A two-photon fluorescence multicolor imaging system and method

Abstract

This invention provides a two-photon fluorescence multicolor imaging system and method, including a laser component, a dichroic mirror, a data acquisition component, a stage, a filter, a photomultiplier tube, and an imaging device. A first optical path connects the laser component and the dichroic mirror; a second optical path connects the dichroic mirror and the stage; the data acquisition component is positioned on the second optical path; and a third optical path connects the dichroic mirror and the imaging device. The third optical path extends from the dichroic mirror through the filter and the photomultiplier tube to the imaging device. The laser component generates excitation light; the dichroic mirror transmits the excitation light and reflects fluorescence signals; the filter transmits fluorescence signals within a specific wavelength range and filters out stray light signals; and the imaging device exchanges data and processes the data to generate an image. This invention utilizes a single excitation wavelength to achieve two-photon multicolor imaging, effectively simplifying optical path complexity and reducing construction costs.

Description

Technical Field

[0001] This invention relates to the field of optical microscope imaging, and in particular to a two-photon fluorescence multicolor imaging system and method. Background Technology

[0002] In 1990, Denk's research group experimentally verified the feasibility of two-photon fluorescence microscopy based on the two-photon excitation principle, greatly expanding the application of optical microscopy in deep tissue imaging. Renowned for its large imaging depth, high image signal-to-noise ratio, and low-range phototoxicity and photobleaching properties, two-photon fluorescence microscopy has rapidly become a powerful tool for researchers both domestically and internationally in biological and medical research, achieving a series of significant results in neuroscience, detection of major diseases, and immunology. With the deepening of medical and biological research, the need for intuitive and clear visualization of different intracellular structures and the study of interactions between different cells has become increasingly urgent, making multicolor microscopy of significant practical importance for in vivo optical imaging. Currently, achieving multicolor imaging in two-photon fluorescence microscopy requires a large number of optical components, making the entire optical path very bulky and significantly increasing the cost of the entire optical system, which is detrimental to widespread practical applications. Summary of the Invention

[0003] The purpose of this invention is to at least partially address the shortcomings of the prior art and provide a two-photon fluorescence multicolor imaging system and method.

[0004] In a first aspect, embodiments of the present invention provide a two-photon fluorescence multicolor imaging system, including a laser component, a dichroic mirror, a data acquisition component, a stage, a filter, a photomultiplier tube, and an imaging device. A first optical path exists between the laser component and the dichroic mirror; a second optical path exists between the dichroic mirror and the stage; the data acquisition component is disposed on the second optical path; and a third optical path exists between the dichroic mirror and the imaging device. The third optical path extends from the dichroic mirror through the filter and the photomultiplier tube sequentially to the imaging device.

[0005] The laser component is used to generate excitation light;

[0006] The dichroic mirror is used to transmit excitation light and reflect fluorescence signals;

[0007] The acquisition component is used to acquire the fluorescence signal of the sample on the stage;

[0008] The stage is used to place and fix the sample, and to adjust the three-dimensional spatial position of the sample;

[0009] The filter is used to filter out stray light signals while allowing fluorescence signals to pass through a specific wavelength range.

[0010] The photomultiplier tube is used to detect and collect fluorescence signals, perform photoelectric conversion, and enhance the signal.

[0011] The imaging device is used for data exchange and for processing data to generate images.

[0012] Secondly, a two-photon fluorescence multicolor imaging method is also provided, applied to the two-photon fluorescence multicolor imaging system as described in any one of the above claims, the method comprising:

[0013] Different cell structures are stained and labeled, and the labels are excited by two-photon excitation using the same wavelength of excitation light to obtain fluorescence signals;

[0014] The fluorescence signal is filtered to obtain a first image signal, a second image signal, and a third image signal. The first image signal, the second image signal, and the third image signal are then subjected to image subtraction to obtain a first fluorescence signal, a second fluorescence signal, and a third fluorescence signal.

