Two-photon and three-photon microscopic imaging system capable of simultaneously exciting and collecting

Abstract

The invention discloses a microscopic imaging system and a microscope for simultaneously exciting / collecting two-photon and three-photon signals, and belongs to the technical field of optical microscopic imaging. The microimaging system comprises a laser, a microimaging device and a signal acquisition / separation device, and a single erbium-doped fiber laser is used for simultaneously exciting two-photon signals and three-photon signals of unmarked biological tissues. The system respectively excites a third harmonic signal, a two-photon excitation fluorescence signal and a second harmonic signal through 1550nm fundamental frequency light and 775nm double frequency light. The signal acquisition / separation device can acquire the three signals at the same time to realize real-time imaging and detection. The whole microscopic imaging device part is integrated into a handheld small system and is connected with a laser and a signal acquisition / separation device through optical fibers to form a plug-and-play microscopic imaging system, and the plug-and-play microscopic imaging system is suitable for simultaneous observation of two-photon and three-photon signals of unmarked biological tissues.

Description

Technical Field

[0001] The present invention belongs to the technical field of optical microscopic imaging, and in particular relates to a microscopic imaging system and a microscope for simultaneously exciting / collecting two-photon and three-photon signals. Background Art

[0002] Two-photon microscopy technology has the advantages of optical sectioning ability, high sensitivity to biochemical information, high spatial resolution, and low lethality to biological tissues. It is an important means of non-invasive, high-resolution imaging of living tissues and cells, and is also an important research tool in the field of bioengineering. Existing two-photon microscopy systems mainly collect two-photon excitation fluorescence and second harmonics of biological tissues. The main source of two-photon excitation fluorescence is biological molecules containing exogenous or endogenous fluorescence, and the main source of second harmonics is components containing collagen. Three-photon microscopy technology is an important research tool in brain science because it uses longer wavelengths of excitation light and penetrates deeper into biological tissues. The three-photon excitation fluorescence signal collected by the three-photon microscopy system comes from biological molecules containing exogenous or endogenous fluorescence, and can specifically image the interface between aqueous media and lipids and mineralized structures. Simultaneous microscopic imaging of two-photon and three-photon signals can obtain more comprehensive tissue biochemical information.

[0003] However, two-photon microscopy and three-photon microscopy require excitation by femtosecond pulses in different wavelength ranges, which puts forward new requirements for the generation and transmission of lasers. One type of existing technology is based on a microscope platform and uses two laser sources to respectively excite two-photon and three-photon signals, which greatly increases the cost of the laser. Although another type of existing technology only uses a single laser source for two-photon and three-photon imaging, it is suitable for three-photon imaging, so the laser wavelength is longer, and biological tissues need to be stained to excite two-photon excitation fluorescence signals, and it is impossible to directly image unlabeled biological tissues.

[0004] Therefore, a microscopic imaging system that can simultaneously excite / collect endogenous two-photon and three-photon signals of unlabeled (unstained) biological tissues is needed to enable rapid noninvasive examination of living tissues in actual clinical applications. Summary of the invention

[0005] In order to overcome the shortcomings of the above-mentioned prior art, the purpose of the present invention is to provide a microscopic imaging system for simultaneously exciting / collecting two-photon and three-photon signals. The system obtains a dual-wavelength excitation source based on a single wavelength laser, and can simultaneously excite / collect two-photon excitation fluorescence, second harmonic signals and third harmonic signals of unlabeled biological tissues.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions: The first object of the present invention is to disclose a microscopic imaging system for simultaneously exciting / collecting two-photon and three-photon signals, comprising: a laser, a microscopic imaging device and a signal collection / separation device; the microscopic imaging device and the laser are connected via an optical fiber, and the microscopic imaging device and the signal collection / separation device are connected via an optical fiber; A phase delay plate, a frequency doubling module, a reflector, a spectrometer and a micro objective lens are sequentially arranged in the microscopic imaging device; the phase delay plate is used to adjust the phase of the laser light emitted by the laser to obtain the laser light with a predetermined polarization direction; the frequency doubling module is used to frequency-multiply the laser light with the predetermined polarization direction to generate a short-wavelength excitation pulse with a wavelength half of the wavelength of the laser light emitted by the laser; the phase delay plate includes a quarter-wave plate and a half-wave plate sequentially arranged; a dimming device is also arranged in the microscopic imaging device.

