrared zone II fluorescent dyes, the visible light confocal and near-infrared zone II confocal imaging modules can be turned on at the same time, and the acquisition system can simultaneously acquire the output signals of the four detectors of the photomultiplier tubes PMT1, 5, 6, and 7 and image them, realizing the four-channel confocal synchronous imaging function;

[0026] Non-linear imaging:

[0027] Turn on the power of the wavelength tunable femtosecond laser Femtosecond Laser, wait for 15 minutes of warm-up time, then set the desired output wavelength and start the laser output; turn on the power of scanners Scanner1, 2, and 3, preset the reverse voltage of photomultiplier tube PMT1 to -500V, and turn on the power of the electric pinhole wheel PH2; turn on the power of photomultiplier tubes PMT2, 3, and 4, and open the control software; place the sample on the stage, turn on the wavelength tunable femtosecond laser Femtosecond Laser Laser output; configure the dichroic mirror DM3 as a two-photon or three-photon dichroic mirror; configure the dichroic mirror DM5 as a combination of a 720LP dichroic mirror and a 720SP filter, so that the slightly leaked excitation light at the dichroic mirror DM3 is further intercepted and most of the signal light is transmitted; the separation of the fluorescence signal and the harmonic signal in the signal light is achieved through the dichroic mirror DM6; configure the dichroic mirror DM7 according to the different needs of fluorescence intensity imaging and fluorescence lifetime imaging. When only fluorescence intensity imaging is performed, insert a The magnetic core with built-in reflector makes all the fluorescence reflect to the photomultiplier tube PMT3. When only fluorescence lifetime imaging is performed, a magnetic core without lens is inserted at the dichroic mirror DM7 to make all the fluorescence transmit to the photomultiplier tube PMT4. When simultaneous imaging of intensity and lifetime is required, a magnetic core with built-in semi-reflective and semi-transparent film is inserted at the dichroic mirror DM7 to make half of the fluorescence go to the photomultiplier tube PMT3 and the other half go to the photomultiplier tube PMT4. Open the imaging software and start scanning to obtain the images corresponding to the output signals of the photomultiplier tubes PMT2, 3, and 4.

[0028] Four-channel simultaneous imaging:

[0029] Turn on the power of the wavelength tunable femtosecond laser Femtosecond Laser, scanners Scanner1, 2, 3, photomultiplier tubes PMT1, 2, 3, 4 and pinhole PH2, and wait for the equipment to be ready; set DM3 to 720SP dichroic mirror. For near-infrared second-zone confocal, the wavelengths of the excitation light and signal light are both longer than 720nm, so both can be reflected at DM3. For nonlinear imaging, the configuration remains unchanged; place the sample on the stage, and connect the signals of the photomultiplier tubes PMT1, 2, 3, 4 to the acquisition system, and start the imaging software to perform scanning. Because the light source, optical path and scanning system are shared, and all are single-point scanning imaging, the software can realize synchronous image reconstruction of the four channels of photomultiplier tubes PMT1, 2, 3, 4, thereby realizing synchronous imaging of the four channels.

[0030] Compared with the prior art, the multimodal fluorescence microscope and the method of use in the present invention have the following beneficial effects:

[0031] Through the above implementation scheme, confocal and wide-field imaging of visible light, confocal and wide-field imaging of the second near-infrared region, and multi-photon intensity / lifetime and harmonic wave imaging based on near-infrared excitation are achieved. The commonly used fluorescence microscopy imaging modality is integrated into a microscope, and the imaging band covers 280-1700nm. As far as we know, this is the widest imaging band that can be achieved by a single microscope at present.

[0032] Based on the present invention, we can achieve wide-band multimodal synchronous in vivo microscopic imaging on a single microscope. Compared with existing common technologies, such as using near-infrared zone 2 dyes to mark blood vessels and then perform single-photon confocal to obtain mouse brain vascular distribution and blood flow, or using green fluorescent protein to mark neurons to obtain mouse brain neuron structure or function information, and using fluorescent lifetime marker dyes to obtain mouse brain microenvironment information, the present invention can achieve synchronous acquisition of mouse brain neuron blood vessels and microenvironment information by a single microscope and diversified markers and spectroscopic strategies, which greatly saves information acquisition time and allows users to synchronously observe the dynamics and mutual connections of neurovascular units. In addition, the present invention also integrates commonly used visible light widefield and confocal imaging, as well as near-infrared zone 2 widefield imaging. Among them, visible light and near-infrared zone 2 widefield imaging can quickly assist users to locate the area required for microscopic observation due to their large field of view and fast imaging speed, and can also be used directly for body imaging; visible light widefield and confocal can also be used for biopsy slice observation of lesions, etc., so the common imaging needs of biomedical laboratories can be met by the present invention in one stop. In summary, the present invention has the advantages of rich functions, high information acquisition efficiency, good comprehensive utilization rate, high application adaptability, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 is a simplified schematic diagram of the overall structure of a multimodal fluorescence microscope in an embodiment of the present invention;

[0034] Figure 2 Schematic diagram of the optical path of the multimodal fluorescence microscope in the near-infrared second zone confocal imaging in an embodiment of the present invention;

[0035] Figure 3 is a schematic diagram of the optical path of a multimodal fluorescence microscope during multiphoton or harmonic imaging under near-infrared excitation in an embodiment of the present invention;

[0036] Figure 4 Schematic diagram of the optical path of the multi-modal fluorescence microscope during near-infrared excited multi-photon fluorescence lifetime imaging in an embodiment of the present invention;

[0037] Figure 5 Schematic diagram of the optical path of the visible light confocal module of the multimodal fluorescence microscope in an embodiment of the present invention;

[0038] Figure 6Schematic diagram of the optical path of the wide-field imaging optical path module of the multimodal fluorescence microscope in an embodiment of the present invention. DETAILED DESCRIPTION

[0039] The embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, but are not to be construed as limiting the present invention. All other embodiments obtained by ordinary technicians in the field without creative work based on the embodiments of the present invention are within the scope of protection of the present invention.

