Fibrillation detection method based on two-photon fluorescence lifetime microscopy and second harmonic generation imaging

Abstract

The present invention discloses a fibrosis detection method based on two-photon fluorescence lifetime microscopy and second harmonic imaging. The method comprises collecting two-photon fluorescence signals and second harmonic signals through a laser scanning microscope; generating and displaying fluorescence lifetime images, second harmonic images and image parameters through an imaging processing terminal according to the two-photon fluorescence signals and second harmonic signals, wherein the image parameters comprise fluorescence lifetime values ​​and the ellipticity ratio of the second harmonic image, and the fluorescence lifetime values ​​and the ellipticity ratio jointly characterize whether fibrosis lesions occur. The present invention adopts two-photon fluorescence lifetime microscopy technology to generate fluorescence lifetime images and fluorescence lifetime values ​​of biological tissues, and adopts second harmonic imaging technology to generate second harmonic images and ellipticity ratios, so that the experimenter can determine whether fibrosis lesions occur in biological tissues by comparing and analyzing the fluorescence lifetime images, second harmonic images, fluorescence lifetime values ​​and ellipticity ratios, thereby improving the accuracy of fibrosis lesion detection.

Description

Technical Field

[0001] The embodiments of the present invention relate to the field of biomedical technology, and in particular to a fibrosis detection method based on two-photon fluorescence lifetime microscopy and second harmonic imaging. Background Art

[0002] Two-photon Fluorescence-Lifetime Imaging Microscopy (TP-FLIM) technology is a nonlinear optical imaging method that uses the combination of lifetime and imaging. Specifically, two photons are absorbed almost simultaneously through a nonlinear polarization process, thereby stimulating fluorescence emission. The image contrast is derived from the measured value of the fluorescence lifetime, which can characterize and analyze biological samples from another dimension of information. The fluorescence lifetime refers to the average time it takes for a large number of fluorescent molecules to decay from an excited state to a ground state.

[0003] Second harmonic generation (SHG) imaging is a nonlinear optical imaging method. Second harmonic signals occur in non-centrosymmetric structures, that is, two photons interact in non-centrosymmetric molecules through a nonlinear polarization process to generate a frequency-doubled second harmonic signal.

[0004] The non-centrosymmetric structure of fibrous collagen makes it the main source of second harmonic signals in biomaterials, and the strength of second harmonic signals in different biological tissues is closely related to the collagen fiber content in the tissue. Collagen fibers have nonlinear optical polarization characteristics and can generate second harmonic signals when exposed to strong light from a laser beam. In the process of pulmonary fibrosis, fibroblasts are activated and transformed into fibroblasts, and these fibroblasts have the ability to produce collagen fibers such as collagen. Type I collagen fibers are mainly produced during pulmonary fibrosis. They are a densely structured fibrin and one of the most common and important types of collagen in the body. Under normal circumstances, type I collagen is mainly present in connective tissue, providing support and structural stability for cells. Abnormal activation and excessive proliferation of fibroblasts are one of the core mechanisms of pulmonary fibrosis. Collagen fibers produced during pulmonary fibrosis accumulate in damaged lung tissue. Excessive accumulation of type I collagen fibers will replace normal lung tissue, leading to fibrosis and structural changes in tissues, forming scars, and ultimately affecting lung function. Summary of the invention

[0005] The embodiment of the present invention provides a fibrosis detection method based on two-photon fluorescence lifetime microscopy imaging and second harmonic generation imaging, aiming to solve the problem of inaccurate detection of fibrosis lesions.

