A multimodal fluorescence microscope and its usage method

Abstract

This invention provides a multimodal fluorescence microscope and its usage method. The microscope includes a microscopic assembly, a near-infrared II excitation optical path, a visible light confocal excitation optical path, a wide-field imaging optical path, a near-infrared II confocal imaging component, a multiphoton intensity / lifetime and harmonic imaging component, and a visible light confocal and wide-field imaging component. The microscopic assembly includes an objective lens, a filter switching component, a sleeve lens, and a transmission / reflection device arranged sequentially from the object side to the image side. The transmission / reflection device can be switched to a dichroic mirror or a reflecting mirror. The near-infrared II excitation optical path, the visible light confocal excitation optical path, and the wide-field imaging optical path are connected to the sleeve lens. The near-infrared II confocal imaging component, the multiphoton intensity / lifetime and harmonic imaging component, and the visible light confocal and wide-field imaging component are all connected to the sleeve lens. This solution achieves visible light confocal and wide-field imaging, near-infrared II confocal and wide-field imaging, and multiphoton intensity / lifetime and harmonic imaging based on near-infrared excitation.

Description

Technical Field

[0001] This invention belongs to the field of optical system technology, and particularly relates to a multimodal fluorescence microscope and its usage method. Background Technology

[0002] Currently developed fluorescence microscopes commonly employ modalities, including wide-field fluorescence microscopy, confocal fluorescence microscopy, two / three-photon fluorescence microscopy, and fluorescence lifetime microscopy. Each of these microscopes has its own characteristics; for example, wide-field fluorescence microscopy offers fast imaging speed, while confocal fluorescence microscopy provides high resolution and optical tomography capabilities—enabling depth resolution. Therefore, multimodal microscopes have emerged on the market, incorporating several of the aforementioned microscopic imaging modalities to combine their advantages and address the needs of different imaging applications.

[0003] In addition, the development of near-infrared II microscopy has been booming over the past decade. Because light in the near-infrared II region has lower scattering and absorption in biological tissues, it can penetrate to greater depths. Therefore, fluorescence microscopy in the near-infrared II region has also seen increasingly rich development in imaging bands, and the imaging modes are gradually becoming consistent with those in the visible light region.

[0004] In related technologies, multimodal fluorescence microscopy imaging modes are relatively limited, encompassing only a finite number of commonly used imaging modes. For example, Nikon's A1R MP from Japan offers single / two-photon and second-harmonic generation scanning imaging, but lacks lifetime imaging capabilities and near-infrared II imaging modes. The Korean IVI M is essentially the same as Nikon's. Leica's TCSSP8 from Germany has two-photon, second-harmonic, and lifetime imaging modules, but it lacks integrated near-infrared II imaging, and as an inverted microscope, it cannot be used for in-situ live microscopy. In the field of near-infrared II imaging, existing technologies include near-infrared II confocal microscopy and wide-field imaging, but their imaging bands are also limited to the near-infrared II region. In other words, currently no microscope can extend its imaging band to a wide range from visible light to the near-infrared II region and is fully compatible with commonly used imaging modes, allowing users to complete almost all necessary imaging experiments on a single microscope. Summary of the Invention

[0005] The technical objective of this invention is to provide a multimodal fluorescence microscope and its usage method, which can achieve confocal and wide-field imaging in the visible light region, confocal and wide-field imaging in the near-infrared region, and multiphoton intensity / lifetime and harmonic imaging based on near-infrared excitation.

[0006] To solve the above-mentioned technical problems, the present invention provides a multimodal fluorescence microscope, including a microscopic assembly, a near-infrared II excitation optical path, a visible light confocal excitation optical path, a wide-field imaging optical path, a near-infrared II confocal imaging component, a multiphoton intensity / lifetime and harmonic imaging component, and a visible light confocal and wide-field imaging component. The microscopic assembly includes an objective lens OL, a filter switching component DM4, a sleeve lens TL1, and a transmission / reflection device arranged sequentially from the object side to the image side. The transmission / reflection device can be switched to a dichroic mirror DM3 or a reflecting mirror M1. The near-infrared II excitation optical path, the visible light confocal excitation optical path, and the wide-field imaging optical path are connected to the sleeve lens TL1. The outer second-zone excitation optical path is used to provide femtosecond lasers with wavelengths of 680nm to 1300nm to the microscopic components. The visible light confocal excitation optical path is used to provide lasers with four confocal bands to the microscopic components. The wide-field imaging optical path is used to provide visible light with a continuous wavelength distribution of 400-700nm or near-infrared light with a wavelength of 900-1450nm to the microscopic components. The near-infrared second-zone confocal imaging component, the multiphoton intensity / lifetime and harmonic 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. The filter switching component DM4 and the transmission-reflection device are used to switch the light transmission state adapted to each of the imaging components.

[0007] Furthermore, the near-infrared II excitation optical path includes a wavelength-tunable femtosecond laser, a reflective beam expander BE, an achromatic lens L1, a pinhole lens 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 sequentially along the optical path. The scanning lens SL1 is anti-transmission in the 700-1700nm range, and the output optical path of the dichroic mirror DM2 faces the transmissive-reflective device. The wavelength-tunable femtosecond laser is continuously tunable from 680nm to 1300nm.

[0008] Furthermore, the first scanning module includes scanners 1 and 2, achromatic lens L3, achromatic lens L4, and scanner 3.

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

[0010] Further, the multiphoton intensity / lifetime and harmonic imaging assembly includes a short-reflecting dichroic mirror DM5, a collimating lens L6, a dichroic mirror DM6, a dichroic mirror DM7, and a filter Fresnel lens F1 and a photomultiplier tube PMT2 arranged sequentially along the optical path direction of the transflection device away from the sleeve lens TL1. It also includes a filter Fresnel lens F1 and a photomultiplier tube PMT2 in the transmission direction of the dichroic mirror DM6, a filter Fresnel lens F2 and a photomultiplier tube PMT3 in the reflection direction of the dichroic mirror DM7, and a filter Fresnel lens F3 and a photomultiplier tube PMT4 in the transmission direction of the dichroic mirror DM7. For near-infrared excited multiphoton or harmonic imaging, the transflection device switches to the dichroic mirror DM3, and the filter Fresnel lens F1 and the photomultiplier tube PMT4 are arranged in sequence along the optical path direction away from the sleeve lens TL1. Each of the aforementioned Fresnel lenses F2 consists of a filter and a Fresnel lens. The photomultiplier tube PMT2 is responsible for second / third harmonic imaging, and the photomultiplier tube PMT3 is responsible for two / three-photon imaging. The detection wavelengths of the photomultiplier tubes PMT2 and PMT3 are 380-720nm. For near-infrared excited multiphoton fluorescence lifetime imaging, the transmission and reflection 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 accurate lifetime measurement. The Fresnel lens F3 consists of a filter and a Fresnel lens, and the detection wavelength of the photomultiplier tube PMT4 is 280-720nm.

