Multi-photon imaging synchronous multi-region data acquisition method and multi-photon imaging system

Abstract

The invention relates to a multi-photon imaging synchronous multi-region data acquisition method and a multi-photon imaging system, and the method comprises the steps: building a multi-photon imaging system through employing a two-photon laser and a three-photon laser with set parameters, and carrying out the focusing depth adjustment of two photons and three photons; the synchronous signal generated by the three-photon laser is subjected to phase reversal processing, and then an acousto-optic modulator of the synchronous two-photon laser is activated to alternately output exciting light; and after frequency multiplication is performed on a synchronizing signal generated by the three-photon laser, the synchronizing signal starts from the acquisition card to drive the photomultiplier, and multi-region two-photon and three-photon imaging signals are synchronously acquired. Compared with the prior art, multi-plane imaging is realized while the imaging quality is ensured, and the risk of light damage to a biological sample is reduced by an alternative excitation strategy.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of multi-photon microscopy, and in particular to a multi-photon imaging synchronous multi-region data acquisition method and a multi-photon imaging system. BACKGROUND

[0002] Multiphoton Microscopy (MPM) is a powerful imaging technique that has revolutionized the field of neuroscience by enabling the imaging of the living brain. Since its first application in the mid-1990s, it has become a key tool for unraveling the complexity of the brain. The importance of MPM lies in its non-invasive and deep-tissue imaging capabilities compared to traditional fluorescence and confocal microscopy. By using longer-wavelength laser sources, MPM reduces scattering and absorption, allowing the laser to penetrate deeper into biological tissue, often up to several hundred micrometers or even 1 millimeter, while minimizing photodamage to surrounding tissues. Furthermore, the technology utilizes nonlinear optical processes, i.e., the simultaneous absorption of two or more photons, enabling highly selective excitation at specific locations, significantly improving spatial resolution.

[0003] In neuroscience research, MPM has become a cornerstone for understanding the structure and function of the brain. It enables scientists to observe the communication between nerve cells in the living brain in real-time, revealing the mechanisms of neurotransmitter release and neuronal action potential propagation. This technology is crucial for mapping neural networks, analyzing neural circuit dynamics, and understanding how the brain processes information. For example, by labeling specific types of nerve cells or neuronal connections, researchers can observe their activity during learning and memory processes or functional changes in disease states.

[0004] MPM also has important applications in observing the vascular structure and hemodynamics of the brain. Neuroscientists can monitor blood supply and metabolic status of the brain, understand the phenomenon of neurovascular coupling, and its role in neurodegenerative diseases and brain injury. In addition, with the advancement of fluorescent dyes and light-sensitive protein technology, combined with multi-photon excitation, researchers can genetically encode or fluorescently label individual neurons, allowing for more precise manipulation and measurement.

[0005] In summary, multiphoton microscopy not only greatly enhances the understanding of the structure and function of the living brain, but also drives the advancement of experimental methods in neuroscience, which has far-reaching significance for future treatment of neurodegenerative diseases and other brain diseases. With the continuous progress and innovation of multi-photon imaging technology, its application prospects in neuroscience research will be even broader.

[0006] Defects and deficiencies of the prior art:

[0007] 1) Limited amount of information: In the multi-photon imaging technology, the intrinsic "optical sectioning" effect of the nonlinear optical response is a significant feature, combined with laser point scanning imaging, to achieve accurate imaging of a specific focal plane. However, the inherent single-plane imaging characteristics of this technology have certain restrictions on the acquisition of information in the axial direction. The acquisition of data is limited to a single two-plane, thereby hindering the continuous dynamic understanding of the sample in the three-dimensional spatial structure.

[0008] 2) Limited imaging depth: While maintaining the imaging quality, improving the imaging depth is also an important challenge faced by the current multi-photon imaging technology. The classical two-photon imaging method is usually limited to an imaging depth of several hundred microns. Although the three-photon imaging technology successfully advances the imaging depth boundary to about 1.2 millimeters by using longer wavelength photons, the imaging effect of the shallow structure still needs to be optimized.