[0006] In order to solve the above technical problems, the present invention provides a fibrosis detection method based on two-photon fluorescence lifetime microscopy imaging and second harmonic imaging, which is applied to an imaging system, wherein the imaging system includes a laser scanning microscope and an imaging processing terminal connected to the laser scanning microscope, and is characterized in that it includes: collecting two-photon fluorescence signals and second harmonic signals excited in biological tissue under laser beam irradiation by the laser scanning microscope; generating a fluorescence lifetime image, a second harmonic image and image parameters by the imaging processing terminal according to the two-photon fluorescence signal and the second harmonic signal, and displaying the fluorescence lifetime image, the second harmonic image and the image parameters, wherein the image parameters include a fluorescence lifetime value corresponding to the fluorescence lifetime image and an ellipse ratio corresponding to the second harmonic image, and the fluorescence lifetime value and the ellipse ratio jointly represent whether fibrosis lesions occur; if the imaging processing terminal receives an adjustment instruction, it obtains a color code corresponding to the adjustment instruction, and displays the fluorescence lifetime image and the lifetime value according to the adjusted color code, wherein the color code corresponds to the fluorescence lifetime value. The technical solution of the embodiment of the present invention adopts two-photon fluorescence lifetime microscopy technology to generate a fluorescence lifetime image of biological tissue and a fluorescence lifetime value corresponding to the fluorescence lifetime image, and adopts second harmonic imaging technology to generate a second harmonic image and an ellipse ratio corresponding to the second harmonic image. Experimental personnel can determine whether fibrotic lesions occur in biological tissue by comparing and analyzing the fluorescence lifetime image, the second harmonic image, the fluorescence lifetime value and the ellipse ratio, thereby improving the accuracy of fibrotic lesion detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying any creative work.

[0008] Figure 1 A schematic flow chart of a method for detecting fibrosis based on two-photon fluorescence lifetime microscopy and second harmonic generation imaging provided in an embodiment of the present invention;

[0009] Figure 2 is a schematic structural diagram of a laser scanning microscope provided by an embodiment of the present invention;

[0010] Figure 3 A schematic diagram of a second harmonic image and a fluorescence lifetime image of a lung tissue slice with fibrosis caused by paraquat poisoning provided by an embodiment of the present invention;

[0011] Figure 4A schematic diagram of the morphological structure and ellipticity ratio of collagen fibers provided in an embodiment of the present invention;

[0012] Figure 5 A schematic diagram of a second harmonic image and a fluorescence lifetime image of a lung tissue slice with virus-induced fibrosis provided in an embodiment of the present invention;

[0013] Figure 6 A schematic diagram of the morphological structure and ellipticity ratio of another group of collagen fibers provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0014] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0015] It should be understood that when used in this specification and the appended claims, the terms "include" and "comprises" indicate the presence of described features, integers, steps, operations, elements and / or components, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components and / or combinations thereof.

[0016] It should also be understood that the terms used in this specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. As used in the specification of the present invention and the appended claims, unless the context clearly indicates otherwise, the singular forms "a", "an" and "the" are intended to include plural forms.

[0017] It should be further understood that the term "and / or" used in the present description and the appended claims refers to any and all possible combinations of one or more of the associated listed items, and includes these combinations.

[0018] As used in this specification and the appended claims, the term "if" may be interpreted as "when" or "upon" or "in response to determining" or "in response to detecting," depending on the context. Similarly, the phrases "if it is determined" or "if [described condition or event] is detected" may be interpreted as meaning "upon determination" or "in response to determining" or "upon detection of [described condition or event]" or "in response to detecting [described condition or event]," depending on the context.

[0019] Figure 1A flow chart of a fiberization detection method based on two-photon fluorescence lifetime microscopy and second harmonic imaging provided in an embodiment of the present invention is applied to an imaging system, wherein the imaging system includes a laser scanning microscope and an imaging processing terminal connected to the laser scanning microscope. The method includes steps S110-S130:

[0020] S110, collecting two-photon fluorescence signals and second harmonic signals excited in biological tissues under the irradiation of the laser beam through the laser scanning microscope.