[0040] In the description of the present invention, it is to be understood that the terms “center”, “longitudinal”, “lateral”, “length”, “width”, “thickness”, “up”, “down”, “front”, “back”, “left”, “right”, “vertical”, “horizontal”, “top”, “bottom”, “inside”, “outside”, “clockwise”, “counterclockwise”, “axial”, “circumferential”, “radial”, etc., indicating orientations or positional relationships based on the orientations or positional relationships shown in the accompanying drawings, are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be understood as limiting the present invention.

[0041] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. In the description of the present invention, the meaning of "plurality" is two or more, unless otherwise clearly and specifically defined.

[0042] In this embodiment, if Figure 1As shown, a multimodal fluorescence microscope is provided, including a microscope component, a near-infrared second-zone excitation light path, a visible light confocal excitation light path, a wide-field imaging light path, a near-infrared second-zone confocal imaging component, a multiphoton intensity / lifetime and harmonic wave imaging component, and a visible light confocal and wide-field imaging component; the microscope component includes an objective lens OL, a filter switching component DM4, a sleeve lens TL1, and a transflection device arranged in sequence from the object side to the image side, and the transflection device can be switched to a dichroic mirror DM3 or a reflector M1; the near-infrared second-zone excitation light path, the visible light confocal excitation light path, and the wide-field imaging light path are connected to the sleeve lens TL1, and the near-infrared second-zone excitation light path is connected to the visible light confocal excitation light path. The invention is used to provide femtosecond laser with a wavelength of 680nm to 1300nm to the microscope component, the visible light confocal excitation optical path is used to provide lasers of four confocal bands to the microscope component, and the wide-field imaging optical path is used to provide visible light with a continuously distributed wavelength band of 400-700nm or near-infrared light with a switchable wavelength of 900-1450nm to the microscope component; the near-infrared second-zone confocal imaging component, the multi-photon intensity / lifetime and harmonic wave imaging component, and the visible light confocal and wide-field imaging components are all connected to the sleeve lens TL1 to receive the returned signal light, and the filter switching component DM4 and the transflective device are used to switch the light transmission state adapted to each imaging component.

[0043] Through the above implementation scheme, confocal and wide-field imaging of visible light, confocal and wide-field imaging of the second near-infrared region, and multi-photon intensity / lifetime and harmonic imaging based on near-infrared excitation are realized. Specifically, it includes visible light confocal imaging, visible light wide-field imaging; near-infrared second region confocal imaging, near-infrared second region wide-field imaging; two- / three-photon fluorescence intensity imaging, two- / three-photon fluorescence lifetime imaging; second / third harmonic imaging. The above modalities are all integrated into one microscope, and multiple modalities can be imaged synchronously. The imaging band is from 280nm to 1700nm, covering the imaging band of most fluorescent probes. As far as we know, this is the widest imaging band that can be achieved by a single microscope.

[0044] The near-infrared zone II excitation optical path includes a wavelength tunable femtosecond laser, a reflective beam expander BE, an achromatic lens L1, a pinhole PH1, an achromatic lens L2, a long-pass dichroic mirror DM1, a first scanning module, a scanning lens SL1, and a dichroic mirror DM2, which are arranged in sequence along the optical path. The scanning lens SL1 is anti-reflective at 700-1700nm, and the output optical path of the dichroic mirror DM2 is toward the transflective device. The wavelength tunable femtosecond laser is continuously adjustable at a wavelength of 680nm to 1300nm. The first scanning module includes scanners Scanner1, 2, achromatic lenses L3, achromatic lenses L4, and scanner Scanner3.

[0045] The near-infrared zone II excitation light route uses a wavelength tunable femtosecond laser as the light source, and the wavelength is continuously adjustable from 680nm to 1300nm. This laser can be used as a light source for near-infrared zone II confocal imaging, as well as for two-photon / three-photon fluorescence intensity imaging, two-photon / three-photon fluorescence lifetime imaging, and second / third harmonic imaging, and the wavelength covers the excitation band of commonly used probes. After the laser outputs the laser, it is expanded without chromatic aberration through a reflective beam expander BE, and then filtered by a 100μm pinhole PH1 after being focused by an achromatic lens L1. After filtering, it is collimated by an achromatic lens L2 and then incident on a long-pass dichroic mirror DM1. The dichroic mirror DM1 is installed on a magnetic dichroic mirror mount, which can be flexibly replaced according to the actual excitation / signal bands. The excitation light is reflected at the dichroic mirror DM1, and the signal light is transmitted. The reflected excitation light enters the scanning module, which is composed of Scanner1, 2, 3 and a pair of achromatic lenses L3L4, and is combined with the visible light excitation light path at the dichroic mirror DM2 after the scanning lens SL1 with anti-reflection in the second near-infrared region. The beam combining position is near the focal plane of SL1.

[0046] Furthermore, the near-infrared second-zone confocal imaging assembly includes a long-pass filter, a reflector M2, an achromatic lens L5, a pinhole PH2, a multimode optical fiber MMF1 and a photomultiplier tube PMT1 arranged in sequence on the optical path away from the long-pass dichroic mirror DM1, and the pinhole PH2 is on the focal plane of the achromatic lens L5.

[0047] Furthermore, the multi-photon intensity / lifetime and harmonic imaging component includes a short-reflecting dichroic mirror DM5, a collimating lens L6, a dichroic mirror DM6, a dichroic mirror DM7, and a filtering Fresnel lens F1 and a photomultiplier tube PMT2 in the transmission direction of the dichroic mirror DM6, a filtering Fresnel lens F2 and a photomultiplier tube PMT3 in the reflection direction of the dichroic mirror DM7, and a filtering Fresnel lens F3 and a photomultiplier tube PMT4 in the transmission direction of the dichroic mirror DM7; wherein, for near-infrared excited multi-photon or harmonic imaging, the transflective device is switched to the dichroic mirror DM3, the filtering Fresnel lens F1 and the filtering Fresnel lens F2 are each composed of a filter and a Fresnel lens, and the photomultiplier tube PMT2 is responsible for second / third harmonic imaging, photomultiplier tube PMT3 is responsible for two- / three-photon imaging, and the detection band of photomultiplier tubes PMT2 and PMT3 is 380-720nm; for multi-photon fluorescence lifetime imaging excited by near-infrared, the transflection device is switched to dichroic mirror DM3, photomultiplier tube PMT3 is a counting detector, and the time-correlated single-photon counting method (TCSPC) is used to achieve accurate lifetime measurement. The filtering Fresnel lens F3 consists of a filter and a Fresnel lens, and the detection band of photomultiplier tube PMT4 is 280-720nm; for the multi-channel synchronous imaging mode excited by near-infrared, a short-pass dichroic mirror is used at the dichroic mirror DM3 to split the excitation light and the longer single-photon signal, and the two-photon fluorescence and harmonic signal are transmitted.