[0015] The obtained first fluorescence signal, second fluorescence signal and third fluorescence signal are used to reconstruct a three-color fluorescence image.

[0016] The beneficial effects of this invention are as follows: This invention provides a two-photon fluorescence multicolor imaging system and method. A laser component emits excitation light of the same wavelength, which is then transmitted through a dichroic mirror to excite the sample. After the acquisition component collects the fluorescence signal emitted by the sample, it is reflected by the dichroic mirror, then selected by bandpass filters of different wavelengths, and finally detected and amplified by a photomultiplier tube before being transmitted to the imaging device. This invention utilizes a single excitation wavelength to achieve two-photon multicolor imaging, effectively simplifying the optical path complexity and reducing costs. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 This is an optical path diagram of a two-photon fluorescence multicolor imaging system provided in an embodiment of the present invention;

[0019] Figure 2 This is a schematic diagram of the two-photon fluorescence multicolor imaging method provided in an embodiment of the present invention;

[0020] Figure 3This invention provides an embodiment of a two-photon trichromatic imaging pattern based on a single excitation wavelength in commercial multicolor cell samples.

[0021] Figure 4 This is a flowchart of the steps of the two-photon fluorescence multicolor imaging method provided in the embodiments of the present invention;

[0022] Figure 5 This is a first sub-flowchart of the two-photon fluorescence multicolor imaging method provided in an embodiment of the present invention;

[0023] Figure 6 This is a second sub-flowchart of the two-photon fluorescence multicolor imaging method provided in an embodiment of the present invention. Detailed Implementation

[0024] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] It should be understood that, when used in this specification and the appended claims, the terms "comprising" and "including" indicate the presence of the described features, integrals, steps, operations, elements and / or components, but do not exclude the presence or addition of one or more other features, integrals, steps, operations, elements, components and / or collections thereof.

[0026] It should also be understood that the terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” are intended to include the plural forms unless the context clearly indicates otherwise.

[0027] 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.

[0028] Please see Figure 1This invention provides a two-photon fluorescence multicolor imaging system, including a laser component 1, a dichroic mirror 2, a acquisition component 3, a stage 4, a filter 5, a photomultiplier tube 6, and an imaging device 7. A first optical path exists between the laser component 1 and the dichroic mirror 2, a second optical path exists between the dichroic mirror 2 and the stage 4, the acquisition component 3 is disposed on the second optical path, and a third optical path exists between the dichroic mirror 2 and the imaging device 7. The third optical path extends from the dichroic mirror 2 through the filter 5 and the photomultiplier tube 6 to the imaging device 7. The laser component 1 generates excitation light; the dichroic mirror 2 transmits the excitation light and reflects fluorescence signals; the acquisition component 3 acquires fluorescence signals from the sample on the stage 4; the stage 4 places and fixes the sample, adjusting its three-dimensional spatial position; the filter 5 transmits fluorescence signals within a specific wavelength range and filters out stray light signals; the photomultiplier tube 6 detects and collects fluorescence signals, performs photoelectric conversion, and enhances the signal; the imaging device 7 exchanges data and processes the data to generate images.

[0029] In this embodiment, the laser component 1 emits excitation light of the same wavelength, which is reflected by the dichroic mirror 2. The signal light is then selected by the bandpass filter 5 of different wavelengths, and after being detected and amplified by the photomultiplier tube 6, it is transmitted to the imaging device 7. That is, multicolor imaging of two photons is achieved by using a single excitation wavelength, which can effectively simplify the complexity of the optical path and reduce the cost.

[0030] Furthermore, the laser assembly 1 includes a laser emission source 11, a first collimating lens 12, a second collimating lens 13, a first lens 14, and a second lens 15. The first optical path extends from the laser emission source 11 through the first collimating lens 12, the second collimating lens 13, the first lens 14, and the second lens 15 to the dichroic mirror 2. The laser emission source 11 generates excitation light; the first collimating lens 12 and the second collimating lens 13 focus and expand the excitation beam during fiber coupling; and the first lens 14 and the second lens 15 focus and collimate the laser spot. A single-mode optical fiber 16 connects the first collimating lens 12 and the second collimating lens 13.