[0021] In the embodiment of the present invention, Figure 2 As shown, the laser scanning microscope includes a laser Laser, which is an ultrashort pulse sapphire laser, and the ultrashort pulse sapphire laser is used to generate a laser beam with a wavelength of 840nm. The laser scanning microscope also includes an objective lens with a magnification of 60 times, and the objective lens is used to obtain the two-photon fluorescence signal and the second harmonic signal excited in the biological tissue (i.e., the sample Sample). The laser scanning microscope also includes a two-photon fluorescence signal detection optical path, and the two-photon fluorescence signal detection optical path is Figure 2 The yellow and blue light paths are shown in the figure, wherein the yellow light path is the TP-FLIM mode, and the TP-FLIM mode is used to detect the fluorescence lifetime value of the two-photon fluorescence signal, and the blue light path is the TPEF (Two-photon Excitation Fluorescence) mode, and the TPEF mode is used to detect the intensity of the two-photon fluorescence signal. The laser scanning microscope also includes a flip mirror. In an embodiment of the present invention, the flip mirror is Figure 2 The midpoint arc represents a dichroic mirror DM, one of which is used to switch the TPEF mode and the TP-FLIM mode of the two-photon fluorescence signal detection optical path. The laser scanning microscope also includes a second harmonic signal detection optical path, that is, Figure 2 The optical path of the SHG mode is marked in purple, and the second harmonic signal detection optical path is used to detect the second harmonic signal. Photomultiplier tubes PMT are set as detectors on the optical paths of the TPEF mode, TP-FLIM mode and SHG mode, and the photomultiplier tubes PMT are used to detect the two-photon fluorescence signal and the second harmonic signal. In addition, the laser scanning microscope also includes a spectrometer, an optical fiber, a focusing lens, a bandpass filter, a reflector and a short-pass filter, such as Figure 2 As shown, the spectrometer is Spectrometer, the optical fiber is Fiber, the focusing lens is FL, the bandpass filter is PPF, the reflector is M and the short-pass filter is SPF, all of which are existing technologies or conventional technical means and do not need to be specially set in the embodiments of the present invention.

[0022] S120. Generate a fluorescence lifetime image, a second harmonic image, and image parameters through the imaging processing terminal according to the two-photon fluorescence signal and the second harmonic signal, and display the fluorescence lifetime image, the second harmonic image, and the image parameters, wherein the image parameters include a fluorescence lifetime value corresponding to the fluorescence lifetime image and an ellipse ratio corresponding to the second harmonic image, and the fluorescence lifetime value and the ellipse ratio together represent whether fibrosis lesions occur.

[0023] In an embodiment of the present invention, the imaging processing terminal includes a data acquisition card and a time-correlated single photon counter, wherein the data acquisition card is used to generate a second harmonic image and the ellipticity ratio according to the second harmonic signal, and the time-correlated single photon counter is used to generate the fluorescence lifetime image and the fluorescence lifetime value according to the two-photon fluorescence signal. The imaging processing terminal also displays the generated fluorescence lifetime image and the second harmonic image independently, displays the fluorescence lifetime value and the fluorescence lifetime image in correspondence, and displays the ellipticity ratio and the second harmonic image in correspondence. It should be noted that, in an embodiment of the present invention, adjusting the two-photon fluorescence signal detection optical path to the TPEF mode can obtain a fluorescence intensity image at the image processing terminal.

[0024] S130. If the imaging processing terminal receives an adjustment instruction, it obtains a color code corresponding to the adjustment instruction, and displays the fluorescence lifetime image and the lifetime value according to the adjusted color code, wherein the color code corresponds to the fluorescence lifetime value.

[0025] In an embodiment of the present invention, the experimenter can adjust some experimental parameters in the imaging processing terminal according to the needs, for example, adjust the range of color coding to obtain different fluorescence lifetime images. Specifically, the adjustment instruction includes at least a first adjustment instruction and a second adjustment instruction; the fluorescence lifetime image includes at least a first group of fluorescence lifetime images and a second group of fluorescence lifetime images; the lifetime value includes at least a first group of fluorescence lifetime values ​​and a second group of fluorescence lifetime values; if the imaging processing terminal receives the first adjustment instruction, it obtains the first group of color coding corresponding to the first adjustment instruction, and displays the first group of fluorescence lifetime images and the first group of fluorescence lifetime values ​​according to the adjusted first group of color coding; if the imaging processing terminal receives the second adjustment instruction, it obtains the second group of color coding corresponding to the second adjustment instruction, and displays the second group of fluorescence lifetime images and the second group of fluorescence lifetime values ​​according to the adjusted second group of color coding. The experimenter determines whether fibrotic lesions occur in the biological tissue by comparing and analyzing the obtained multiple groups of fluorescence lifetime images, the second harmonic images, the fluorescence lifetime values ​​and the ellipticity ratios, thereby improving the accuracy of fibrotic lesion detection.