[0048] Specifically, the near-infrared zone II excitation light is reflected at the dichroic mirror DM2 and reaches a dichroic mirror DM3 or a reflector M1. When single-photon confocal imaging is performed, the transflective device can be switched to the reflector M1, and the signal light returns along the original path of the excitation light path, and after exiting the first scanning module, it is separated from the excitation light at the dichroic mirror DM1. This process is also called unscanning imaging. When multi-photon or harmonic imaging is performed, the transflective device uses a short-pass dichroic mirror DM3, and the signal light with a shorter wavelength is separated from the near-infrared zone II excitation light here, and then enters the multi-photon or harmonic imaging channel. Since the signal light does not pass through the first scanning module again at this time, this process is also called non-unscanning imaging. Therefore, the dichroic mirror DM3 and the reflector M1 are installed in different magnetic dichroic mirror mounts, and can be installed in rotation according to actual needs. After the combined light passes through the sleeve lens TL1, it passes through the filter wheel DM4 in the fluorescent arm of the microscope frame and enters the objective lens. When performing point scanning imaging, the filter wheel DM4 needs to be turned to an empty position, that is, directly transmitting; when performing wide-field imaging, the filter wheel DM4 needs to be turned to other positions where filter groups are installed.

[0049] For near-infrared second-zone confocal imaging, the returned signal light passes through the dichroic mirror DM1 and a long-pass filter, is reflected by the reflector M2, is focused by the achromatic lens L5, and is filtered by the pinhole PH2 placed at the focal plane of L5. The filtered light is coupled into the multimode optical fiber MMF1 and docked with the optical fiber interface of the photomultiplier tube PMT1 to achieve near-infrared second-zone confocal imaging. The pinhole PH2 is an electric pinhole wheel that can select 16 pinhole sizes from 25μm to 2mm for different levels of filtering. The specific optical path used is as follows: Figure 2 shown.

[0050] For near-infrared excited multi-photon or harmonic imaging, the signal light passes through the dichroic mirror DM3 and is reflected by the short-reflection dichroic mirror DM5 to the collimating lens L6. The collimated signal light becomes parallel light and is split by the dichroic mirrors DM6 and 7. The photomultiplier tube PMT2 is responsible for second / third harmonic imaging, and the photomultiplier tube PMT3 is responsible for two / three-photon imaging. The filtering Fresnel lens F1 and the filtering Fresnel lens F2 are both composed of a filter and a Fresnel lens. The filter is responsible for filtering the signal light to improve the signal-to-noise ratio. The Fresnel lens has a very short focal length and can effectively focus the parallel light onto the photosensitive surface of the PMT in a compact space. This part of the optical path is shown in the figure. Figure 3 shown.

[0051] For near-infrared excited multiphoton fluorescence lifetime imaging, it is similar to near-infrared excited multiphoton or harmonic imaging, except that the detector is changed to photomultiplier tube PMT4, which is a counting detector and uses time-correlated single photon counting (TCSPC) to achieve accurate lifetime measurement. The position information required by this method is synchronously given to the TCSPC acquisition card by the control program. F3 has the same structure and function as F1 and F2. In addition, DM7 can be replaced by a half-reflective half-mirror. With the support of sample marking, synchronous imaging of PMT1, 2, 3, and 4 can be achieved, realizing the optical path such as Figure 4 As shown. As far as we know, this is the method with the most wavelengths and modes that can be achieved by single-channel excitation.

[0052] For the multi-channel synchronous imaging mode of near-infrared excitation, the sample needs to be multi-labeled so that multiple wavelengths of signals can be output under a single excitation wavelength. A short-pass dichroic mirror is used at the dichroic mirror DM3 to split the light, and the excitation light and longer single-photon signals are reflected, while two-photon fluorescence and harmonic signals are transmitted. For example, the sample is double-labeled with GFP green fluorescent protein and near-infrared second-zone quantum dot dyes. GFP can be labeled in rat brain neurons, and quantum dots can be labeled in blood vessels. After that, under the excitation state of 920nm, GFP emits two-photon fluorescence signals and is captured by PMT3 and 4, respectively, to achieve two-photon intensity and lifetime imaging; quantum dots emit single-photon fluorescence signals and are captured by PMT1, to achieve single-photon confocal imaging; collagen and fibers in the sample can emit second harmonic SHG signals and be captured by PMT2, to achieve harmonic imaging. Therefore, under the appropriate labeling and filter selection, this system can achieve synchronous in vivo microscopic imaging of four modes of single-wavelength excitation. As far as we know, no microscope has been able to do this in the past.

[0053] For near-infrared excited dual / multi-photon fluorescence lifetime imaging, since the existing fluorescence lifetime measurement technology is mainly based on TCSPC, this technology has the advantages of high precision and good time resolution, but the technology is too slow, and the limited photon count rate limits its fastest imaging speed, making it difficult to truly synchronize with dual / multi-photon imaging, etc., and it often takes dozens of frames of scanning to superimpose a usable lifetime image. Therefore, in some embodiments, the scheme of the present application can use Pulse-Sampling technology for lifetime measurement. This method requires the use of a high-speed digitizer and a high-speed PMT to directly obtain the fluorescence decay curve after a single excitation pulse and obtain the lifetime result. The time accuracy of this method is limited by the sampling rate of the digitizer and the time jitter of the PMT, which can be about 300ps, but considering the lifetime length of the fluorescent probes involved in actual biological imaging - 1ns or more, the time accuracy of this method is fully sufficient and can meet actual needs.