[0031] In this embodiment, the first collimating lens 12 is a 4X / 0.1 collimating lens, the second collimating lens 13 is a collimating beam expander, and the single-mode fiber 16 is a single-mode polarization-maintaining fiber. Specifically, the single excitation beam is generated by a femtosecond laser with a wavelength tunable and a pulse frequency of 80MHz. Since the original beam spot is strip-shaped, in order to improve the beam spot quality, a 4X / 0.1 collimating lens is used to couple the excitation beam into the single-mode polarization-maintaining fiber. The single-mode polarization-maintaining fiber is used to shape the excitation beam to obtain the preset light, which is then transmitted to a specific collimating beam expander through the first optical path, and the fundamental mode beam spot is output through it.

[0032] In this embodiment, the first lens 14 and the second lens 15 form a 4f system. After the fundamental mode spot is scaled and expanded by the 4f system, the spot size matches the size of the scanning galvanometer 31 lens, which facilitates subsequent scanning by the scanning galvanometer 31. Of course, in other embodiments, a 4f system composed of multiple pairs of lenses can also be used. This is not limited here, as long as the fundamental mode spot is scaled and expanded by the 4f system so that the spot size matches the size of the scanning galvanometer 31 lens.

[0033] Furthermore, the acquisition component 3 includes a scanning galvanometer 31, a scanning lens 32, a tube mirror 33, and a microscope objective 34. The second optical path passes sequentially from the dichroic mirror 2 through the scanning galvanometer 31, the scanning lens 32, the tube mirror 33, and the microscope objective 34 to the stage 4. The scanning galvanometer 31 is used to perform two-dimensional scanning of the sample; the scanning lens 32 is used to focus the excitation beam; the tube mirror 33 is used to expand the excitation beam and adapt it to the light transmission aperture of the microscope objective 34; and the microscope objective 34 is used to focus the spatially overlapping excitation light and simultaneously collect the fluorescence signal reflected back from the sample.

[0034] In this embodiment, the stage 4 is a three-dimensional stage, and the scanning galvanometer 31 is driven by a motor to perform XY scanning of the light spot for two-dimensional planar imaging. The microscope objective 34 has a magnification of 100x and a numerical aperture of 1.4 to improve the energy utilization of the light beam. At the same time, it makes full use of the numerical aperture of the microscope objective 34. After the beam is expanded by the scanning lens 32 and the tube lens 33, it is focused on the sample by the microscope objective 34. The sample is excited and generates a fluorescence signal. The signal light is collected by the microscope objective 34 and reflected by the dichroic mirror 2.

[0035] Furthermore, a third lens 8 is provided between the filter 5 and the photomultiplier tube 6.

[0036] In this embodiment, after selecting bandpass filters of different wavelengths to pass the signal light, the beam is focused into the photomultiplier tube 6 through the third lens 8 to convert the fluorescence signal into an electrical signal.

[0037] Furthermore, the imaging device 7 includes a data acquisition card 71 and an imaging module 72. One end of the data acquisition card 71 is communicatively connected to the photomultiplier tube 6, and the other end of the data acquisition card 71 is communicatively connected to the imaging module 72.

[0038] In this embodiment, the data acquisition card 71 is used for data exchange and sending data to the imaging module 72; the imaging module 72 is used for controlling the imaging software, storing experimental data, and processing image data, etc.; wherein, the imaging module 72 is a computer. Of course, in some other embodiments, the imaging module 72 may also be other devices that can control the imaging software and have storage and processing functions, which are not limited here.