[0009] 3) Significant difference in three-photon and two-photon laser pulse frequency: While pursuing the imaging quality, the influence of the excitation light pulse frequency on the potential optical damage of the sample must be fully considered. In particular, in biological samples, due to the more significant absorption of long-wavelength light by water and other biological media, the excitation light source required for three-photon imaging usually operates at a lower pulse frequency, which is significantly lower than the typical operating frequency of two-photon imaging technology. Due to the significant difference in the pulse frequency of the operation of the two technologies, this causes a significant technical challenge when synchronously collecting two-photon and three-photon imaging data. SUMMARY

[0010] The purpose of the present application is to overcome the defects of the prior art and provide a multi-photon imaging synchronous multi-region data acquisition method and a multi-photon imaging system, which ensures the imaging quality while realizing multi-plane imaging, and the alternating excitation strategy reduces the risk of optical damage to biological samples.

[0011] The purpose of the present application can be achieved by the following technical solutions:

[0012] According to a first aspect of the present application, a multi-photon imaging synchronous multi-region data acquisition method is provided, comprising:

[0013] A multi-photon imaging system is built using a two-photon laser and a three-photon laser with set parameters, and two-photon and three-photon focusing depth adjustment is performed.

[0014] The synchronous signal generated by the three-photon laser is processed in reverse to activate the acousto-optic modulator of the synchronous two-photon laser, and the excitation light is alternately output; the synchronous signal generated by the three-photon laser is multiplied to drive the photomultiplier tube of the acquisition card, and the multi-region two-photon and three-photon imaging signals are synchronously collected.

[0015] Preferably, the wavefront modulation technique is used for adjusting the focusing depth of the two-photon and three-photon, specifically, a spatial light modulator is used to shape the wavefront curvature of the incident laser, the propagation path of the light beam in the sample is controlled, a three-photon or two-photon optical path is added to adjust the focusing depth of the light beam, and the two-photon and three-photon excitation light is focused on different depth layers of the sample.

[0016] Preferably, a zoom lens is used to adjust the focusing depth of the two-photon and three-photon.

[0017] Preferably, a refractive index gradient lens is used to adjust the focusing depth of the two-photon and three-photon.

[0018] Preferably, a plurality of refractive index gradient lenses are combined or the axial position of a single refractive index gradient lens is adjusted to obtain focus points at different depths.

[0019] Preferably, an optical focusing adjustment device is used to adjust the focusing depth of the two-photon and three-photon, specifically, a lens group with a magnification of 1 is installed on a displacement table, the distance between the lenses is changed to adjust the light beam focus position, and the two-photon and three-photon excitation light is focused on different depth layers of the sample.

[0020] Preferably, the reverse circuit and the analog frequency multiplication circuit are used to process the synchronization signal generated by the three-photon laser in reverse and frequency multiplication, respectively, and the frequency multiplication signal is used as the trigger signal of the acquisition card to drive the photomultiplier tube, and the multi-region two-photon and three-photon imaging signals are synchronously collected.

[0021] Preferably, the digital signal processor is used to process the synchronization signal generated by the three-photon laser in reverse and frequency multiplication, specifically:

[0022] The synchronization signal generated by the three-photon laser is digitized by an analog-to-digital converter and sent to a digital signal processor, and the digital signal processor receives all the signal samples and multiplies them by -1 to achieve reverse;

[0023] The non-fundamental frequency components of the synchronization signal generated by the three-photon laser are removed by mathematical means, the fundamental frequency components are copied, and their amplitudes are increased as needed, and then the digital signal is converted back to an analog signal by a digital-to-analog converter, the converted analog signal is used as the trigger signal of the acquisition card to drive the photomultiplier tube, and the multi-region two-photon and three-photon imaging signals are synchronously collected.

[0024] The sampling rate of the analog-to-digital converter and the digital-to-analog converter is more than twice the target frequency.

[0025] Preferably, a digital logic circuit is set up in a field programmable logic gate array to receive the synchronization signal generated by the three-photon laser, and each input data bit is inverted to achieve signal reversal.