[0054] Furthermore, the visible light confocal excitation optical path includes a four-wavelength laser module, a single-mode optical fiber SMF, a 10x objective lens L7, a multi-bandpass dichroic mirror DM8, a second scanning module, a scanning lens SL2 and a dichroic mirror DM2 arranged in sequence along the optical path. The four-wavelength laser module includes four lasers with wavelengths of 405nm, 488nm, 561nm and 638nm. The lasers of each laser are respectively coupled to the single-mode optical fiber SMF through a dichroic mirror. The scanning lens SL2 is enhanced in the visible light band, and the dichroic mirror DM2 is also used for combining with the near-infrared second zone excitation optical path. The second scanning module includes a scanner Scanner4,5, an achromatic lens L8, a 2-inch elliptical reflector M3, an achromatic lens L9 and a scanner Scanner6 arranged in sequence.

[0055] The visible light confocal and wide-field imaging assembly includes a reflecting mirror M4, a focusing lens L10, a pinhole PH3, a multimode optical fiber MMF2, a focusing lens L11, a dichroic mirror DM9, a dichroic mirror DM10, a filtering Fresnel lens F4 and a photomultiplier tube PMT5 on the reflected light path of the dichroic mirror DM9, a filtering Fresnel lens F5 and a photomultiplier tube PMT6 on the reflected light path of the dichroic mirror DM10, and a filtering Fresnel lens F6 and a photomultiplier tube PMT7 on the transmitted light path of the dichroic mirror DM10, which are arranged in sequence on the light path of the multi-bandpass dichroic mirror DM8 away from the 10x objective lens L7. The photomultiplier tubes PMT5, PMT6 and PMT7 constitute three imaging channels to detect the blue band, the green band and the red band respectively.

[0056] Specifically, the excitation light path of the visible light confocal module uses a 4-wavelength laser as the light source, with wavelengths of 405nm, 488nm, 561nm, and 638nm. These four wavelengths cover the commonly used excitation bands of visible light confocal. After the 4 wavelengths are combined by the dichroic mirror, they are coupled to the single-mode fiber SMF by the achromatic lens. The core of the single-mode fiber is the excitation pinhole. After the light from the optical fiber is collimated by the 10x objective lens L7, it is incident on a multi-bandpass dichroic mirror DM8. The excitation light is reflected at the dichroic mirror DM8, and the signal light is transmitted. The reflected excitation light enters the second scanning module, which is composed of scanners Scanner4, 5, 6, a pair of achromatic lenses L8L9, and a 2-inch elliptical reflector M3. After the scanning lens SL2 with visible light band transmission, it is combined with the near-infrared second zone excitation light path at the dichroic mirror DM2. The combined light is reflected by the reflector M1 and enters the sleeve lens, and then passes through the filter wheel DM4 in the fluorescence arm of the microscope frame and enters the objective lens OL. When performing point scanning imaging, the filter wheel DM4 needs to be turned to the empty position, that is, directly transmitted; when performing wide-field imaging, the filter wheel DM4 needs to be turned to other positions where filter groups are installed. After the returned signal light is transmitted at the dichroic mirror DM8, it is reflected by the reflector M4 to the focusing lens L10. The pinhole PH3 is the same as the pinhole PH2, both of which are 16-position adjustable pinhole wheels. The signal light is filtered and coupled into the multimode fiber MMF2 and collimated by the focusing lens L11 at the light outlet. The photomultiplier tubes PMT5, 6, and 7 constitute three imaging channels, detecting the blue band, green band, and red band respectively, and the dichroic mirrors DM9 and 10 complete the light splitting. The configuration of the filter Fresnel lens F4F5F6 is similar to that of the filter Fresnel lens F1F2F3. The Fresnel lenses used are the same, but different filters are configured according to different imaging bands. Therefore, the excitation optical path can simultaneously realize 3-channel imaging. Figure 5 shown.

[0057] Furthermore, the microscope assembly includes a frame fluorescent arm on which a filter switching assembly DM4 is arranged, the frame fluorescent arm is provided with an excitation light source entrance corresponding to the position of the filter switching assembly DM4, and the wide-field imaging optical path includes a white light LED or a near-infrared LED switchably installed at the excitation light source entrance, the white light LED excitation band can be continuously distributed in 400-700nm, and the optional wavelength of the near-infrared LED is 900-1450nm. The visible light confocal and wide-field imaging assembly includes a short-reflecting dichroic mirror DM5 and a switchable camera on the optical path of the transflective device away from the filter switching assembly DM4, and the switchable camera can be switched to a visible light camera and a near-infrared camera, the detection band of the visible light camera is 380-1000nm, and the detection band of the near-infrared camera is 900-1700nm.

[0058] Specifically, the light inlet of the fluorescence arm of the microscope stand is the entrance of the excitation light source for wide-field imaging. When a white light LED is installed, the excitation band can be continuously distributed in 400-700nm. The specific band to be used for excitation is determined by the screening of the excitation filter group; when a near-infrared LED is installed, fluorescence excitation in the near-infrared zone II can be performed. There are currently a variety of near-infrared LEDs on the market. This solution currently configures an LED with a wavelength of 950nm. Both the LED light outlet and the light inlet of the fluorescence arm are SM1 threads, so the white light LED and the near-infrared LED can be flexibly replaced with the help of a pluggable SM1 lens sleeve according to actual imaging needs. Since the bands of visible wide-field imaging and near-infrared zone II wide-field imaging are different, we have configured a filter combination of three channels of red, green, and blue and a long-pass filter combination of near-infrared zone II at the filter wheel DM4 of the fluorescence arm. Wide-field imaging of different bands requires turning the wheel to switch to the corresponding filter combination, that is, the filter box at the filter wheel DM4 may be empty, or any configuration of red, green, blue, and near-infrared. The signal light is transmitted through the filter wheel and focused on the imaging camera through a tube lens that is consistent with point scanning imaging. In addition, the interfaces between the visible light camera and the near-infrared camera and the system are made pluggable, so that the camera can be flexibly replaced when imaging at different wavelengths. Figure 6 shown.