[0007] The signal acquisition / separation device is used to receive the light signal emitted by the microscopic imaging device and perform multi-dimensional photon counting measurement, convert the light signal into an electrical signal output, so as to obtain a multi-contrast biological image with two-photon fluorescence signal, second harmonic signal and third harmonic signal at the same time.

[0008] Preferably, the frequency doubling module includes a first converging lens, a nonlinear medium and a second collimating lens; the nonlinear medium is arranged between the first converging lens and the second collimating lens; the rear focal plane of the first converging lens coincides with the front focal plane of the second collimating lens, and the nonlinear medium is located on the rear focal plane of the first converging lens.

[0009] Preferably, the material of the nonlinear medium is lithium niobate crystal or potassium titanyl phosphate crystal.

[0010] Preferably, the reflector is a scanning galvanometer and the beam splitter is a dichroic filter.

[0011] Preferably, the laser and the microscopic imaging device are connected by a single-mode optical fiber with a built-in optical fiber collimator, and the optical fiber collimator of the single-mode optical fiber includes a pigtail of the single-mode optical fiber and a first collimating lens, and the phase delay plate is located on the output light path of the first collimating lens; the beam splitter is located on the reflected light path of the reflector; and the micro-objective lens is located on the transmitted light path of the beam splitter or the reflected light path of the reflector.

[0012] Preferably, the dimming device is a second converging lens arranged between the reflector and the beam splitter, a second collimating lens located between the micro-objective lens and the beam splitter, and a third converging lens located on the reflected light path of the beam splitter; the rear focus of the second converging lens, the front focus of the second collimating lens on the reflected light path of the reflector and the front focus of the third converging lens on the reflected light path of the beam splitter coincide with each other; the rear focus of the second converging lens is located at the beam splitter; and the third converging lens is connected to the signal acquisition / separation device via a multimode optical fiber.

[0013] Preferably, the signal acquisition / separation device has parallel signal detection modules, including a second harmonic signal detection module, a two-photon fluorescence signal detection module, a third harmonic signal detection module and a two-photon fluorescence signal supplementary detection module; Each parallel signal detection module has a spectrometer and a photoelectric signal conversion device; the spectrometer sends the second harmonic signal, the two-photon fluorescence signal, the third harmonic signal and the two-photon supplementary fluorescence signal to the corresponding detection module respectively; the photoelectric signal conversion device converts the spectrometer signal of the corresponding detection module to obtain an electrical signal; The signal acquisition / separation device also includes a data acquisition card, which analyzes the electrical signal through an algorithm program to obtain a multi-contrast biological image with two-photon fluorescence signals, second harmonic signals and third harmonic signals.

[0014] Preferably, the signal acquisition / separation device has a third collimating lens, which is connected to the microscopic imaging module through a multimode optical fiber, and a second dichroic filter, a third dichroic filter, a fourth dichroic filter and a fifth bandpass filter are sequentially arranged on the transmission light path of the third collimating lens; A second band-pass filter, a first photomultiplier tube and a first photon counter are arranged on the reflected light path of the second dichroic filter; a third band-pass filter, a second photomultiplier tube and a second photon counter are arranged on the reflected light path of the third dichroic filter; a fourth band-pass filter, a third photomultiplier tube and a third photon counter are arranged on the reflected light path of the fourth dichroic filter; a fifth band-pass filter, a fourth photomultiplier tube and a fourth photon counter are arranged on the transmitted light path of the fourth dichroic filter.

[0015] Preferably, a first bandpass filter is further provided on the optical path between the second dichroic filter and the third collimating lens.

[0016] Preferably, the laser is an erbium-doped fiber laser.

[0017] The second object of the present invention is to provide a microscope whose microscopic imaging device is positioned unspecifically relative to other devices of the microscope and can be directly applied to the direct measurement of the surface of an unlabeled object to be tested, without the need to separate the sample from the object to be tested or to perform any staining on the object to be tested.

[0018] Compared with the prior art, the present invention has the following beneficial effects: The present invention discloses a microscopic imaging system for simultaneously exciting / collecting two-photon and three-photon signals. The doubled frequency light and the remaining fundamental frequency light generated after being doubled by a frequency doubling device can simultaneously excite two-photon signals and three-photon signals, wherein the doubled frequency light with a wavelength of 775 nm after being doubled by a frequency doubling crystal excites two-photon signals (intrinsic two-photon excitation fluorescence of biological tissue, second harmonic), and the fundamental frequency light with a wavelength of 1550 nm excites three-photon signals (third harmonic), thereby realizing a single laser system to simultaneously excite two-photon signals (two-photon excitation fluorescence and second harmonic signals) and three-photon signals (third harmonic signals) of label-free biological tissue, making the entire microscope system compact and low-cost.