[0026] The digital phase-locked loop module is arranged in the field programmable logic gate array, a synchronization signal generated by a three-photon laser is multiplied, and the multiplied signal is output from a pin of the field programmable logic gate array, so that the multiplied signal is used as a trigger signal of a collection card to drive a photomultiplier tube, and multi-region two-photon and three-photon imaging signals are synchronously collected.

[0027] According to a second aspect of the present application, a multi-photon imaging system is provided, which uses any of the above methods to synchronously collect multi-region data.

[0028] Compared with the prior art, the present application has the following beneficial effects:

[0029] 1) The present application combines two-photon and three-photon imaging technologies, effectively utilizes the high-quality output of two-photon imaging on shallow structures and the advantages of three-photon imaging on deep imaging, realizes the ability to select multiple depth levels without adjusting the optical path, and significantly enhances the operation flexibility.

[0030] 2) Excitation light alternation output control: the alternation output of two-photon and three-photon excitation light is realized through the timely scheduling of trigger signals, the alternation excitation strategy reduces the risk of light damage to biological samples, and the dynamic range and accuracy of experimental monitoring are improved by improving the signal-to-noise ratio of collected data; at the same time, thanks to the characteristics of working at high repetition frequency, high-speed imaging is realized, and the imaging efficiency and safety of the sample are improved.

[0031] 3) The synchronization signal of the three-photon laser is used as the trigger signal of the two-photon laser, the output of the two-photon laser is scheduled, and the synchronization of two-photon and three-photon signals by a single photomultiplier tube is made possible; and the use of a single photomultiplier tube greatly simplifies the imaging system architecture, reduces costs, and facilitates system expansion and maintenance.

[0032] 4) The focusing depth of two-photon and three-photon is adjusted by using an optical focusing adjustment method, precise focus adjustment can be realized by using a displacement table, and stable focusing adjustment can be obtained by physically moving the lens, which is suitable for long-time imaging; the focusing depth of two-photon and three-photon is adjusted by using a wavefront modulation method, which can realize rapid and mechanical movement-free focus adjustment; the focusing depth of two-photon and three-photon is adjusted by using an electrically tunable focus lens, which has a small volume and is easy to integrate into existing optical systems; the focusing depth of two-photon and three-photon is adjusted by using a refractive index gradient lens, which has a small volume and is easy to use in a compact space.

[0033] 5) An analog frequency doubling circuit is used to frequency-multiply the synchronization signal generated by the three-photon laser for signal acquisition. The circuit structure is relatively simple, easy to implement and debug, and the required components are cheap and low-cost. A digital signal processor is used to frequency-multiply the synchronization signal generated by the three-photon laser, which can accurately control the frequency and phase of the signal, and the parameters can be directly adjusted through programming, which is more convenient. A digital phase-locked loop is used to frequency-multiply the synchronization signal generated by the three-photon laser for signal acquisition, which can provide a highly stable frequency-multiplied signal. In addition, the field programmable logic gate array provides extremely high flexibility and programmability, and can implement complex digital logic. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 is a flow chart of the method of the present invention;

[0035] Figure 2 Schematic diagram of the structure of a multi-photon imaging system in an embodiment;

[0036] Figure 3 This is a signal diagram collected in the embodiment;

[0037] Figure 1: 1-three-photon laser; 2, 3-magnifying lens group; 4-reflecting mirror; 5-two-photon laser; 6-acousto-optic modulator, 7, 8-lens group; 9, 10-magnifying lens; 11-dichroic mirror; 12-scanning galvanometer; 13-scanning lens; 14-reflecting mirror; 15-tube lens; 16-dichroic mirror; 17-reflecting mirror; 18-objective lens; 19-lens; 20-filter; 21-filter; 22-photomultiplier tube. DETAILED DESCRIPTION

[0038] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts should fall within the scope of protection of the present invention.

[0039] Example

[0040] like Figure 1 As shown, this embodiment provides a method for synchronous multi-region data acquisition of multi-photon imaging, including:

[0041] Use two-photon lasers and three-photon lasers with set parameters to build a multiphoton imaging system, and adjust the two-photon and three-photon focusing depth;

[0042] The synchronization signal generated by the three-photon laser is inverted to activate the acousto-optic modulator of the synchronized two-photon laser to output the excitation light alternately;

[0043] The synchronization signal generated by the three-photon laser is multiplied to drive the photomultiplier tube, and the multi-region two-photon and three-photon imaging signals are synchronously collected.