[0059] The present invention also provides a method for using a multimodal fluorescence microscope, which is applied to a multimodal fluorescence microscope as described in any of the above items. The microscope can flexibly set corresponding lenses at DM1, DM3, DM4, DM5, DM6, and DM7 according to actual imaging. The lens described in DM4 is installed in the filter switching wheel in the fluorescence arm of the microscope frame, and there are 6 usable slots. Slots 1-3 are respectively configured for violet light excitation / blue light imaging, blue light excitation / green light imaging, and yellow light excitation / red light imaging. Slot 4 is near-infrared zone 1 excitation (750-980nm) / near-infrared zone 2 imaging (1000-1700nm). Slots 5 and 6 are not provided with lenses, and light can pass directly through; the lenses described in DM1, 3, 5, 6, and 7 are installed in the magnetic core of the magnetic dichroic mirror cube, the mounting seat of the cube is fixed and unchanged, and the magnetic core is pluggable, so different types of lenses can be installed in the magnetic core and configured on mounting seats at different positions according to imaging needs to achieve corresponding functions.

[0060] Classification of imaging modalities of the present invention: The present invention can be divided into two categories: wide-field fluorescence microscopy and point scanning fluorescence microscopy. The specific distinction is shown in the following table:

[0061] Table 1: Classification of imaging modalities

[0062]

[0063] The specific usage includes:

[0064] Visible widefield imaging mode:

[0065] A white light LED is configured at the entrance of the excitation light source, and its output spectrum covers 400nm-700nm. First, turn on the white light LED, and rotate the filter switching component DM4 to any one of the slots 1 to 3. The filter at this position corresponds to the fluorescence emission color of the sample; then turn on the visible light camera, and set the dichroic mirror DM3 and the dichroic mirror DM5 to empty magnetic cores to ensure the passage of light. Then, place the sample on the stage, and adjust the height of the stage to achieve focus. Focusing is achieved by observing the image of the camera;

[0066] Near-infrared wide-field imaging in the second region:

[0067] For this mode, the usage is basically the same as that of the visible light wide field. A near-infrared LED is configured at the entrance of the excitation light source, and its luminous band is adapted to the dye marking situation. For example, if the dye needs to be optimally excited at 940nm, we can install a near-infrared LED with a central wavelength of 940nm; replace the visible light camera with a near-infrared camera, and then rotate the filter switching assembly DM4 to slot 4, and set the dichroic mirror DM3 and dichroic mirror DM5 to an empty magnetic core to ensure the passage of light. Then place the sample on the stage, adjust the height of the stage to achieve focus, and focus is achieved by observing the image of the camera;

[0068] The optical paths of the above two modes are as follows: Figure 6 shown.

[0069] Visible light confocal imaging:

[0070] This microscope is equipped with a 4-wavelength continuous laser module, which contains a 405nm, 488nm, 561nm, and 638nm continuous light laser. They are combined in the same module and output after coupling through a single-mode fiber SMF. When multi-color imaging, it is generally necessary to turn on lasers of multiple wavelengths to excite different fluorescent dyes separately. The operation steps when using this mode are as follows: turn on the light source of the four-wavelength laser module and turn on the laser output of the corresponding wavelength; turn on the power of scanners Scanner4, 5, 6, and turn on the power of the electric pinhole wheel PH3 and photomultiplier tubes PMT5, 6, 7; insert a magnetic core with a reflector at the dichroic mirror DM3 to achieve the function of full reflection; rotate the dichroic mirror DM4 to slot No. 5 to allow all light to pass; place the sample on the stage, open the imaging software to start scanning, and adjust the left and right position and height of the sample, as well as the size of the pinhole PH3 according to the image results obtained by the scan; realize the synchronous acquisition of the three-way signals of the photomultiplier tubes PMT5, 6, and 7 through multiple acquisition channels to achieve three-color synchronous imaging;

[0071] Near-infrared zone 2 confocal imaging:

[0072] The imaging of this mode is different from visible light confocal imaging. Its usage is as follows: turn on the power of the near-infrared second zone photomultiplier tube PMT1 and wait for 1 hour of cooling time; turn on the power of the wavelength tunable femtosecond laser Femtosecond Laser and wait for 15 minutes of warm-up time, then set the required output wavelength and start the laser output; turn on the power of scanners Scanner1, 2, and 3, preset the reverse voltage of the photomultiplier tube PMT1 to -500V, and turn on the power of the electric pinhole wheel PH2; insert a magnetic core with a reflector at the dichroic mirror DM3 to achieve the function of full reflection, and rotate the dichroic mirror DM4 to slot 5 to allow all light to pass through; place the sample on the stage and open the imaging software to start scanning and imaging; this mode does not have the ability of multi-color synchronous imaging, because The light source can only output one wavelength of laser at a time, and there is only one corresponding detector. If multi-color imaging is required, switch the magnetic core at the dichroic mirror DM1, configure the dichroic mirror / filter required for the corresponding band excitation / imaging, and then perform imaging in batches. When the sample is labeled with both visible light fluorescent dyes and near-infrared zone II fluorescent dyes, the visible light confocal and near-infrared zone II confocal imaging modules can be turned on at the same time, and the acquisition system can simultaneously collect the output signals of the four detectors of the photomultiplier tubes PMT1, 5, 6, and 7 and image them, realizing the four-channel confocal synchronous imaging function. At this time, the optical path is as follows: Figure 5 shown.