[0011] For near-infrared excited multi-channel synchronous imaging mode, a short-pass dichroic mirror is used at the dichroic mirror DM3 to split the light, reflecting the excitation light and the longer single-photon signal, while transmitting the two-photon fluorescence and harmonic signal.

[0012] Furthermore, the visible light confocal excitation optical path includes a four-wavelength laser module, a single-mode 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 sequentially along the optical path. The four-wavelength laser module includes four lasers with wavelengths of 405nm, 488nm, 561nm, and 638nm. The laser light from each laser is coupled to the single-mode fiber SMF through the dichroic mirror. The scanning lens SL2 enhances the transmission in the visible light band, and the dichroic mirror DM2 is also used for beam combining with the near-infrared II excitation optical path.

[0013] Furthermore, the second scanning module includes Scanner4,5, achromatic lens L8, 2-inch elliptical mirror M3, achromatic lens L9, and Scanner6 arranged in sequence.

[0014] Furthermore, the visible light confocal and wide-field imaging assembly includes a mirror M4, a focusing lens L10, a pinhole PH3, a multimode fiber MMF2, a focusing lens L11, a dichroic mirror DM9, a dichroic mirror DM10, a Fresnel filter lens F4 and a photomultiplier tube PMT5 on the reflected light path of the multi-bandpass dichroic mirror DM9, a Fresnel filter lens F5 and a photomultiplier tube PMT6 on the reflected light path of the dichroic mirror DM10, and a Fresnel filter lens F6 and a photomultiplier tube PMT7 on the transmitted light path of the dichroic mirror DM10. The photomultiplier tubes PMT5, PMT6, and PMT7 constitute three imaging channels to detect the blue, green, and red bands, respectively.

[0015] Furthermore, the microscopic assembly includes a lens frame fluorescence arm that houses the filter switching assembly DM4. The lens frame fluorescence arm has an excitation source inlet corresponding to the position of the filter switching assembly DM4. The wide-field imaging optical path includes a white LED or a near-infrared LED that can be switched and installed at the excitation source inlet. The excitation wavelength of the white LED can be continuously distributed between 400-700nm, and the peak wavelength of the near-infrared LED is between 900-1450nm.

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

[0017] Furthermore, a method of using a multimodal fluorescence microscope is provided, applicable to any of the multimodal fluorescence microscopes described above, comprising:

[0018] Visible wide-field imaging mode:

[0019] A white LED with an output spectrum covering 400nm-700nm is configured at the excitation source inlet. First, the white LED is turned on, and the filter switching component DM4 is rotated to the filter position corresponding to the fluorescence emission color of the sample. Then, the visible light camera is turned on, and the dichroic mirrors DM3 and DM5 are set to empty magnetic cores to ensure light transmission. Subsequently, the sample is placed on the stage, and the height of the stage is adjusted to achieve focus. Focusing is achieved by observing the image from the camera.

[0020] Near-infrared II wide-field imaging:

[0021] Near-infrared LEDs are configured at the excitation light source inlet, with their emission bands adapted to the dye marking conditions. The visible light camera is replaced with a near-infrared camera, and the filter switching assembly DM4 is rotated to slot 4. The dichroic mirrors DM3 and DM5 are set to empty magnetic cores to ensure light transmission. The sample is then placed on the stage, and the height of the stage is adjusted to achieve focusing. Focusing is achieved by observing the image from 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 Scanner4,5,6 and the power of the motorized pinhole wheel PH3 and photomultiplier tubes PMT5,6,7; insert the magnetic core with the reflector into the dichroic mirror DM3 to achieve complete reflection; rotate the dichroic mirror DM4 to slot 5 to allow all light to pass through; 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 scanned image results; achieve synchronous acquisition of the three signals of photomultiplier tubes PMT5,6,7 through multiple acquisition channels to achieve three-color synchronous imaging;

[0024] Near-infrared two-region confocal imaging:

[0025] Turn on the power to the near-infrared II photomultiplier tube PMT1 and wait for 1 hour of cooling time; turn on the wavelength-tunable femtosecond laser. Turn on the laser power and wait for a 15-minute warm-up time. Then set the desired output wavelength and start the laser output. Turn on the power of Scanner1, 2, and 3, preset the reverse voltage of PMT1 to -500V, and turn on the power of the motorized pinhole wheel PH2. Insert the magnetic core with the reflector into the dichroic mirror DM3 to achieve full reflection. Rotate the dichroic mirror DM4 to slot 5 to allow all light to pass through. Place the sample on the stage and start scanning and imaging with the imaging software. If multicolor imaging is required, switch the magnetic core at the dichroic mirror DM1 and configure the dichroic mirror / filter required for excitation / imaging of the corresponding wavelength. Then perform imaging in stages. When the sample is labeled with both visible fluorescent dye and near-infrared II fluorescent dye, the visible light confocal and near-infrared II confocal imaging modules can be turned on simultaneously. The acquisition system can simultaneously acquire the output signals of the four detectors of PMT1, 5, 6, and 7 and image them, realizing the four-channel confocal synchronous imaging function.

[0026] Nonlinear imaging:

[0027] Turn on the power to the wavelength-tunable femtosecond laser and wait for a 15-minute warm-up time. Then, set the desired output wavelength and start laser output. Turn on the power to Scanners 1, 2, and 3, preset the reverse voltage of photomultiplier tube PMT1 to -500V, and turn on the power to the motorized pinhole wheel PH2. Turn on the power to photomultiplier tubes PMT2, 3, and 4, and open the control software. Place the sample on the stage and turn on the wavelength-tunable femtosecond laser. Laser output; DM3 is configured as a two-photon or three-photon dichroic mirror; DM5 is configured as a combination of a 720LP dichroic mirror and a 720SP filter, further intercepting the excitation light that slightly leaks at DM3 and allowing most of the signal light to pass through; DM6 is used to separate the fluorescence signal and harmonic signal in the signal light; DM7 is configured according to the different needs of fluorescence intensity imaging and fluorescence lifetime imaging. When only fluorescence intensity imaging is performed, a filter is inserted at DM7. The magnetic core with a built-in reflector reflects all the fluorescence toward photomultiplier tube PMT3. When only fluorescence lifetime imaging is performed, a magnetic core without a mirror is inserted at dichroic mirror DM7, which transmits all the fluorescence toward 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 at dichroic mirror DM7, so that half of the fluorescence goes to photomultiplier tube PMT3 and half goes to photomultiplier tube PMT4. Open the imaging software, start scanning, and you can acquire the images corresponding to the output signals of photomultiplier tubes PMT2, 3, and 4.