[0026] In the embodiment of the present invention, lung tissue slices in biological tissues are used as samples, and experiments are carried out to detect pulmonary fibrosis according to the above method. It should be noted that the collagen fibers in experiments 1 to 4 mainly refer to type I collagen fibers.

[0027] Experiment 1: Detect the degree of fibrosis in different areas of the same lung tissue slice.

[0028] In this experiment, the lung tissue slices are lung tissue slices with fibrosis caused by paraquat (PQ) poisoning. The color coding in the fluorescence lifetime image is adjusted in the image processing terminal, and the color coding is set to the fluorescence lifetime value range of 120-450ps to obtain Figure 3 The fluorescence lifetime image shown and the fluorescence lifetime values ​​corresponding to the fluorescence lifetime image, the pseudo color in the fluorescence lifetime image ranges from yellow corresponding to low lifetime to blue corresponding to high lifetime. Figure 3 Schematic diagram of the second harmonic generation image and fluorescence lifetime image of lung tissue slices with fibrosis caused by PQ poisoning, as shown in Figure 3As shown, a, b and c in A are images obtained by two-photon fluorescence lifetime microscopy and second harmonic imaging of the first area of ​​the lung tissue slice with fibrosis caused by PQ poisoning, d, e and f are images obtained by two-photon fluorescence lifetime microscopy and second harmonic imaging of the second area of ​​the lung tissue slice with fibrosis caused by PQ poisoning, wherein a and d are second harmonic images, b and e are fluorescence intensity images, c and f are fluorescence lifetime images, and B is a fluorescence lifetime curve histogram corresponding to c and f, wherein the ordinate of the fluorescence lifetime curve histogram represents intensity, and the abscissa represents fluorescence lifetime value.

[0029] It can be seen from the second harmonic images a and d that the positions with second harmonic signals in a and d correspond to the high lifetime areas in c and f, respectively, and the positions without second harmonic signals in a and d correspond to the low lifetime areas in c and f, respectively, and the second harmonic signals in d are more and stronger than those in a, indicating that in the lung tissue sections with fibrosis caused by PQ poisoning, the amount of excessive collagen fiber deposition in the second area is more than that in the first area, that is, the degree of fibrosis in the second area is more serious than that in the first area.

[0030] From the colors of the fluorescence lifetime images c and f, we can see that there are more low-lifetime components in c, and more high-lifetime components in f, and the average fluorescence lifetime value corresponding to c is 276.54ps, and the average fluorescence lifetime value corresponding to f is 314.14ps, and the average fluorescence lifetime value corresponding to f is higher than that of c. From the fluorescence lifetime curve histogram corresponding to c and f, we can see that c corresponds to two peaks, namely the low-lifetime peak and the high-lifetime peak, and there is a very obvious distinction between the high and low lifetimes, while f only corresponds to one high-lifetime peak, and the high-lifetime components corresponding to f are more than those in c, indicating that the average fluorescence lifetime value corresponding to f is higher than that of c, further indicating that in the lung tissue sections with fibrosis caused by PQ poisoning, the amount of excessive deposition of collagen fibers in the second area is higher than that in the first area, that is, the degree of fibrosis in the second area is more serious than that in the first area.

[0031] The above analysis based on second harmonic generation images and fluorescence lifetime images shows that the stronger the second harmonic generation signal is, the higher the average fluorescence lifetime value is, the greater the amount of excessive collagen fiber deposition is, and the more severe the degree of pulmonary fibrosis is.

[0032] Experiment 2: Comparison of normal lung tissue sections and fibrotic lung tissue sections.

[0033] In this experiment, the lung tissue sections are normal lung tissue sections and lung tissue sections with fibrosis caused by PQ poisoning. Figure 4 is a schematic diagram of the morphological structure and elliptical ratio of collagen fibers, as shown in Figure 4As shown, a is a second harmonic image of a normal lung tissue slice, e is a schematic diagram of the ellipse ratio of a, and the ellipse ratio AR value is equal to 0.5, b, c and d are second harmonic images of different areas of lung tissue slices with fibrosis caused by PQ poisoning, f, g and h are schematic diagrams of the ellipse ratios of b, c and d, respectively, and the AR values ​​of f, g and h are 0.68, 0.77 and 0.80, respectively, which are all greater than the AR value of e. It can be seen that the larger the AR value, the more excessive collagen fiber deposition and the more severe the degree of lung fibrosis.