[0073] Non-Linear Imaging:

[0074] Nonlinear imaging and near-infrared zone 2 confocal imaging share most of the optical paths. When using this mode: turn on the power of the wavelength tunable femtosecond laser Femtosecond Laser, wait for 15 minutes of warm-up time, then set the required output wavelength and start the laser output; turn on the power of scanners Scanner1, 2, 3, preset the reverse voltage of the photomultiplier tube PMT1 to -500V, and turn on the power of the electric pinhole wheel PH2; turn on the power of the photomultiplier tubes PMT2, 3, 4, and open the control software; place the sample on the stage, turn on the wavelength tunable femtosecond laser Femtosecond Laser, and wait for 15 minutes of warm-up time, then set the required output wavelength and start the laser output; turn on the power of scanners Scanner1, 2, 3, and ... and set the reverse voltage of the photomultiplier tube PMT1 to The output of Laser; configure the dichroic mirror DM3 as a two-photon or three-photon dichroic mirror, such as 720SP. The dichroic mirror can transmit light with a wavelength shorter than 720nm and reflect light with a wavelength longer than 720nm. In this way, the long-wavelength excitation light can be reflected to the sample at DM3, and the short-wavelength signal light can be transmitted from DM3 to the detection light path; configure the dichroic mirror DM5 as a combination of a 720LP dichroic mirror and a 720SP filter, so that the excitation light slightly leaked at the dichroic mirror DM3 is further intercepted and most of the signal light is transmitted; the separation of the fluorescence signal and the harmonic signal in the signal light is achieved through the dichroic mirror DM6. The basic principle is that under the same excitation wavelength, the wavelength of the harmonic signal will be shorter than that of the fluorescence signal. Therefore, DM6 will be set as a short-pass dichroic mirror to transmit the harmonic signal with a shorter wavelength. The signal is transmitted to the harmonic detector PMT2, and the fluorescence signal with a longer wavelength is reflected to DM7; the dichroic mirror DM7 needs to be configured according to the difference between fluorescence intensity imaging and fluorescence lifetime imaging. When only fluorescence intensity imaging is performed, a magnetic core with a built-in reflector is inserted into the dichroic mirror DM7 to reflect all the fluorescence to the photomultiplier tube PMT3. When only fluorescence lifetime imaging is performed, a magnetic core without a lens is inserted into the dichroic mirror DM7 to transmit all the fluorescence to the photomultiplier tube PMT4. When simultaneous imaging of intensity and lifetime is required, a magnetic core with a built-in semi-reflective and semi-transparent film is inserted into the dichroic mirror DM7 to send half of the fluorescence to the photomultiplier tube PMT3 and the other half to the photomultiplier tube PMT4. Open the imaging software and start scanning to obtain the images corresponding to the output signals of the photomultiplier tubes PMT2, 3, and 4. The optical path is as follows Figure 3 shown.

[0075] Four-channel simultaneous imaging:

[0076] When the sample has multiple labels, such as labels required for multi-photon imaging and labels required for near-infrared zone 2 single-photon confocal, the microscope can achieve 4-channel synchronous imaging. The specific implementation method is: turn on the power of the wavelength tunable femtosecond laser Femtosecond Laser, scanner Scanner1,2,3, photomultiplier tubes PMT1,2,3,4 and pinhole PH2, and wait for the equipment to be ready; set DM3 to 720SP dichroic mirror. For near-infrared zone 2 confocal, the wavelengths of excitation light and signal light are both longer than 720nm, so both can be reflected at DM3. For nonlinear imaging, the configuration does not change; place the sample on the stage, and connect the signals of photomultiplier tubes PMT1,2,3,4 to the acquisition system, start the imaging software to perform scanning, because the light source, optical path and scanning system are shared, and all are single-point scanning imaging, so the software can achieve synchronous image reconstruction of the four channels of photomultiplier tubes PMT1,2,3,4, thereby achieving four-channel synchronous imaging. The optical path is as follows: Figure 4 shown.

[0077] Based on the present invention, we can achieve wide-band multimodal synchronous in vivo microscopic imaging on a single microscope. Compared with existing common technologies, such as using near-infrared zone 2 dyes to mark blood vessels and then perform single-photon confocal to obtain mouse brain vascular distribution and blood flow, or using green fluorescent protein to mark neurons to obtain mouse brain neuron structure or function information, and using fluorescent lifetime marker dyes to obtain mouse brain microenvironment information, the present invention can achieve synchronous acquisition of mouse brain neuron blood vessels and microenvironment information by a single microscope and diversified markers and spectroscopic strategies, which greatly saves information acquisition time and allows users to synchronously observe the dynamics and mutual connections of neurovascular units. In addition, the present invention also integrates commonly used visible light widefield and confocal imaging, as well as near-infrared zone 2 widefield imaging. Among them, visible light and near-infrared zone 2 widefield imaging can quickly assist users to locate the area required for microscopic observation due to their large field of view and fast imaging speed, and can also be used directly for body imaging; visible light widefield and confocal can also be used for biopsy slice observation of lesions, etc., so the common imaging needs of biomedical laboratories can be met by the present invention in one stop. In summary, the present invention has the advantages of rich functions, high information acquisition efficiency, good comprehensive utilization rate, high application adaptability, etc.

[0078] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A multimodal fluorescence microscope, characterized in that: It includes a microscope component, a near-infrared second-zone excitation optical path, a visible light confocal excitation optical path, a wide-field imaging optical path, a near-infrared second-zone confocal imaging component, a multiphoton intensity / lifetime and harmonic wave imaging component, and a visible light confocal and wide-field imaging component; The microscope assembly includes an objective lens OL, a filter switching assembly DM4, a tube lens TL1 and a transflective device arranged in sequence from the object side to the image side, and the transflective device can be switched to a dichroic mirror DM3 or a reflective mirror M1; The near-infrared second-zone excitation optical path, the visible light confocal excitation optical path and the wide-field imaging optical path are connected to the tube lens TL1, the near-infrared second-zone excitation optical path is used to provide a femtosecond laser with a wavelength of 680nm to 1300nm to the microscope component, the visible light confocal excitation optical path is used to provide a laser with four confocal bands to the microscope component, and the wide-field imaging optical path is used to provide a visible light with a continuous distribution of 400-700nm or a near-infrared light with a wavelength of 900-1450nm to the microscope component; The near-infrared two-zone confocal imaging component, the multiphoton intensity / lifetime and harmonic wave imaging component, and the visible light confocal and wide-field imaging component are all connected to the tube lens TL1 to receive the returned signal light, and the filter switching component DM4 and the transflective device are used to switch the light transmission state adapted to each of the imaging components.