[0028] Four-channel simultaneous imaging:

[0029] Turn on the power to the wavelength-tunable femtosecond laser, scanners 1, 2, and 3, photomultiplier tubes (PMTs) 1, 2, 3, and 4, and the pinhole PH2, and wait for the equipment to be ready. Set DM3 to a 720SP dichroic mirror. For near-infrared confocal imaging, the wavelengths of both the excitation and signal light are longer than 720 nm, so they can both be reflected at DM3. For nonlinear imaging, the configuration remains unchanged. Place the sample on the stage and connect the signals from PMTs 1, 2, 3, and 4 to the acquisition system. Start the imaging software to perform scanning. Since 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 PMTs 1, 2, 3, and 4, thereby achieving synchronous imaging of 4 channels.

[0030] Compared with existing technologies, the multimodal fluorescence microscope and its usage method in this invention have the following advantages:

[0031] Through the above implementation scheme, confocal and wide-field imaging in the visible light region, confocal and wide-field imaging in the near-infrared II region, and multiphoton intensity / lifetime and harmonic imaging based on near-infrared excitation were achieved. Commonly used fluorescence microscopy imaging modes were integrated into a single microscope, and the imaging band covers 280-1700nm, which, to our knowledge, is currently the widest imaging band achievable by a single microscope.

[0032] Based on this invention, we can achieve wide-band multimodal simultaneous in vivo microscopy imaging using a single microscope. Compared to existing common techniques, such as using near-infrared 2-zone dyes to label blood vessels and then performing single-photon confocal microscopy to obtain the distribution of blood vessels and blood flow in the mouse brain, or using green fluorescent protein to label neurons to obtain structural or functional information of mouse brain neurons, or using fluorescent lifetime labeling dyes to obtain information about the microenvironment of the mouse brain, this invention can achieve simultaneous acquisition of information about the blood vessels and microenvironment of mouse brain neurons using a single microscope and diverse labeling and spectroscopic strategies, greatly saving information acquisition time and allowing users to simultaneously observe the dynamics and interrelationships of neurovascular units. In addition, this invention also integrates commonly used visible light wide-field and confocal imaging, as well as near-infrared 2-zone wide-field imaging. Among them, visible light and near-infrared 2-zone wide-field imaging, due to their large field of view and fast imaging speed, can quickly assist users in locating the area to be observed under microscopy, and can also be used directly for volume imaging; visible light wide-field and confocal imaging can also be used for biopsy section observation of lesions, etc. Therefore, this invention can meet the common imaging needs of biomedical laboratories in one stop. In summary, this invention has the advantages of rich functionality, high information acquisition efficiency, good comprehensive utilization rate, and high application adaptability. Attached Figure Description

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

[0034] Figure 2 This is a schematic diagram of the optical path of the multimodal fluorescence microscope during near-infrared two-region confocal imaging in an embodiment of the present invention;

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

[0036] Figure 4 This is a schematic diagram of the optical path of the multimodal fluorescence microscope in near-infrared excited multiphoton fluorescence lifetime imaging in an embodiment of the present invention;

[0037] Figure 5 This is a schematic diagram of the visible light confocal module of the multimodal fluorescence microscope in an embodiment of the present invention;

[0038] Figure 6This is a schematic 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 Implementation

[0039] Embodiments of the present invention are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0040] In the description of this invention, it should be understood that the terms "center," "longitudinal," "lateral," "length," "width," "thickness," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," "clockwise," "counterclockwise," "axial," "circumferential," and "radial," etc., indicating the orientation or positional relationship, are based on the orientation or positional relationship shown in the accompanying drawings and are only for the convenience of describing this invention and simplifying the description, and are not intended to indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of this invention.

[0041] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of this invention, "a plurality of" means two or more, unless otherwise explicitly specified.

[0042] In this embodiment, as Figure 1As shown, a multimodal fluorescence microscope is provided, including a microscopic assembly, a near-infrared II excitation optical path, a visible light confocal excitation optical path, a wide-field imaging optical path, a near-infrared II confocal imaging component, a multiphoton intensity / lifetime and harmonic imaging component, and a visible light confocal and wide-field imaging component. The microscopic assembly includes an objective lens OL, a filter switching component DM4, a sleeve lens TL1, and a transmission-reflection device arranged sequentially from the object side to the image side. The transmission-reflection device can be switched to a dichroic mirror DM3 or a reflecting mirror M1. The near-infrared II excitation optical path, the visible light confocal excitation optical path, and the wide-field imaging optical path are connected to the sleeve lens TL1. The near-infrared II excitation optical path uses... The system provides femtosecond lasers with wavelengths from 680nm to 1300nm to the microscopic components. The visible light confocal excitation optical path is used to provide lasers in four confocal bands to the microscopic components. The wide-field imaging optical path is used to provide visible light with a continuous wavelength distribution of 400-700nm or near-infrared light with a switchable wavelength of 900-1450nm to the microscopic components. The near-infrared two-zone confocal imaging component, the multiphoton intensity / lifetime and harmonic imaging component, as well as the visible light confocal and wide-field imaging components are all connected to the sleeve lens TL1 to receive the returned signal light. The filter switching component DM4 and the transmission-reflection 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 in the visible light region, confocal and wide-field imaging in the near-infrared II region, and multiphoton intensity / lifetime and harmonic imaging based on near-infrared excitation were achieved. Specifically, this includes visible light confocal imaging, visible light wide-field imaging; near-infrared II confocal imaging, near-infrared II wide-field imaging; two / three-photon fluorescence intensity imaging, two / three-photon fluorescence lifetime imaging; and second / third harmonic imaging. All of these modes are integrated into a single microscope, and multiple modes can be imaged simultaneously. The imaging band ranges from 280nm to 1700nm, covering the imaging bands of most fluorescent probes. To our knowledge, this is currently the widest imaging band achievable with a single microscope.