[0019] The microscopic imaging system for simultaneously exciting / collecting two-photon and three-photon signals disclosed in the present invention can simultaneously collect two-photon signals (two-photon excitation fluorescence, second harmonic) and three-photon signals (third harmonic) of unlabeled biological tissues with the same signal collection / separation optical path, thereby minimizing imaging time and reducing laser damage to biological tissues. In this system, the acquisition modules of different signals are synchronously controlled by a data acquisition card and a computer, which can realize simultaneous excitation / collection of two-photon signals and three-photon signals of unlabeled biological tissues, and can realize the fusion and synchronization of light stimulation, point scanning imaging and line scanning imaging, and can perform optogenetic regulation of cells at a mesoscopic scale and with subcellular resolution, and can quantitatively observe the structural and functional information of biological tissues.

[0020] Furthermore, the microscope based on the microscopic imaging system of the present invention is composed of three parts: a laser, a microscopic imaging device, and a signal acquisition / separation device, and all the discrete optical elements of the microscopic imaging device can be integrated into a handheld small microscope system, and the three parts are connected by optical fibers, so that the entire microscope system is plug-and-play. This design significantly improves the flexibility of the microscopic imaging device, and the microscopic imaging device can move freely without being restricted by traditional fixed connections, thereby allowing activities within a wider range of operations, and can be directly applied to direct measurement of the surface of unlabeled objects to be tested, without the need to separate samples from the objects to be tested or to perform any staining on the objects to be tested. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 A schematic diagram of the structure of a microscopic imaging system for simultaneously exciting / collecting two-photon and three-photon signals provided by an embodiment of the present invention; Among them: 1- erbium-doped fiber laser, 2- single-mode fiber, 3- first collimating lens, 4- quarter-wave plate, 5- half-wave plate, 6- first converging lens, 7- periodically poled lithium niobate crystal, 8- second collimating lens, 9- scanning galvanometer, 10- second converging lens, 11- first dichroic filter, 12- second collimating lens, 13- miniature objective lens, 14- third converging lens, 15- multimode fiber, 16- third collimating lens, 17- first bandpass filter, 18- second dichroic filter Plate, 19-second bandpass filter, 20-first photomultiplier tube, 21-first photon counter, 22-third dichroic filter, 23-third bandpass filter, 24-second photomultiplier tube, 25-second photon counter, 26-fourth dichroic filter, 27-fourth bandpass filter, 28-third photomultiplier tube, 29-third photon counter, 30-fifth bandpass filter, 31-fourth photomultiplier tube, 32-fourth photon counter, 33-data acquisition card, 34-computer. DETAILED DESCRIPTION

[0022] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only 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 should fall within the scope of protection of the present invention.

[0023] It should be noted that the terms "first", "second", etc. in the specification and claims of the present invention and the above-mentioned drawings are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequence. It should be understood that the data used in this way can be interchanged where appropriate, so that the embodiments of the present invention described herein can be implemented in an order other than those illustrated or described herein. In addition, the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusions, for example, a process, method, system, product or device that includes a series of steps or units is not necessarily limited to those steps or units that are clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.

[0024] The present invention is further described in detail below in conjunction with the accompanying drawings: like Figure 1As shown, the microscopic imaging system for simultaneously exciting / collecting two-photon and three-photon signals disclosed in the present invention comprises: an erbium-doped fiber laser 1, a single-mode optical fiber 2, a first collimating lens 3, a quarter-wave plate 4, a half-wave plate 5, a first converging lens 6, a periodically polarized lithium niobate crystal 7, a second collimating lens 8, a scanning galvanometer 9, a second converging lens 10, a first dichroic filter 11, a second collimating lens 12, a miniature objective lens 13, a third converging lens 14, a multimode optical fiber 15, a third collimating lens 16, a first bandpass Filter 17, second dichroic filter 18, second bandpass filter 19, first photomultiplier tube 20, first photon counter 21, third dichroic filter 22, third bandpass filter 23, second photomultiplier tube 24, second photon counter 25, fourth dichroic filter 26, fourth bandpass filter 27, third photomultiplier tube 28, third photon counter 29, fifth bandpass filter 30, fourth photomultiplier tube 31, fourth photon counter 32, data acquisition card 33 and computer 34. The entire microscope system consists of three parts: an erbium-doped fiber laser 1, a microscopic imaging device part 3-14, and a signal collection / separation device part 16-34. The erbium-doped fiber laser 1 and the microscopic imaging device part 3-14 are connected by a single-mode optical fiber 2, and the microscopic imaging device part 3-14 and the signal collection / separation device part 16-34 are connected by a multi-mode optical fiber 15.