[0039] Please see Figure 2 and Figure 4 , Figure 2 This is a schematic diagram illustrating the principle of two-photon multicolor imaging based on a single excitation wavelength according to the present invention. The dashed lines represent the absorption spectra of each fluorescent dye, and the solid lines represent the emission spectra of each fluorescent dye. Figure 2 The central column, from left to right, represents filter A, filter B, filter C, and the excitation light. Dashed lines from left to right represent the absorption spectra of the three fluorescent probes, while solid lines from left to right represent their emission spectra. Analysis of the absorption spectra of these three fluorescent probes (as shown by the dashed lines) reveals that the absorption coefficients of all three probes at 400 nm are not zero. Therefore, a laser with a wavelength of 800 nm can simultaneously excite all three fluorescent probes, causing them to produce corresponding fluorescence signals.

[0040] Secondly, a two-photon fluorescence multicolor imaging method is also provided, applied to the two-photon fluorescence multicolor imaging system described above, the method comprising:

[0041] S110, different structures of cells are stained and labeled, and the labels are excited by two-photon excitation using the same wavelength of excitation light to obtain fluorescence signals;

[0042] In this step, DAPI is a fluorescent dye used in multiphoton microscopy and commonly used for labeling cell nuclei. It is frequently used with STED's commercial "Abberior" probe, which exhibits excellent bleach resistance and photostability. Commercial multicolor cell samples use different dyes to stain different cell structures, specifically: the fluorescent probe STARORANGE labels the Golgi apparatus; the fluorescent probe STARGREEN labels microtubule structures; and the fluorescent probe DAPI labels nuclear structures. Since the absorption coefficients of all three fluorescent probes at 400 nm are not zero, this embodiment uses pulsed light with a wavelength of 800 nm, which can excite... Figure 2 The three different fluorescence signals are shown.

[0043] S120. Filter the fluorescence signal to obtain a first image signal, a second image signal and a third image signal, and perform image subtraction on the first image signal, the second image signal and the third image signal to obtain a first fluorescence signal, a second fluorescence signal and a third fluorescence signal.

[0044] In this step, since the emission spectrum of the second image completely covers the emission spectrum of the first image, it is impossible to directly use filter 5 to distinguish between the two signals. Therefore, the first fluorescence signal, the second fluorescence signal, and the third fluorescence signal are obtained by subtracting the images.

[0045] S130. The obtained first fluorescence signal, second fluorescence signal and third fluorescence signal are used to reconstruct a three-color fluorescence image.

[0046] Furthermore, image subtraction includes: I GREEN =I B -αI DAPI I ORANGE =I C -βI B Among them, I DAPI I represents the signal strength of the first image signal. B I represents the signal strength of the second image signal. C I represents the signal strength of the third image signal. DAPI I represents the signal intensity of the first fluorescence signal. GREEN I represents the signal intensity of the second fluorescence signal. ORANGE Let α be the signal intensity of the third fluorescence signal, and β be the difference coefficients. The value of the difference coefficient is determined by the sum of the emission spectral rates under the corresponding filters, i.e., α = ∑E B (λ) / ∑E A (λ), β=∑E C (λ) / ∑E B (λ), where E is the emission coefficient.

[0047] In this embodiment, since the selected filter 5 has different throughput for each fluorescence signal, the intensity of the obtained two-photon image is adjusted by using an adjustment coefficient, which can more completely separate the cell structures of each part.

[0048] Further, please refer to Figure 5 The fluorescence signal is filtered to obtain a first image signal, a second image signal, and a third image signal. Image subtraction is then performed on the first image signal, the second image signal, and the third image signal to obtain a first fluorescence signal, a second fluorescence signal, and a third fluorescence signal, including:

[0049] S121. A first image signal is obtained through the first filtering process. The first image signal contains only the first fluorescence signal that marks the cell nuclear structure information.

[0050] In this step, filter 5 is divided into filter A, filter B and filter C. Under the premise of three selectable bandpass filters, filter A is used to perform the first filtering process to obtain the first image signal. This image only contains the fluorescence signal emitted by the fluorescent probe DAPI that marks the cell nuclear structure information, that is, the first fluorescence signal.