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

[0045] The near-infrared II excitation light route uses a wavelength-tunable femtosecond laser as the light source, with the wavelength continuously adjustable from 680nm to 1300nm. This laser can be used as a light source for near-infrared II confocal imaging, as well as for two / three-photon fluorescence intensity imaging, two / three-photon fluorescence lifetime imaging, and second / third harmonic imaging, and its wavelength covers the excitation band of commonly used probes. After the laser output is emitted, it undergoes chromatic beam expansion by a reflective beam expander BE, then is focused by an achromatic lens L1, and filtered by a 100μm pinhole PH1. 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 mounted on a magnetic dichroic mirror mount, which can be flexibly replaced according to different excitation / signal bands. The excitation light is reflected at the dichroic mirror DM1, while the signal light is transmitted. The reflected excitation light enters the scanning module, which consists of Scanner1, 2, 3 and a pair of achromatic lenses L3 and L4. After passing through the near-infrared II anti-reflection scanning lens SL1, the light is combined with the visible light excitation light path at the dichroic mirror DM2. The beam combining point is near the focal plane of SL1.

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

[0047] Furthermore, the multiphoton intensity / lifetime and harmonic imaging assembly includes, sequentially arranged along the optical path direction away from the sleeve lens TL1 of the transflection device, a short-reflecting dichroic mirror DM5, a collimating lens L6, a dichroic mirror DM6, a dichroic mirror DM7, and a filter Fresnel lens F1 and a photomultiplier tube PMT2 in the transmission direction of the dichroic mirror DM6, a filter Fresnel lens F2 and a photomultiplier tube PMT3 in the reflection direction of the dichroic mirror DM7, and a filter Fresnel lens F3 and a photomultiplier tube PMT4 in the transmission direction of the dichroic mirror DM7. For near-infrared excited multiphoton or harmonic imaging, the transflection device switches to the dichroic mirror DM3. The filter Fresnel lenses F1 and F2 each consist of a filter and a Fresnel lens, and the photomultiplier tube... PMT2 is responsible for second / third harmonic imaging, and PMT3 is responsible for two / three-photon imaging. The detection bands of PMT2 and PMT3 are 380-720nm. For near-infrared excited multiphoton fluorescence lifetime imaging, the transmission-reflection device is switched to a dichroic mirror DM3. PMT3 is a counting detector, and the time-correlated single-photon counting method (TCSPC) is used to achieve accurate lifetime measurement. The filter Fresnel lens F3 consists of a filter and a Fresnel lens. The detection band of PMT4 is 280-720nm. For near-infrared excited multi-channel synchronous imaging mode, a short-pass dichroic mirror is used at the dichroic mirror DM3 to split the light. The excitation light and the longer single-photon signal are reflected, while the two-photon fluorescence and harmonic signals are transmitted.

[0048] Specifically, the near-infrared II excitation light is reflected at the dichroic mirror DM2 and then reaches a dichroic mirror DM3 or a reflecting mirror M1. When performing single-photon confocal imaging, the transmission-reflection device can be switched to reflecting mirror M1. The signal light returns along the original excitation path and, after exiting the first scanning module, is split with the excitation light at the dichroic mirror DM1. This process is also called unscanning imaging. When performing multiphoton or harmonic imaging, the transmission-reflection device uses a short-pass dichroic mirror DM3. The shorter wavelength signal light separates from the near-infrared II excitation light here and then enters the multiphoton or harmonic imaging channel. Since the signal light does not re-pass through the first scanning module, this process is also called non-unscanning imaging. Therefore, the dichroic mirror DM3 and reflecting mirror M1 are installed in different magnetic dichroic mirror mounting bases and can be rotated according to actual needs. After beam combining, the light passes through the sleeve lens TL1, then through the filter wheel DM4 in the microscope frame's fluorescence arm, and finally enters the objective lens. When performing point scan imaging, the filter wheel DM4 needs to be rotated to the empty position, i.e., direct transmission; when performing wide field imaging, the filter wheel DM4 needs to be rotated to another position where filter groups are installed.

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

[0050] For near-infrared excited multiphoton or harmonic imaging, the signal light, after passing through the dichroic mirror DM3, is reflected by the short-reflecting dichroic mirror DM5 to the collimating lens L6. The collimated signal light becomes parallel light and is then split by dichroic mirrors DM6 and DM7. The photomultiplier tube PMT2 is responsible for second / third harmonic imaging, and the photomultiplier tube PMT3 is responsible for two / three-photon imaging. Both the Fresnel filter lenses F1 and F2 consist of a filter and a Fresnel lens. The filter filters the signal light, improving the signal-to-noise ratio; the Fresnel lens, due to its short focal length, can effectively focus parallel light onto the photosensitive surface of the PMT within a compact space. This part of the optical path is as follows: Figure 3 As shown.

[0051] For near-infrared excited multiphoton fluorescence lifetime imaging, the process is similar to near-infrared excited multiphoton or harmonic imaging, except the detector is replaced by a photomultiplier tube (PMT4). This detector is a counting detector, and time-correlated single-photon counting (TCSPC) is used to achieve accurate lifetime measurement. The position information required by this method is synchronously provided to the TCSPC acquisition card by the control program. F3 has the same configuration and function as F1 and F2. Furthermore, DM7 can be replaced by a semi-reflective mirror. With sample marking support, simultaneous imaging of PMT1, 2, 3, and 4 can be achieved, realizing an optical path similar to... Figure 4 As shown. To our knowledge, this is currently the method that can achieve the most bands and modes with single-channel excitation energy.

[0052] For near-infrared excitation multi-channel synchronous imaging, the sample needs to be multiple-labeled to output signals of multiple wavelengths under a single excitation wavelength. A short-pass dichroic mirror is used at the DM3 dichroic mirror for spectral dispersion, reflecting the excitation light and the longer single-photon signal, while transmitting two-photon fluorescence and harmonic signals. For example, the sample is dual-labeled with GFP (green fluorescent protein) and near-infrared II quantum dot dyes. GFP can label mouse brain neurons, and quantum dots label blood vessels. Then, under 920nm excitation, GFP emits a two-photon fluorescence signal and is captured by PMT3 and PMT4, achieving two-photon intensity and lifetime imaging, respectively; quantum dots emit a single-photon fluorescence signal and are captured by PMT1, achieving single-photon confocal imaging; collagen and fibers in the sample can emit second-harmonic SHG signals and be captured by PMT2, achieving harmonic imaging. Therefore, with appropriate labeling and filter selection, this system can achieve simultaneous in vivo microscopy imaging of four modes under single-wavelength excitation, something no microscope has previously been able to do.

[0053] For near-infrared excited two / multiphoton fluorescence lifetime imaging, existing fluorescence lifetime measurement techniques mainly rely on TCSPC. While this technique boasts high accuracy and good temporal resolution, its slow speed and limited photon count rate restrict its fastest imaging speed, making it difficult to achieve true synchronization with two / multiphoton imaging. Often, dozens of scans are required to create a usable lifetime image. Therefore, in some embodiments, the scheme of this application can use Pulse-Sampling technology for lifetime measurement. This method requires a high-speed digitizer and a high-speed PMT to directly acquire the fluorescence decay curve after a single excitation pulse and derive the lifetime result. The temporal accuracy of this method is limited by the digitizer sampling rate and PMT timing jitter, reaching approximately 300 ps. However, considering the lifetime length of fluorescent probes involved in actual biological imaging—1 ns or more—the temporal accuracy of this method is sufficient and meets practical requirements.