[0034] Experiment 3 detected the degree of fibrosis in different areas of the same lung tissue slice.

[0035] In this experiment, the lung tissue slice is a lung tissue slice caused by virus fibrosis, wherein the virus may be COVID-19. The color coding in the fluorescence lifetime image is adjusted in the image processing terminal, and the color coding is set to the fluorescence lifetime value range of 100-300ps to obtain Figure 5 The fluorescence lifetime image shown and the fluorescence lifetime value corresponding to the fluorescence lifetime image. Figure 5 Second harmonic generation image and fluorescence lifetime image of a lung tissue slice with fibrosis caused by a virus, such as Figure 5 As shown, a, b and c in A are the second harmonic image and fluorescence lifetime image of the first area in a lung tissue section with virus-induced fibrosis, d, e and f are the second harmonic image and fluorescence lifetime image of the second area in a lung tissue section with virus-induced fibrosis, wherein a and d are second harmonic images, b and e are fluorescence intensity images, c and f are fluorescence lifetime images, and B is a histogram of fluorescence lifetime curves corresponding to c and f.

[0036] In this experiment, the second harmonic signals presented by the second harmonic images a and b are not much different, that is, the severity of fibrosis cannot be determined based on the second harmonic images, so the fluorescence lifetime value is analyzed to distinguish the severity of fibrosis. From the colors of c and f, it can be seen that there are more low-lifetime components in c, and more high-lifetime components in f, and the average fluorescence lifetime value corresponding to c is 165.8ps, and the average fluorescence lifetime value corresponding to f is 190.8ps, and the average fluorescence lifetime value corresponding to f is higher than that of c. From the fluorescence lifetime curve histogram corresponding to c and f, it can be seen that both c and f have only one peak, and the fluorescence lifetime value corresponding to the peak of f is significantly higher than the fluorescence lifetime value corresponding to the peak of c, indicating that in a lung tissue section with fibrosis caused by a virus, the amount of excessive deposition of collagen fibers in the second area is greater than that in the first area, that is, the degree of fibrosis in the second area is more serious than that in the first area.

[0037] Experiment 4: Comparison of normal lung tissue sections and fibrotic lung tissue sections.

[0038] In this experiment, the lung tissue sections are normal lung tissue sections and lung tissue sections with fibrosis caused by a virus. Figure 6 is a schematic diagram of the morphological structure and elliptical ratio of collagen fibers, as shown in Figure 6 As shown, a is the second harmonic image of a normal lung tissue slice, e is the ellipse ratio schematic diagram of a, and the ellipse ratio AR value is equal to 0.5, b, c and d are the second harmonic images of different areas of a lung tissue slice with fibrosis caused by a virus, f, g and h are the ellipse ratio schematic diagrams of b, c and d, respectively, and the AR values ​​of f, g and h are 0.63, 0.68 and 0.88, respectively, which are all greater than the AR value of e, indicating that the larger the AR value, the greater the amount of excessive collagen fiber deposition and the more severe the degree of lung fibrosis.

[0039] From Experiments 1 to 4, it can be seen that the more collagen fibers there are, the greater the fluorescence lifetime value. However, since collagen fibers also exist in normal human lungs, it is not possible to determine whether the lungs have lesions (such as fibrosis caused by poisoning or viral infection) based on the detected fluorescence lifetime value. It is also necessary to combine the second harmonic image and the AR value corresponding to the second harmonic image to determine whether the lungs have lesions. If the fluorescence lifetime value in the lung tissue section is detected to be longer and the AR value corresponding to the second harmonic image is greater than 0.5, it is determined that fibrosis has occurred in the lungs. On this basis, the higher the fluorescence lifetime value, the more collagen fibers are excessively deposited and the more severe the degree of lung fibrosis, which improves the accuracy of fibrosis lesion detection.

[0040] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can easily think of various equivalent modifications or replacements within the technical scope disclosed by the present invention, and these modifications or replacements should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention shall be based on the protection scope of the claims.