2. The multimodal fluorescence microscope according to claim 1, characterized in that: The near-infrared second zone excitation optical path includes a wavelength tunable femtosecond laser, a reflective beam expander BE, an achromatic lens L1, a pinhole PH1, an achromatic lens L2, a long-pass dichroic mirror DM1, a first scanning module, a scanning lens SL1 and a dichroic mirror DM2 arranged in sequence along the optical path. The scanning lens SL1 increases transmittance at 700-1700nm, the output optical path of the dichroic mirror DM2 is toward the transflective device, and the wavelength tunable femtosecond laser is continuously adjustable at a wavelength of 680nm to 1300nm.

3. The multimodal fluorescence microscope according to claim 2, characterized in that: The first scanning module includes scanners Scanner1, 2, an achromatic lens L3, an achromatic lens L4 and a scanner Scanner3.

4. The multimodal fluorescence microscope according to claim 2, characterized in that: The near-infrared two-zone confocal imaging assembly includes a long-pass filter, a reflector M2, an achromatic lens L5, a pinhole PH2, a multimode optical fiber MMF1 and a photomultiplier tube PMT1 arranged in sequence on the optical path away from the long-pass dichroic mirror DM1, and the pinhole PH2 is on the focal plane of the achromatic lens L5.

5. The multimodal fluorescence microscope according to claim 2, characterized in that: The multi-photon intensity / lifetime and harmonic wave imaging assembly comprises a short reflection dichroic mirror DM5, a collimating lens L6, a dichroic mirror DM6, a dichroic mirror DM7, and a filtering Fresnel lens F1 and a photomultiplier tube PMT2 in the transmission direction of the dichroic mirror DM6, a filtering Fresnel lens F2 and a photomultiplier tube PMT3 in the reflection direction of the dichroic mirror DM7, and a filtering Fresnel lens F3 and a photomultiplier tube PMT4 in the transmission direction of the dichroic mirror DM7, which are arranged in sequence along the optical path direction of the transflection device away from the sleeve lens TL1; Wherein, for near-infrared excited multi-photon or harmonic imaging, the transflective device is switched to a dichroic mirror DM3, the filtering Fresnel lens F1 and the filtering Fresnel lens F2 are both composed of a filter and a Fresnel lens, the photomultiplier tube PMT2 is responsible for second / third harmonic imaging, the photomultiplier tube PMT3 is responsible for two / three-photon imaging, and the detection band of the photomultiplier tube PMT2 and the photomultiplier tube PMT3 is 380-720nm; For near-infrared excited multi-photon fluorescence lifetime imaging, the transflective device is switched to the dichroic mirror DM3, the photomultiplier tube PMT3 is a counting detector, and the time-correlated single photon counting method (TCSPC) is used to achieve accurate lifetime measurement. The filtering Fresnel lens F3 consists of a filter and a Fresnel lens, and the detection band of the photomultiplier tube PMT4 is 280-720nm. For the multi-path synchronous imaging mode of near-infrared excitation, a short-pass dichroic mirror is used to split the light at the dichroic mirror DM3, so that the excitation light and longer single-photon signals are reflected, and the two-photon fluorescence and harmonic wave signals are transmitted.

6. The multimodal fluorescence microscope according to claim 1, characterized in that: The visible light confocal excitation optical path includes a four-wavelength laser module, a single-mode optical fiber SMF, a 10x objective lens L7, a multi-bandpass dichroic mirror DM8, a second scanning module, a scanning lens SL2 and a dichroic mirror DM2 which are sequentially arranged along the optical path. The four-wavelength laser module includes four lasers with wavelengths of 405nm, 488nm, 561nm and 638nm. The lasers of each laser are respectively combined and coupled to the single-mode optical fiber SMF through a dichroic mirror. The scanning lens SL2 increases transmittance in the visible light band, and the dichroic mirror DM2 is also used for combining with the near-infrared second-zone excitation optical path.

7. The multimodal fluorescence microscope according to claim 6, characterized in that: The second scanning module includes scanners Scanner4, 5, an achromatic lens L8, a 2-inch elliptical reflector M3, an achromatic lens L9 and a scanner Scanner6 which are arranged in sequence.

8. The multimodal fluorescence microscope according to claim 6, characterized in that: The visible light confocal and wide-field imaging component includes a reflecting mirror M4, a focusing lens L10, a pinhole PH3, a multimode optical fiber MMF2, a focusing lens L11, a dichroic mirror DM9, a dichroic mirror DM10, a filtering Fresnel lens F4 and a photomultiplier tube PMT5 on the reflected light path of the dichroic mirror DM9, a filtering Fresnel lens F5 and a photomultiplier tube PMT6 on the reflected light path of the dichroic mirror DM10, and a filtering Fresnel lens F6 and a photomultiplier tube PMT7 on the transmitted light path of the dichroic mirror DM10, which are arranged in sequence on the light path of the multi-bandpass dichroic mirror DM8 away from the 10x objective lens L7. The photomultiplier tubes PMT5, PMT6 and PMT7 constitute three imaging channels to detect the blue band, the green band and the red band respectively.

9. The multimodal fluorescence microscope according to claim 1, characterized in that: The microscope assembly includes a frame fluorescent arm on which the filter switching assembly DM4 is arranged, the frame fluorescent arm is provided with an excitation light source entrance corresponding to the position of the filter switching assembly DM4, the wide-field imaging optical path includes a white light LED or a near-infrared LED switchably installed at the excitation light source entrance, the white light LED excitation band can be continuously distributed in the range of 400-700nm, and the peak wavelength of the near-infrared LED is between 900-1450nm; The visible light confocal and wide-field imaging component includes a short-reflecting dichroic mirror DM5 and a switchable camera on the optical path of the transflective device away from the filter switching component DM4. The switchable camera can be switched to a visible light camera and a near-infrared camera. The detection band of the visible light camera is 380-1000nm, and the detection band of the near-infrared camera is 900-1700nm.