[0054] Furthermore, the visible light confocal excitation optical path includes a four-wavelength laser module, a single-mode 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 sequentially along the optical path. The four-wavelength laser module includes four lasers with wavelengths of 405nm, 488nm, 561nm, and 638nm. The laser light from each laser is coupled to the single-mode fiber SMF via a dichroic mirror. The scanning lens SL2 provides anti-reflection in the visible light band, and the dichroic mirror DM2 is also used for beam combining with the near-infrared II excitation optical path. The second scanning module includes scanners Scanner4 and 5, an achromatic lens L8, a 2-inch elliptical mirror M3, an achromatic lens L9, and a scanner Scanner6 arranged sequentially.

[0055] The visible light confocal and wide-field imaging assembly includes a mirror M4, a focusing lens L10, a pinhole PH3, a multimode fiber MMF2, a focusing lens L11, a dichroic mirror DM9, a dichroic mirror DM10, a Fresnel filter lens F4 and a photomultiplier tube PMT5 on the reflected light path of the multi-bandpass dichroic mirror DM9, a Fresnel filter lens F5 and a photomultiplier tube PMT6 on the reflected light path of the dichroic mirror DM10, and a Fresnel filter lens F6 and a photomultiplier tube PMT7 on the transmitted light path of the dichroic mirror DM10. The photomultiplier tubes PMT5, PMT6, and PMT7 constitute three imaging channels to detect the blue, green, and red bands, respectively.

[0056] Specifically, the visible light confocal module uses a four-wavelength laser as its excitation source, with wavelengths of 405nm, 488nm, 561nm, and 638nm, covering the commonly used excitation bands for visible light confocal lighting. After being combined by a dichroic mirror, the four wavelengths are coupled to a single-mode fiber (SMF) by an achromatic lens. The core of the single-mode fiber serves as the excitation pinhole. The light exiting the fiber is collimated by a 10x objective lens L7 and then incident on a multi-bandpass dichroic mirror DM8. The excitation light is reflected at DM8, while the signal light is transmitted. The reflected excitation light enters the second scanning module, which consists of scanners 4, 5, and 6, a pair of achromatic lenses L8 and L9, and a 2-inch elliptical mirror M3. After being enhanced with a visible light scanning lens SL2, the light is combined with the near-infrared II excitation light at DM2. The combined light beam is reflected by mirror M1 and enters the sleeve lens, then passes through the filter wheel DM4 in the microscope stand's fluorescence arm before entering the objective lens OL. For point scanning imaging, filter wheel DM4 must be rotated to an empty position for direct transmission; for wide-field imaging, filter wheel DM4 must be rotated to another position where filter groups are installed. The returned signal light is transmitted through dichroic mirror DM8 and reflected by mirror M4 onto focusing lens L10. Pinhole PH3 is identical to pinhole PH2, both being 16-position adjustable pinhole wheels. The filtered signal light is coupled into the multimode fiber MMF2 and collimated by focusing lens L11 at the output port. Photomultiplier tubes PMT5, 6, and 7 form three imaging channels, respectively detecting the blue, green, and red wavelengths, which are then split by dichroic mirrors DM9 and DM10. The Fresnel filters F4, F5, and F6 have similar configurations to those of F1, F2, and F3, using the same Fresnel lenses but different filters for different imaging bands. Therefore, this excitation optical path can simultaneously achieve 3-channel imaging. The visible light confocal module optical path is as follows... Figure 5 As shown.

[0057] Furthermore, the microscopic assembly includes a lens frame fluorescence arm housing a filter switching assembly DM4. The fluorescence arm has an excitation source inlet corresponding to the position of the filter switching assembly DM4. The wide-field imaging optical path includes a white LED or a near-infrared LED that can be switched and mounted at the excitation source inlet. The white LED excitation wavelength can be continuously distributed between 400-700 nm, while the near-infrared LED has a selectable wavelength of 900-1450 nm. 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 transmission-reflection device away from the filter switching assembly DM4. The switchable camera can be switched between a visible light camera and a near-infrared camera. The visible light camera has a detection wavelength of 380-1000 nm, and the near-infrared camera has a detection wavelength of 900-1700 nm.

[0058] Specifically, the light inlet of the fluorescence arm on the microscope stand is the excitation source inlet for wide-field imaging. When a white LED is installed, the excitation wavelength can be continuously distributed between 400-700nm, and the specific wavelength used for excitation is determined by the selection of the excitation filter set. When a near-infrared LED is installed, fluorescence excitation in the near-infrared II region can be performed. Currently, there are several near-infrared LEDs available on the market, and this solution currently uses a 950nm wavelength LED. Both the LED light outlet and the fluorescence arm light inlet are SM1 threads, so the white LED and near-infrared LED can be flexibly replaced according to actual imaging needs using the pluggable SM1 lens sleeve. Since the wavelengths of visible light wide-field imaging and near-infrared II wide-field imaging are different, we have configured a combination of red, green, and blue channels and a near-infrared II long-pass filter at the filter wheel DM4 on the fluorescence arm. Wide-field imaging of different wavelengths requires rotating the wheel to switch to the corresponding filter combination. That is, the filter box at the filter wheel DM4 may be empty or any of the red, green, blue, and near-infrared configurations. The signal light is transmitted through the filter wheel and focused onto the imaging camera via a sleeve lens, consistent with point scan imaging. Furthermore, the interfaces between the visible light camera and the near-infrared II camera and the system are both designed to be pluggable, allowing for flexible camera replacement for wide-field imaging at different wavelengths. The wide-field imaging optical path module is as follows: Figure 6 As shown.

[0059] This invention also provides a method for using a multimodal fluorescence microscope, applicable to the multimodal fluorescence microscope described in any of the preceding claims. The microscope allows for flexible lens configuration at DM1, DM3, DM4, DM5, DM6, and DM7 based on actual imaging requirements. The lens at DM4 is installed in a filter switching wheel within the fluorescence arm of the microscope holder, with six usable slots. Slots 1-3 are configured for violet excitation / blue light imaging, blue light excitation / green light imaging, and yellow light excitation / red light imaging, respectively. Slot 4 is for near-infrared I region excitation (750-980nm) / near-infrared II region imaging (1000-1700nm). Slots 5 and 6 are unconfigured, allowing direct light transmission. The lenses at DM1, 3, 5, 6, and 7 are installed in the magnetic core of a magnetic dichroic mirror cube. The cube's mounting base is fixed, while the magnetic core is removable. Therefore, different types of lenses can be installed in the magnetic core and configured on different mounting positions according to imaging needs to achieve the corresponding functions.