[0025] 1. Microscopic imaging device 1) Structure of the microscopic imaging device The following are arranged in sequence: a first collimating lens 3, a quarter wave plate 4, a half wave plate 5, a first converging lens 6, a periodically polarized lithium niobate crystal 7, a second collimating lens 8, a scanning galvanometer 9, a second converging lens 10, a first dichroic filter 11, a second collimating lens 12, a micro-objective lens 13, and a third converging lens 14, to form a microscopic imaging device. Among them, the quarter wave plate 4 and the half wave plate 5 constitute a phase delay plate module, and the first converging lens 6, the periodically polarized lithium niobate crystal 7, and the second collimating lens 8 constitute a frequency doubling module. The frequency doubling module frequency-doubles the 1550 nm wavelength laser beam emitted by the erbium-doped fiber laser 1 after being transmitted through the single-mode optical fiber 2 and adjusted in phase by the phase delay plate, to obtain a doubled frequency light with a wavelength of 775 nm and a residual fundamental frequency light with a wavelength of 1550 nm. The doubled frequency light and the remaining fundamental frequency light are reflected by the reflector, namely the scanning galvanometer 9, and are split by the spectrometer, namely the first two-phase filter 11. During this period, the doubled frequency light and the remaining fundamental frequency light are adjusted in optical path by the second converging lens 10 and the second collimating lens 12, and then act on the sample to be tested through the micro-objective lens 13. The doubled frequency light with a wavelength of 775 nm excites the endogenous two-photon excitation fluorescence signal and the second harmonic signal in the unlabeled biological tissue, and the remaining fundamental frequency light with a wavelength of 1550 nm excites the third harmonic signal in the unlabeled biological tissue. The microscopic imaging device constitutes a handheld small microscope module, which has a non-specific position relative to other modules of the entire microscopic imaging system.

[0026] 2) When the microscopic imaging device is working The 1550 nm wavelength laser emitted by the erbium-doped fiber laser 1 enters the first collimating lens 3 through the single-mode optical fiber 2 and becomes a parallel beam. The polarization state is adjusted by the quarter-wave plate 4 and the half-wave plate 5. The laser passes through the first converging lens 6 and is focused on the periodically polarized lithium niobate crystal 7 to generate a double frequency light with a wavelength of 775 nm and a residual fundamental frequency light with a wavelength of 1550 nm. The laser passes through the second collimating lens 8 and becomes a parallel beam and enters the scanning galvanometer 9 for scanning. The scanning beam passes through the second converging lens 10, the first dichroic filter 11, and the second collimating lens 12 and then enters the micro-objective lens 13 and is focused on the biological tissue for light stimulation, which excites and generates two-photon signals and three-photon signals, that is, two-photon fluorescence signals, second harmonic signals, and third harmonic signals. The signal is collected by the micro-objective lens 13 and passes through the second collimating lens 12. It is reflected by the first dichroic filter 11 and enters the third converging lens 14 and then converges to the multimode optical fiber 15.

[0027] 2. Signal acquisition / separation device After the two-photon fluorescence signal, the second harmonic signal and the third harmonic signal converge to the multimode optical fiber 15 , they are transformed into parallel light beams by the third collimating lens 16 and then pass through the first bandpass filter 17 to filter out the sample reflected light.

[0028] 1) Second harmonic signal acquisition device The structure is as follows: a second dichroic filter 18, a second bandpass filter 19, a first photomultiplier tube 20, and a first photon counter 21 constitute a second harmonic signal acquisition module; During operation: the filtered light beam portion after passing through the first bandpass filter 17 is reflected by the second dichroic filter 18, filtered by the second bandpass filter 19, detected by the first photomultiplier tube 20, amplified and shaped by the first photon counter 21, and then the signal is collected and processed by the data acquisition card 33 and the computer 34 to realize imaging observation of the second harmonic signal in the label-free biological tissue.