10. A method for using a multimodal fluorescence microscope, applied to the multimodal fluorescence microscope according to any one of claims 1 to 9, characterized in that: include: Visible widefield imaging mode: A white light LED is configured at the entrance of the excitation light source, and its output spectrum covers 400nm-700nm. First, turn on the white light LED and rotate the filter switching component DM4 to the filter position corresponding to the fluorescence emission color of the sample; then turn on the visible light camera, and set the dichroic mirror DM3 and the dichroic mirror DM5 to empty magnetic cores to ensure the passage of light. Then, place the sample on the stage and adjust the height of the stage to achieve focus. Focus is achieved by observing the image of the camera; Near-infrared wide-field imaging in the second region: A near-infrared LED is configured at the entrance of the excitation light source, and its luminous band is adapted to the dye marking situation; the visible light camera is replaced with a near-infrared camera, and then the filter switching component DM4 is rotated to the 4-slot position, and the dichroic mirror DM3 and the dichroic mirror DM5 are set to empty magnetic cores to ensure the passage of light, and then the sample is placed on the stage, and the height of the stage is adjusted to achieve focusing, which is achieved by observing the image of the camera; Visible light confocal imaging: Turn on the light source of the four-wavelength laser module and turn on the laser output of the corresponding wavelength; turn on the power of scanners Scanner4, 5, 6, and turn on the power of the electric pinhole wheel PH3 and the photomultiplier tubes PMT5, 6, 7; insert a magnetic core with a reflector at the dichroic mirror DM3 to achieve the function of full reflection; rotate the dichroic mirror DM4 to slot No. 5 to allow all light to pass; place the sample on the stage, open the imaging software to start scanning, and adjust the left and right position and height of the sample, as well as the size of the pinhole PH3 according to the image results obtained by the scan; realize the synchronous acquisition of the three-way signals of the photomultiplier tubes PMT5, 6, and 7 through multiple acquisition channels to achieve three-color synchronous imaging; Near-infrared zone 2 confocal imaging: Turn on the power of the near-infrared second-zone photomultiplier tube PMT1 and wait for 1 hour of cooling time; turn on the wavelength tunable femtosecond laser Femtosecond Turn on the power of the Laser, wait for 15 minutes of warm-up time, then set the required output wavelength and start the laser output; turn on the power of scanners Scanner1, 2, and 3, preset the reverse voltage of the photomultiplier tube PMT1 to -500V, and turn on the power of the electric pinhole wheel PH2; insert the magnetic core with a reflector at the dichroic mirror DM3 to achieve the function of full reflection, and rotate the dichroic mirror DM4 to slot 5 to allow all light to pass through; place the sample on the stage and open the imaging software to start scanning and imaging; if multi-color imaging is required, switch the magnetic core at the dichroic mirror DM1, configure the dichroic mirror / filter required for excitation / imaging of the corresponding band, and then image in batches; when the sample is marked with both visible light fluorescent dyes and near-infrared zone II fluorescent dyes, the visible light confocal and near-infrared zone II confocal imaging modules can be turned on at the same time, and the acquisition system can simultaneously acquire the output signals of the four detectors of the photomultiplier tubes PMT1, 5, 6, and 7 and image them, realizing the four-channel confocal synchronous imaging function; Non-linear imaging: Turn on the power of the wavelength tunable femtosecond laser Femtosecond Laser, wait for 15 minutes of warm-up time, then set the required output wavelength and start the laser output; turn on the power of scanners Scanner1, 2, and 3, preset the reverse voltage of the photomultiplier tube PMT1 to -500V, and turn on the power of the electric pinhole wheel PH2; Turn on the power of photomultiplier tubes PMT2, 3, and 4, and open the control software; place the sample on the stage, and turn on the output of the wavelength-tunable femtosecond laser Femtosecond Laser; configure the dichroic mirror DM3 as a two-photon or three-photon dichroic mirror; configure the dichroic mirror DM5 as a combination of a 720LP dichroic mirror and a 720SP filter, so that the slightly leaked excitation light at the dichroic mirror DM3 is further intercepted, and most of the signal light is transmitted; separate the fluorescence signal and the harmonic signal in the signal light through the dichroic mirror DM6; according to The difference between fluorescence intensity imaging and fluorescence lifetime imaging requires the configuration of a dichroic mirror DM7. When only fluorescence intensity imaging is performed, a magnetic core with a built-in reflector is inserted into the dichroic mirror DM7 to reflect all the fluorescence to the photomultiplier tube PMT3. When only fluorescence lifetime imaging is performed, a magnetic core without a lens is inserted into the dichroic mirror DM7 to transmit all the fluorescence to the photomultiplier tube PMT4. When simultaneous imaging of intensity and lifetime is required, a magnetic core with a built-in semi-reflective and semi-transparent film is inserted into the dichroic mirror DM7 to send half of the fluorescence to the photomultiplier tube PMT3 and the other half to the photomultiplier tube PMT4. Open the imaging software and start scanning to obtain the image corresponding to the output signal of the photomultiplier tubes PMT2, 3, and 4; Four-channel simultaneous imaging: Turn on the power of the wavelength tunable femtosecond laser Femtosecond Laser, scanners Scanner1, 2, 3, photomultiplier tubes PMT1, 2, 3, 4 and pinhole PH2, and wait for the equipment to be ready; set DM3 to 720SP dichroic mirror. For near-infrared second-zone confocal, the wavelengths of the excitation light and signal light are both longer than 720nm, so both can be reflected at DM3. For nonlinear imaging, the configuration remains unchanged; place the sample on the stage, and connect the signals of the photomultiplier tubes PMT1, 2, 3, 4 to the acquisition system, and start the imaging software to perform scanning. Because the light source, optical path and scanning system are shared, and all are single-point scanning imaging, the software can realize synchronous image reconstruction of the four channels of photomultiplier tubes PMT1, 2, 3, 4, thereby realizing synchronous imaging of the four channels.

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