[0060] Imaging modality classification of this invention: This invention can be divided into two main categories: wide-field fluorescence microscopy and point-scan fluorescence microscopy, as shown in the table below:

[0061] Table 1: Imaging Modality Classification Table

[0062]

[0063] The specific usage instructions include:

[0064] Visible wide-field imaging mode:

[0065] A white LED with an output spectrum covering 400nm-700nm is configured at the excitation source inlet. First, the white LED is turned on, and the filter switching component DM4 is rotated to any one of slots 1 to 3. The filter at this position corresponds to the fluorescence emission color of the sample. Next, the visible light camera is turned on, and the dichroic mirrors DM3 and DM5 are set to empty magnetic cores to ensure light transmission. Then, the sample is placed on the stage, and the height of the stage is adjusted to achieve focus. Focusing is achieved by observing the image from the camera.

[0066] Near-infrared II wide-field imaging:

[0067] For this mode, the usage method is basically the same as that for the visible light wide field. A near-infrared LED is configured at the excitation source inlet, with its emission band adapted to the dye marking conditions. For example, if the dye requires optimal excitation at 940nm, a near-infrared LED with a center wavelength of 940nm can be installed. The visible light camera is replaced with a near-infrared camera, and the filter switching assembly DM4 is rotated to slot 4. The dichroic mirrors DM3 and DM5 are set to empty magnetic cores to ensure light transmission. The sample is then placed on the stage, and the stage height is adjusted to achieve focus. Focusing is achieved by observing the camera image.

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

[0069] Visible light confocal imaging:

[0070] This microscope is equipped with a 4-wavelength continuous laser module, which contains one continuous laser each with wavelengths of 405nm, 488nm, 561nm, and 638nm, combined within the same module and output through single-mode fiber (SMF) coupling. For multicolor imaging, it is generally necessary to turn on multiple wavelength lasers to excite different fluorescent dyes separately. The operating steps for 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 the scanners Scanner4, 5, and 6, and turn on the power of the electric pinhole wheel PH3 and photomultiplier tubes PMT5, 6, and 7; insert the magnetic core with the reflector into the dichroic mirror DM3 to achieve the function of complete reflection; rotate the dichroic mirror DM4 to slot 5 to allow all light to pass through; 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 scanned image results; achieve synchronous acquisition of the three signals of photomultiplier tubes PMT5, 6, and 7 through multiple acquisition channels to achieve three-color synchronous imaging;

[0071] Near-infrared two-region confocal imaging:

[0072] This mode of imaging differs from visible light confocal imaging. Its operation is as follows: Turn on the power to the near-infrared II photomultiplier tube (PMT1) and wait for a 1-hour cooling time; turn on the power to the wavelength-tunable femtosecond laser and wait for a 15-minute warm-up time, then set the desired output wavelength and start laser output; turn on the power to scanners 1, 2, and 3, preset the reverse voltage of PMT1 to -500V, and turn on the power to the motorized pinhole wheel PH2; insert the magnetic core containing the reflector into the dichroic mirror DM3 to achieve complete reflection; rotate the dichroic mirror DM4 to slot 5 to allow all light to pass through; place the sample on the stage and start the imaging software to begin scanning; this mode does not have multicolor synchronous imaging capability, therefore... The light source can only output laser light of one wavelength at a time, and there is only one corresponding detector. For multicolor imaging, the magnetic core at the dichroic mirror DM1 is switched, and the dichroic mirror / filter required for excitation / imaging of the corresponding wavelength is configured, followed by multiple imaging operations. When the sample is labeled with both visible and near-infrared II fluorescent dyes, the visible confocal and near-infrared II confocal imaging modules can be activated simultaneously. The acquisition system can simultaneously acquire and image the output signals of the four photomultiplier tube detectors PMT1, 5, 6, and 7, achieving four-channel confocal synchronous imaging. At this time, the optical path is as follows: Figure 5 As shown.

[0073] Nonlinear imaging:

[0074] Nonlinear imaging and near-infrared region 2 confocal imaging share most of the optical path. When using this mode: turn on the power of the wavelength-tunable femtosecond laser and wait for a 15-minute warm-up time, then set the desired output wavelength and start laser output; turn on the power of scanners 1, 2, and 3, preset the reverse voltage of photomultiplier tube PMT1 to -500V, and turn on the power of the motorized 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 wavelength-tunable femtosecond laser. Laser output; The dichroic mirror DM3 is configured as a two-photon or three-photon dichroic mirror, such as a 720SP. This dichroic mirror allows light with wavelengths shorter than 720nm to pass through and light with wavelengths longer than 720nm to reflect. This allows the long-wavelength excitation light to be reflected to the sample at DM3, while the short-wavelength signal light is transmitted from DM3 to the detector path. The dichroic mirror DM5 is configured as a combination of a 720LP dichroic mirror and a 720SP filter, further intercepting the slightly leaked excitation light at DM3 and allowing most of the signal light to pass through. The dichroic mirror DM6 separates the fluorescence and harmonic signals in the signal light. The basic principle is that at the same excitation wavelength, the wavelength of the harmonic signal is shorter than that of the fluorescence signal. Therefore, DM6 is set as a short-pass dichroic mirror, allowing the shorter-wavelength harmonic signal to pass through. The fluorescence signal is transmitted to the harmonic detector PMT2, while longer wavelength fluorescence signals are reflected to DM7. The dichroic mirror DM7 is configured according to the different needs of fluorescence intensity imaging and fluorescence lifetime imaging. When only fluorescence intensity imaging is required, a magnetic core with a built-in reflector is inserted into the dichroic mirror DM7 to reflect all the fluorescence to photomultiplier tube PMT3. When only fluorescence lifetime imaging is required, a magnetic core without a mirror is inserted into the dichroic mirror DM7 to transmit all the fluorescence to photomultiplier tube PMT4. When simultaneous intensity and lifetime imaging is required, a magnetic core with a built-in semi-reflective and semi-transparent film is inserted into the dichroic mirror DM7, so that half of the fluorescence goes to photomultiplier tube PMT3 and half goes to photomultiplier tube PMT4. The imaging software is opened, and scanning is started to acquire the images corresponding to the output signals of photomultiplier tubes PMT2, 3, and 4. The optical path is as follows: Figure 3 As shown.