[0051] S122. A second image signal is obtained through a second filtering process, and the first image signal and the second image signal are subjected to image subtraction processing to obtain a second fluorescence signal containing the information of the labeled microtubule structure.

[0052] In this step, filter B is used for a second filtering process to obtain the second image signal. This second image signal contains both microtubule structural information and cell nucleus structural information, meaning there is crosstalk between these two fluorescence signals. Using I... GREEN =I B -αI DAPI By subtracting α times the first image signal from the second image signal using ImageJ software, a separate second fluorescence signal can be obtained.

[0053] S123. The third image signal is obtained through the third filtering process, and the second and third image signals are subtracted to obtain the third fluorescence signal containing the Golgi structure information.

[0054] In this step, filter C is used for a third filtering process to obtain the third image signal. This third image signal contains structural information of the microtubules, the cell nucleus, and the Golgi apparatus; that is, crosstalk occurs between these three fluorescence signals. Using I... GREEN =I B -αI DAPI I ORANGE =I C -βI B Based on dual-color imaging, by subtracting α times the first image signal from the second image signal and β times the second image signal from the third image signal, the individual Golgi structure, namely the third fluorescence signal, can be separated.

[0055] Further, please refer to Figure 6 The obtained first fluorescence signal, second fluorescence signal, and third fluorescence signal are used to reconstruct a three-color fluorescence image, including:

[0056] S131. Microtubule structures and cell nuclear structures are superimposed using image reconstruction methods to obtain a two-color fluorescence image that distinguishes morphological structures and spatial locations using different colors.

[0057] In this step, the first and second fluorescence signals are obtained separately by image subtraction, realizing the signal separation between the microtubule structure and the cell nuclear structure. Then, the two structures are superimposed by a method similar to digital image reconstruction, which can realize two-photon two-color imaging that distinguishes morphological structure and spatial position by different colors, to obtain a two-color fluorescence image.

[0058] S132. Using image reconstruction, the Golgi apparatus structure is superimposed with a two-color fluorescence image to obtain a three-color fluorescence image that distinguishes morphological structure and spatial location using different colors.

[0059] In this step, based on two-photon two-color imaging, a separate Golgi structure, namely the third fluorescence signal, is obtained by image subtraction. Finally, image reconstruction technology is used to achieve two-photon three-color imaging to obtain a three-color fluorescence image.

[0060] Please see Figure 3 Two-photon trichromatic imaging based on a single excitation wavelength was achieved in commercial multicolor cell samples. Specifically, the experimental conditions in this embodiment are as follows: the excitation wavelength is 800 nm, the measured laser power in front of the objective is 18 mW, a 100X / 1.4 microscope objective is used, the imaging field of view is 50 μm, and in the emission spectrum of the DAPI probe, the signal throughput of filter A (ET460 / 30M) is 20.2 (i.e., the sum of the emission spectral rates of the DAPI probe in the range of 445 nm to 475 nm), and the signal throughput of filter B (ET525 / 40M) is 29.1 (i.e., the sum of the emission spectral rates of the DAPI probe in the range of 500 nm to 540 nm). Therefore, the difference coefficient α is 0.7. In the emission spectrum of the STARGREEN probe, the signal throughput of filter C (ET585 / 40M) is 5.9 (i.e., the sum of the emission spectral rates of the STARGREEN probe in the range of 565nm to 605nm), and the throughput of filter B is 31.7 (i.e., the sum of the emission spectral rates of the STARGREEN probe in the range of 500nm to 540nm). The calculated difference coefficient β is 0.2. Fluorescence signals from three cellular structures—the Golgi apparatus, microtubules, and the nucleus—can be observed simultaneously using the ET585 / 40M bandpass filter. Figure 3 (Figure I in the text). Figure 3 Figure II shows how fluorescence signals from both microtubules and the nucleus can be obtained simultaneously using a bandpass filter ET525 / 40M. Figure 3 Figure V shows the individual nucleus signals obtained using an ET460 / 30M bandpass filter. The structures were then distinguished one by one using spectral separation, as follows: First, using... Figure 3 Figure II in Figure 3 The subtraction of Figure V in the diagram yields the fluorescence signal of individual cell microtubules. Figure 3 (Figure IV in the middle); then through Figure 3 Figure I in Figure 3 The fluorescence signal of the individual Golgi apparatus is obtained by subtracting the phase from Figure II. Figure 3 (Figure III in the text);