[0029] 2) Two-photon excitation fluorescence signal acquisition device The structure is as follows: a third dichroic filter 22, a third bandpass filter 23, a second photomultiplier tube 24, and a second photon counter 25 constitute an endogenous two-photon excitation fluorescence signal acquisition module; During operation: the filtered light beam after passing through the first bandpass filter 17 is transmitted through the second dichroic filter 18 and then reflected by the third dichroic filter 22, and then filtered by the third bandpass filter 23 and detected by the second photomultiplier tube 24, and then amplified and shaped by the second photon counter 25, and then the data acquisition card 33 and the computer 34 collect and process the signal, so as to realize imaging observation of endogenous two-photon excitation fluorescence signals in label-free biological tissues.

[0030] 3) Third harmonic signal acquisition device The structure is as follows: a fourth dichroic filter 26, a fourth bandpass filter 27, a third photomultiplier tube 28, and a third photon counter 29 constitute a third harmonic signal acquisition module; During operation: the filtered light beam after passing through the first band-pass filter 17 is transmitted through the second dichroic filter 18 and the third dichroic filter 22 in sequence, reflected by the fourth dichroic filter 26, and then filtered by the fourth band-pass filter 27 to be detected by the third photomultiplier tube 28, and then amplified and shaped by the third photon counter 29, and then the data acquisition card 33 and the computer 34 collect and process the signal to realize imaging observation of the third harmonic signal in the label-free biological tissue.

[0031] 4) Two-photon fluorescence signal supplementary detection module The structure is as follows: the fifth bandpass filter 30, the fourth photomultiplier tube 31, and the fourth photon counter 32 constitute an endogenous two-photon fluorescence signal supplementary detection module; During operation: the filtered light beam after passing through the first band-pass filter 17 is transmitted through the second dichroic filter 18, the third dichroic filter 22, and the fourth dichroic filter 26 in sequence, filtered by the fifth band-pass filter 30, detected by the fourth photomultiplier tube 31, and then amplified and shaped by the fourth photon counter 32, and then the signal is collected and processed by the data acquisition card 33 and the computer 34, so as to realize imaging observation of endogenous two-photon excitation fluorescence signals in label-free biological tissues.

[0032] In this system, the acquisition modules of different signals are synchronously controlled by the data acquisition card 33 and the computer 34, which can realize the simultaneous excitation / acquisition of two-photon signals and three-photon signals of label-free biological tissues. The scanning galvanometer 9, the first photon counter 21, the second photon counter 25, and the third photon counter 29 are synchronously controlled by the data acquisition card 33 and the computer 34, which can realize the fusion and synchronization of light stimulation, point scanning imaging, and line scanning imaging, and can perform optogenetic regulation of cells at a mesoscopic scale and with subcellular resolution, and can also quantitatively observe the structural and functional information of biological tissues.

[0033] The above contents are only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. A microscopic imaging system for simultaneously exciting / collecting two-photon and three-photon signals, characterized in that: It includes a laser, a microscopic imaging device and a signal collection / separation device; the microscopic imaging device and the laser are connected via an optical fiber, and the microscopic imaging device and the signal collection / separation device are connected via an optical fiber; The microscopic imaging device is provided with a phase delay plate, a frequency doubling module, a reflector, a beam splitter and a micro objective lens in sequence; the phase delay plate is used to adjust the phase of the laser light emitted by the laser to obtain the laser light with a predetermined polarization direction, and the frequency doubling module is used to perform frequency doubling on the laser light with a predetermined polarization direction to generate a short-wavelength excitation pulse with a wavelength half the wavelength of the laser light emitted by the laser; the microscopic imaging device is also provided with a dimming device; The signal acquisition / separation device is used to receive the light signal emitted by the microscopic imaging device and perform multi-dimensional photon counting measurement, convert the light signal into an electrical signal output, so as to obtain a multi-contrast biological image with two-photon fluorescence signal, second harmonic signal and third harmonic signal at the same time.

2. The microscopic imaging system for simultaneously exciting / collecting two-photon and three-photon signals according to claim 1, characterized in that: The frequency doubling module comprises a first converging lens (6), a nonlinear medium (7) and a second collimating lens (8); the rear focal plane of the first converging lens (6) coincides with the front focal plane of the second collimating lens (8), and the nonlinear medium (7) is located on the rear focal plane of the first converging lens.