[0075] Four-channel simultaneous imaging:

[0076] When a sample has multiple markings, such as those required for both multiphoton imaging and near-infrared 2-zone single-photon confocal imaging, this microscope can achieve simultaneous 4-channel imaging. The specific method is as follows: Turn on the power to the wavelength-tunable femtosecond laser, scanners 1, 2, and 3, photomultiplier tubes (PMTs) 1, 2, 3, and 4, and the pinhole PH2, and wait for the equipment to be ready. Set DM3 to a 720SP dichroic mirror. For near-infrared 2-zone confocal imaging, the wavelengths of both the excitation and signal light are 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 from PMTs 1, 2, 3, and 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, the software can simultaneously reconstruct the images from the four channels of PMTs 1, 2, 3, and 4, thus achieving simultaneous 4-channel imaging. The optical path is as follows: Figure 4 As shown.

[0077] Based on this invention, we can achieve wide-band multimodal simultaneous in vivo microscopy imaging using a single microscope. Compared to existing common techniques, such as using near-infrared 2-zone dyes to label blood vessels and then performing single-photon confocal microscopy to obtain the distribution of blood vessels and blood flow in the mouse brain, or using green fluorescent protein to label neurons to obtain structural or functional information of mouse brain neurons, or using fluorescent lifetime labeling dyes to obtain information about the microenvironment of the mouse brain, this invention can achieve simultaneous acquisition of information about the blood vessels and microenvironment of mouse brain neurons using a single microscope and diverse labeling and spectroscopic strategies, greatly saving information acquisition time and allowing users to simultaneously observe the dynamics and interrelationships of neurovascular units. In addition, this invention also integrates commonly used visible light wide-field and confocal imaging, as well as near-infrared 2-zone wide-field imaging. Among them, visible light and near-infrared 2-zone wide-field imaging, due to their large field of view and fast imaging speed, can quickly assist users in locating the area to be observed under microscopy, and can also be used directly for volume imaging; visible light wide-field and confocal imaging can also be used for biopsy section observation of lesions, etc. Therefore, this invention can meet the common imaging needs of biomedical laboratories in one stop. In summary, this invention has the advantages of rich functionality, high information acquisition efficiency, good comprehensive utilization rate, and high application adaptability.

[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 within the protection scope of the present invention.

Claims

1. A multimodal fluorescence microscope, characterized in that, It includes microscopic components, near-infrared II excitation optical path, visible light confocal excitation optical path, wide-field imaging optical path, near-infrared II confocal imaging component, multiphoton intensity / lifetime and harmonic imaging component, and visible light confocal and wide-field imaging component; The microscope assembly includes an objective lens OL, a filter switching assembly DM4, a sleeve lens TL1, and a transmission and reflection device arranged sequentially from the object side to the image side. The transmission and reflection device can be switched to a dichroic mirror DM3 or a reflecting mirror M1. The near-infrared II excitation optical path, the visible light confocal excitation optical path, and the wide-field imaging optical path are connected to the sleeve lens TL1. The near-infrared II excitation optical path is used to provide the microscopic component with femtosecond laser with a wavelength of 680nm to 1300nm. The visible light confocal excitation optical path is used to provide the microscopic component with laser in four confocal bands. The wide-field imaging optical path is used to provide the microscopic component with visible light or near-infrared light with a wavelength of 900-1450nm in a continuously distributed band of 400-700nm. The near-infrared 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 arranged sequentially along the optical path. The scanning lens SL1 is anti-transmission in the 700-1700nm range, and the output optical path of the dichroic mirror DM2 faces the transflection device. The wavelength-tunable femtosecond laser is continuously tunable from 680nm to 1300nm. The near-infrared two-zone confocal imaging component, the multiphoton intensity / lifetime and harmonic imaging component, and the visible light confocal and wide-field imaging component are all connected to the sleeve lens TL1 to receive the returned signal light. The filter switching component DM4 and the transmission-reflection device are used to switch the light transmission state adapted to each of the imaging components. The multiphoton intensity / lifetime and harmonic imaging assembly includes a short-reflecting dichroic mirror DM5, a collimating lens L6, a dichroic mirror DM6, a dichroic mirror DM7, and a filter Fresnel lens F1 and a photomultiplier tube PMT2 arranged sequentially along the optical path direction away from the sleeve lens TL1 of the transflection device; a filter Fresnel lens F2 and a photomultiplier tube PMT3 arranged in the transmission direction of the dichroic mirror DM6; and a filter Fresnel lens F3 and a photomultiplier tube PMT4 arranged in the reflection direction of the dichroic mirror DM7. For near-infrared excited multiphoton or harmonic imaging, the transmission-reflection device is switched to a dichroic mirror DM3. The Fresnel filter lens F1 and the Fresnel filter lens F2 are each composed of a filter and a Fresnel lens. The photomultiplier tube PMT2 is responsible for second / third harmonic imaging, and the photomultiplier tube PMT3 is responsible for two / three-photon imaging. The detection wavelength of the photomultiplier tube PMT2 and the photomultiplier tube PMT3 is 380-720nm. For near-infrared excited multiphoton fluorescence lifetime imaging, the transmission-reflection 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 applied to achieve accurate lifetime measurement. The filter Fresnel lens F3 consists of a filter and a Fresnel lens, and the photomultiplier tube PMT4 has a detection band of 280-720nm. For near-infrared excited multi-channel synchronous imaging mode, a short-pass dichroic mirror is used at the dichroic mirror DM3 to split the light, reflecting the excitation light and the longer single-photon signal, while transmitting the two-photon fluorescence and harmonic signal.

2. The multimodal fluorescence microscope according to claim 1, characterized in that, The first scanning module includes scanners Scanner1,2, achromatic lens L3, achromatic lens L4, and scanner3.

3. 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 fiber MMF1, and a photomultiplier tube PMT1 arranged sequentially on the optical path away from the long-pass dichroic mirror DM1. The pinhole PH2 is located on the focal plane of the achromatic lens L5.

4. The multimodal fluorescence microscope according to claim 3, characterized in that, The visible light confocal excitation optical path includes a four-wavelength laser module, a single-mode 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 sequentially along the optical path. The four-wavelength laser module includes four lasers with wavelengths of 405nm, 488nm, 561nm, and 638nm. The laser light from each laser is coupled to the single-mode fiber SMF through the dichroic mirror. The scanning lens SL2 enhances the transmission in the visible light band, and the dichroic mirror DM2 is also used for beam combining with the near-infrared II excitation optical path.

5. The multimodal fluorescence microscope according to claim 4, characterized in that, The second scanning module includes Scanner4,5, achromatic lens L8, 2-inch elliptical mirror M3, achromatic lens L9, and Scanner6 arranged in sequence.