[0061] The fluorescence signals of the three cellular structures can be separated by subtracting two simple images pairwise. Finally, the individual Golgi apparatus, microtubules, and nucleus are obtained. Figure 3 Figure III in Figure 3 Figure IV in Figure 3 (Image V in the image) is overlaid by color channel, using different colors to distinguish different structures, such as Figure 3 As shown in Figure VI. (and...) Figure 3 Compared to Figure I in the previous version, the relative spatial positions of the various parts of the cell structure can be seen intuitively, effectively reducing the impact of spectral crosstalk without losing relevant information about each part of the structure.

[0062] In summary, the two-photon fluorescence multicolor imaging system and method provided by this invention utilizes spectral separation technology to effectively reduce the impact of spectral crosstalk on image quality, achieving two-photon multicolor microscopy based on a single excitation wavelength. This method is based on two-photon fluorescence imaging, relying solely on filters to achieve two-photon multicolor microscopy on the basis of a conventional two-photon microscope. Furthermore, it does not place excessive demands on the characteristics of the fluorescent dye itself; only an appropriate filter combination needs to be selected based on its fluorescence emission spectrum.

[0063] In the above embodiments, the descriptions of each embodiment have different focuses. For parts not described in detail in a certain embodiment, please refer to the relevant descriptions of other embodiments.

[0064] The above is a description of the technical solution provided by the present invention. For those skilled in the art, based on the ideas of the embodiments of the present invention, there will be changes in the specific implementation methods and application scope. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A two-photon fluorescence multicolor imaging system, characterized by, The system includes a laser component, a dichroic mirror, a data acquisition component, a stage, a filter, a photomultiplier tube, and an imaging device. A first optical path exists between the laser component and the dichroic mirror; a second optical path exists between the dichroic mirror and the stage; the data acquisition component is disposed on the second optical path; and a third optical path exists between the dichroic mirror and the imaging device. This third optical path extends from the dichroic mirror through the filter and the photomultiplier tube to the imaging device. The laser component is used to generate excitation light; The dichroic mirror is used to transmit excitation light and reflect fluorescence signals; The acquisition component is used to acquire the fluorescence signal of the sample on the stage; The stage is used to place and fix the sample, and to adjust the three-dimensional spatial position of the sample; The filter is used to filter out stray light signals while allowing fluorescence signals to pass through a specific wavelength range. The photomultiplier tube is used to detect and collect fluorescence signals, perform photoelectric conversion, and enhance the signal. The imaging device is used for data exchange and data processing to generate images. The process of data processing to generate images includes: filtering the fluorescence signal to obtain a first image signal, a second image signal, and a third image signal; and performing image subtraction on the first image signal, the second image signal, and the third image signal to obtain a first fluorescence signal, a second fluorescence signal, and a third fluorescence signal. The first image signal contains only the first fluorescence signal. The fluorescence signal is obtained by staining and labeling different cell structures and then exciting the labels with two-photon excitation light of the same wavelength. The image subtraction includes: I GREEN = I B - αI DAPI , I ORANGE =I C -βI B ;in, I DAPI The signal strength of the first image signal. I B The signal strength of the second image signal. I C The signal strength of the third image signal. I GREEN The signal intensity of the second fluorescence signal. I ORANGE The signal intensity of the third fluorescence signal. α and β The difference coefficient is determined by the sum of the emission spectral rates under the corresponding filters. α =∑ E B (λ) / ∑ E A (λ), β =∑ E C (λ) / ∑ E B (λ), where E The emission coefficient is used to reconstruct a three-color fluorescence image by combining the first fluorescence signal, the second fluorescence signal, and the third fluorescence signal.