Claims

1. A fiberization detection method based on two-photon fluorescence lifetime microscopy and second harmonic imaging, applied to an imaging system, wherein the imaging system comprises a laser scanning microscope and an imaging processing terminal connected to the laser scanning microscope, characterized in that: include: The laser scanning microscope is used to collect two-photon fluorescence signals and second harmonic signals excited in biological tissues under the irradiation of the laser beam; Generate a fluorescence lifetime image, a second harmonic image and image parameters through the imaging processing terminal according to the two-photon fluorescence signal and the second harmonic signal, and display the fluorescence lifetime image, the second harmonic image and the image parameters, wherein the image parameters include a fluorescence lifetime value corresponding to the fluorescence lifetime image and an ellipse ratio corresponding to the second harmonic image, and if the fluorescence lifetime value becomes longer and the ellipse ratio corresponding to the second harmonic image is greater than a preset value, it is determined that fibrosis occurs; If the imaging processing terminal receives an adjustment instruction, it obtains a color code corresponding to the adjustment instruction, and displays the fluorescence lifetime image and the lifetime value according to the adjusted color code, wherein the color code corresponds to the fluorescence lifetime value.

2. The method according to claim 1, characterized in that The adjustment instruction includes at least a first adjustment instruction and a second adjustment instruction; the fluorescence lifetime image includes at least a first group of fluorescence lifetime images and a second group of fluorescence lifetime images; the lifetime value includes at least a first group of fluorescence lifetime values ​​and a second group of fluorescence lifetime values; if the imaging processing terminal receives the adjustment instruction, it obtains a color code corresponding to the adjustment instruction, and displays the fluorescence lifetime image and the lifetime value according to the adjusted color code, including: If the imaging processing terminal receives the first adjustment instruction, it obtains the first group of color codes corresponding to the first adjustment instruction, and displays the first group of fluorescence lifetime images and the first group of fluorescence lifetime values ​​according to the adjusted first group of color codes; If the imaging processing terminal receives the second adjustment instruction, it obtains the second group of color codes corresponding to the second adjustment instruction, and displays the second group of fluorescence lifetime images and the second group of fluorescence lifetime values ​​according to the adjusted second group of color codes.

3. The method according to claim 1, characterized in that Displaying the fluorescence lifetime image, the second harmonic generation image, and the image parameters includes: independently displaying the fluorescence lifetime image and the second harmonic generation image; Displaying the fluorescence lifetime value and the fluorescence lifetime image in correspondence; The ellipticity ratio and the second harmonic image are displayed in correspondence.

4. The method according to claim 1, characterized in that: The laser scanning microscope includes a two-photon fluorescence signal detection optical path, and the two-photon fluorescence signal detection optical path includes a TPEF mode and a TP-FLIM mode, wherein the TPEF mode is used to detect the intensity of the two-photon fluorescence signal, and the TP-FLIM mode is used to detect the fluorescence lifetime value of the two-photon fluorescence signal.

5. The method according to claim 4, characterized in that The laser scanning microscope further comprises a flip mirror, and the flip mirror is used to switch between the TPEF mode and the TP-FLIM mode of the two-photon fluorescence signal detection optical path.

6. The method according to claim 1, characterized in that The laser scanning microscope also includes a second harmonic signal detection optical path, and the second harmonic signal detection optical path is used to detect the second harmonic signal.

7. The method according to claim 1, characterized in that The laser scanning microscope also includes a laser, which is an ultrashort pulse sapphire laser. The ultrashort pulse sapphire laser is used to generate a laser beam with a wavelength of 840 nm.

8. The method according to claim 1, characterized in that The laser scanning microscope also includes an objective lens with a magnification of 60 times, and the objective lens is used to obtain the two-photon fluorescence signal and the second harmonic signal excited in the biological tissue.

9. The method according to claim 1, characterized in that: The imaging processing terminal includes a data acquisition card, and the data acquisition card is used to generate a second harmonic image and the ellipse ratio according to the second harmonic signal.

10. The method according to claim 1, characterized in that The imaging processing terminal further includes a time-correlated single-photon counter, which is used to generate the fluorescence lifetime image and the fluorescence lifetime value according to the two-photon fluorescence signal.

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