6. The multimodal fluorescence microscope according to claim 5, characterized in that, The visible light confocal and wide-field imaging assembly includes a mirror M4, a focusing lens L10, a pinhole PH3, a multimode fiber MMF2, a focusing lens L11, a dichroic mirror DM9, a dichroic mirror DM10, a Fresnel filter lens F4 and a photomultiplier tube PMT5 on the reflected light path of the dichroic mirror DM9, a Fresnel filter lens F5 and a photomultiplier tube PMT6 on the reflected light path of the dichroic mirror DM10, and a Fresnel filter lens F6 and a photomultiplier tube PMT7 on the transmitted light path of the dichroic mirror DM10. The photomultiplier tubes PMT5, PMT6, and PMT7 constitute three imaging channels to detect the blue, green, and red bands, respectively.

7. The multimodal fluorescence microscope according to claim 6, characterized in that, The microscopic assembly includes a lens frame fluorescence arm that houses the filter switching assembly DM4. The lens frame fluorescence arm has an excitation source inlet corresponding to the position of the filter switching assembly DM4. The wide-field imaging optical path includes a white LED or a near-infrared LED that can be switched and installed at the excitation source inlet. The excitation wavelength of the white LED can be continuously distributed between 400-700nm, and the peak wavelength of the near-infrared LED is between 900-1450nm. The visible light confocal and wide-field imaging assembly includes a short-reflective dichroic mirror DM5 and a switchable camera in the optical path of the transflective device away from the filter switching assembly DM4. The switchable camera can be switched between a visible light camera and a near-infrared camera. The visible light camera has a detection band of 380-1000nm, and the near-infrared camera has a detection band of 900-1700nm.

8. A method of using a multimodal fluorescence microscope, applied to the multimodal fluorescence microscope as described in claim 7, characterized in that, include: Visible wide-field imaging mode: A white LED with an output spectrum covering 400nm-700nm is configured at the excitation source inlet. First, the white LED is turned on, and the filter switching component DM4 is rotated to the filter position corresponding to the fluorescence emission color of the sample. Then, the visible light camera is turned on, and the dichroic mirrors DM3 and DM5 are set to empty magnetic cores to ensure light transmission. Subsequently, the sample is placed on the stage, and the height of the stage is adjusted to achieve focus. Focusing is achieved by observing the image from the camera. Near-infrared II wide-field imaging: Near-infrared LEDs are configured at the excitation light source inlet, with their emission bands adapted to the dye marking conditions. The visible light camera is replaced with a near-infrared camera, and the filter switching assembly DM4 is rotated to slot 4. The dichroic mirrors DM3 and DM5 are set to empty magnetic cores to ensure light transmission. The sample is then placed on the stage, and the height of the stage is adjusted to achieve focusing. Focusing is achieved by observing the image from 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 Scanner4,5,6 and the power of the motorized pinhole wheel PH3 and photomultiplier tubes PMT5,6,7; insert the magnetic core with the reflector into the dichroic mirror DM3 to achieve complete reflection; rotate the dichroic mirror DM4 to slot 5 to allow all light to pass through; 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 scanned image results; achieve synchronous acquisition of the three signals of photomultiplier tubes PMT5,6,7 through multiple acquisition channels to achieve three-color synchronous imaging; Near-infrared two-region confocal imaging: Turn on the power to the near-infrared II photomultiplier tube PMT1 and wait for 1 hour of cooling time; turn on the wavelength-tunable femtosecond laser. Turn on the laser power and wait for a 15-minute warm-up time. Then set the desired output wavelength and start the laser output. Turn on the power of Scanner1, 2, and 3, preset the reverse voltage of PMT1 to -500V, and turn on the power of the motorized pinhole wheel PH2. Insert the magnetic core with the reflector into the dichroic mirror DM3 to achieve full reflection. Rotate the dichroic mirror DM4 to slot 5 to allow all light to pass through. Place the sample on the stage and start scanning and imaging with the imaging software. If multicolor imaging is required, switch the magnetic core at the dichroic mirror DM1 and configure the dichroic mirror / filter required for excitation / imaging of the corresponding wavelength. Then perform imaging in stages. When the sample is labeled with both visible fluorescent dye and near-infrared II fluorescent dye, the visible light confocal and near-infrared II confocal imaging modules can be turned on simultaneously. The acquisition system can simultaneously acquire the output signals of the four detectors of PMT1, 5, 6, and 7 and image them, realizing the four-channel confocal synchronous imaging function. Nonlinear imaging: Turn on the power of the wavelength-tunable femtosecond laser and wait for a 15-minute warm-up time. Then set the desired output wavelength and start the laser output. Turn on the power of the scanners 1, 2, and 3, preset the reverse voltage of the photomultiplier tube PMT1 to -500V, and turn on the power of the motorized pinhole wheel PH2. Turn on the power to 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; configure dichroic mirror DM3 as a two-photon or three-photon dichroic mirror; configure dichroic mirror DM5 as a combination of a 720LP dichroic mirror and a 720SP filter, further intercepting the slightly leaked excitation light at dichroic mirror DM3 and allowing most of the signal light to pass through; separate the fluorescence signal and harmonic signal in the signal light using dichroic mirror DM6; according to The different requirements for fluorescence intensity imaging and fluorescence lifetime imaging necessitate 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 toward the photomultiplier tube PMT3. When only fluorescence lifetime imaging is performed, a magnetic core without a mirror is inserted into the dichroic mirror DM7 to transmit all the fluorescence toward 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, so that half of the fluorescence goes to the photomultiplier tube PMT3 and half goes to the photomultiplier tube PMT4. Open the imaging software, start scanning, and you can obtain the images corresponding to the output signals of photomultiplier tubes PMT2,3,4; Four-channel simultaneous imaging: Turn on the power to the wavelength-tunable femtosecond laser, scanners 1, 2, and 3, photomultiplier tubes (PMTs) 1, 2, 3, and 4, and the pinhole PH2, and wait for the equipment to be ready. Set DM3 to a 720SP dichroic mirror. For near-infrared confocal imaging, the wavelengths of both the excitation and signal light are longer than 720 nm, so they can both be reflected at DM3. For nonlinear imaging, the configuration remains unchanged. Place the sample on the stage and connect the signals from PMTs 1, 2, 3, and 4 to the acquisition system. Start the imaging software to perform scanning. Since they share the same light source, optical path, and scanning system, and all are single-point scanning imaging, the software can realize synchronous image reconstruction of the four channels of PMTs 1, 2, 3, and 4, thereby achieving synchronous imaging of four channels.

Images