3. The microscopic imaging system for simultaneously exciting / collecting two-photon and three-photon signals according to claim 2, characterized in that: The material of the nonlinear medium (7) is lithium niobate crystal or potassium titanyl phosphate crystal.

4. The microscopic imaging system for simultaneously exciting / collecting two-photon and three-photon signals according to claim 1, characterized in that: The laser and the microscopic imaging device are connected by a single-mode optical fiber (2) with a built-in optical fiber collimator, the optical fiber collimator of the single-mode optical fiber comprises a pigtail of the single-mode optical fiber and a first collimating lens (3), and the phase delay plate is located on the output light path of the first collimating lens (3); The reflector is located on the outgoing light path of the frequency doubling module, and the beam splitter is located on the reflected light path of the reflector; the micro-objective lens (13) is located on the transmitted light path of the beam splitter and the reflected light path of the reflector.

5. The microscopic imaging system for simultaneously exciting / collecting two-photon and three-photon signals according to claim 4, characterized in that: The dimming device comprises a second converging lens (10) arranged between a reflector and a beam splitter, a second collimating lens (12) located between the beam splitter and a micro-objective lens (13), and a third converging lens (14) located on a reflected light path of the beam splitter; the rear focus of the second converging lens (10), the front focus of the second collimating lens (12) on the reflected light path of the reflector, and the front focus of the third converging lens (14) on the reflected light path of the beam splitter overlap; the rear focus of the second converging lens (10) is located on the beam splitter; and the third converging lens (14) is connected to the signal acquisition / separation device via a multimode optical fiber (15).

6. The microscopic imaging system for simultaneously exciting / collecting two-photon and three-photon signals according to claim 1, characterized in that: The signal acquisition / separation device has parallel multi-dimensional signal detection modules, including a second harmonic signal detection module, a two-photon fluorescence signal detection module, a third harmonic signal detection module and a two-photon fluorescence signal supplementary detection module; Each of the signal detection modules in parallel has a spectrometer and a photoelectric signal conversion device; the spectrometer sends the second harmonic signal, the two-photon fluorescence signal, the third harmonic signal and the two-photon supplementary fluorescence signal to the corresponding detection module respectively; the electrical signal conversion device converts the corresponding spectrometer signals into photoelectric signals to obtain electrical signals; The signal acquisition / separation device also includes a data acquisition card, which analyzes the electrical signal through an algorithm program to obtain a multi-contrast biological image with two-photon fluorescence signals, second harmonic signals and third harmonic signals.

7. The microscopic imaging system for simultaneously exciting / collecting two-photon and three-photon signals according to claim 1, characterized in that: The signal collection / separation device comprises a third collimating lens (16), the third collimating lens (16) being connected to the microscopic imaging module via a multimode optical fiber (15), and a second dichroic filter (18), a third dichroic filter (22), a fourth dichroic filter (26) and a fifth bandpass filter (30) being arranged in sequence on the transmission light path of the third collimating lens (16); A second band-pass filter (19), a first photomultiplier tube (20) and a first photon counter (21) are provided on the reflected light path of the second dichroic filter (18); a third band-pass filter (23), a second photomultiplier tube (24) and a second photon counter (25) are provided on the reflected light path of the third dichroic filter (22); a fourth band-pass filter (27), a third photomultiplier tube (28) and a third photon counter (29) are provided on the reflected light path of the fourth dichroic filter (26); and a fourth photomultiplier tube (31) and a fourth photon counter (32) are provided on the transmitted light path of the fifth band-pass filter (30).

8. The microscopic imaging system for simultaneously exciting / collecting two-photon and three-photon signals according to claim 7, characterized in that: A first bandpass filter (17) is also provided on the optical path between the second dichroic filter (18) and the third collimating lens (16).

9. The microscopic imaging system for simultaneously exciting / collecting two-photon and three-photon signals according to any one of claims 1 to 8, characterized in that: The laser (1) is an erbium-doped fiber laser.

10. A microscope based on the microscopic imaging system for simultaneously exciting / collecting two-photon and three-photon signals according to any one of claims 1 to 9, characterized in that: The position of the microscopic imaging device is unspecific relative to the positions of other devices of the microscope.

Images