2. The two-photon fluorescence multicolor imaging system as described in claim 1, characterized in that, The laser assembly includes a laser emission source, a first collimating lens, a second collimating lens, a first lens, and a second lens. The first optical path extends from the laser emission source sequentially through the first collimating lens, the second collimating lens, the first lens, and the second lens to the dichroic mirror. The laser emission source is used to generate excitation light; The first collimating lens and the second collimating lens are used to focus and expand the excitation beam during fiber coupling. The first lens and the second lens are used to focus and collimate the laser spot.

3. The two-photon fluorescence multicolor imaging system as described in claim 2, characterized in that, A single-mode optical fiber is provided between the first collimating lens and the second collimating lens.

4. The two-photon fluorescence multicolor imaging system as described in claim 1, characterized in that, The acquisition assembly includes a scanning galvanometer, a scanning lens, a tube mirror, and a microscope objective. The second optical path extends from the dichroic mirror, sequentially through the scanning galvanometer, the scanning lens, the tube mirror, and the microscope objective to the stage. The scanning galvanometer is used to perform two-dimensional scanning of the sample; The scanning lens is used to focus the excitation beam; The tube is used to expand the excitation beam and is adapted to the aperture of the microscope objective. The microscope objective is used to focus the spatially overlapping excitation light while simultaneously collecting the fluorescence signal reflected back from the sample.

5. The two-photon fluorescence multicolor imaging system as described in claim 1, characterized in that, A third lens is provided between the filter and the photomultiplier tube.

6. The two-photon fluorescence multicolor imaging system as described in claim 1, characterized in that, The imaging device includes a data acquisition card and an imaging module. One end of the data acquisition card is communicatively connected to the photomultiplier tube, and the other end of the data acquisition card is communicatively connected to the imaging module.

7. A two-photon fluorescence multicolor imaging method, characterized in that, The method, applied to the two-photon fluorescence multicolor imaging system as described in any one of claims 1-6, comprises: Different cell structures are stained and labeled, and the labels are excited by two-photon excitation using the same wavelength of excitation light to obtain fluorescence signals; The fluorescence signal is filtered to obtain a first image signal, a second image signal, and a third image signal. The first image signal, the second image signal, and the third image signal are then subjected to image subtraction to obtain a first fluorescence signal, a second fluorescence signal, and a third fluorescence signal. The obtained first fluorescence signal, second fluorescence signal and third fluorescence signal are used to reconstruct a three-color fluorescence image.

8. The two-photon fluorescence multicolor imaging method as described in claim 7, characterized in that, The step of filtering the fluorescence signal to obtain a first image signal, a second image signal, and a third image signal, and then performing image subtraction on the first image signal, the second image signal, and the third image signal to obtain a first fluorescence signal, a second fluorescence signal, and a third fluorescence signal includes: The first image signal is obtained through the first filtering process. The first image signal contains only the first fluorescence signal that marks the cell nuclear structure information. The second image signal is obtained through a second filtering process, and the first image signal and the second image signal are subtracted to obtain a second fluorescence signal containing the information of the labeled microtubule structure. The third image signal is obtained through a third filtering process, and the second image signal and the third image signal are subjected to image subtraction to obtain a third fluorescence signal containing the Golgi structure information.

9. The two-photon fluorescence multicolor imaging method as described in claim 7, characterized in that, The step of using image reconstruction to obtain a three-color fluorescence image from the obtained first fluorescence signal, second fluorescence signal, and third fluorescence signal includes: By superimposing microtubule structures and cell nuclear structures using image reconstruction methods, a two-color fluorescence image is obtained, which distinguishes morphological structures and spatial locations using different colors. The Golgi apparatus structure is superimposed on the two-color fluorescence image using image reconstruction methods to obtain a three-color fluorescence image that distinguishes morphological structure and spatial location using different colors.

Images