[0044] Next, the method of the embodiment is described in detail.

[0045] I. Selection of multi-photon imaging system parameters

[0046] First, appropriate two-photon and three-photon lasers and acousto-optic modulators need to be selected. The two-photon laser can be a femtosecond laser with a wavelength of about 700-1000 nm, and the repetition frequency is best set to 80 MHz. The pulse width should be between 100 femtoseconds and 2 picoseconds. The laser needs to have a high peak power to ensure sufficient nonlinear absorption efficiency. The three-photon laser is a femtosecond laser with a wavelength of 1300-1700 nm, and the repetition frequency is about 1 MHz. Similarly, a high peak power is required. The acousto-optic modulator is used to precisely control the laser output, including adjusting the time of the laser pulses to ensure precise synchronization of the pulses at different imaging depths.

[0047] II. Two-photon and three-photon focal depth adjustment

[0048] 1. Optical focusing adjustment

[0049] A pair of lenses with a magnification of 1 is used, which is installed on a precision displacement stage. By precisely moving the lenses, the actual length of the optical path is changed. This allows the two beams to focus at different depths as needed. The three-photon or two-photon optical path is added to adjust the focusing depth of the light beam. By changing the distance between the lenses, the focal point position of the light beam can be adjusted, allowing the two-photon and three-photon excitation light to focus on different depth layers of the sample. Precise displacement of the stage allows very precise focal length adjustment, and physically moving the lens allows stable focusing adjustment, which is suitable for long-term imaging.

[0050] 2. Wavefront engineering

[0051] By using wavefront modulation techniques such as spatial light modulators (SLM), the curvature of the incident laser wavefront can be shaped, and the propagation path of the light beam in the sample can be precisely controlled. The three-photon or two-photon optical path is added to adjust the focusing depth of the light beam, which ensures that the two-photon and three-photon excitation light focuses on different depth layers of the sample. Wavefront modulation can achieve rapid and mechanical-free focal point adjustment.

[0052] 3. Zoom lens

[0053] A zoom lens, such as an electrically tunable lens, is added to the three-photon or two-photon optical path to adjust the depth of focus of the beam. This allows the depth of focus to be dynamically adjusted without moving physical components, allowing the two-photon and three-photon excitation light to be focused at different depths in the sample. Electrically tunable lenses are typically small in volume and can be easily integrated into existing optical systems.

[0054] 4. Gradient index lens (GRIN lens)

[0055] A gradient index lens (GRIN lens) can also be added to the three-photon or two-photon optical path to adjust the depth of focus of the beam. This lens has a refractive index that varies along the axis of the lens. A GRIN lens is installed in the optical path, and the appropriate lens length is selected to adjust the focal length. The position of the GRIN lens is adjusted to change the focal point of the axially incident light. The two-photon and three-photon excitation light is focused at different depths in the sample. To obtain different depths of focus, multiple GRIN lenses can be combined or the axial position of a single GRIN lens can be adjusted. GRIN lenses are small in volume and can be easily used in a compact space.

[0056] Three, two-photon and three-photon alternating excitation scheme

[0057] In this embodiment, the pulse repetition frequency of the two-photon laser is typically 80 MHz, corresponding to a time interval of 12.5 ns. The pulse repetition frequency of the three-photon laser is typically 1 MHz, forming a pulse sequence with a period of 1 μs. The three-photon laser synchronization signal is inverted and frequency-doubled to control the acousto-optic modulator AOM in the two-photon laser to achieve two-photon and three-photon alternating excitation, and to trigger the acquisition of the fluorescence signal by the acquisition card. A hardware logic board (such as an FPGA board) or a microcontroller is used as the generation and control center of the synchronization signal. An acousto-optic modulator (AOM) is used to control the excitation of the two-photon laser pulses.

[0058] Four, circuit implementation of the signal acquisition scheme

[0059] 1. Analog frequency doubling circuit

[0060] In the analog frequency doubling circuit, the inverter circuit is configured by an operational amplifier (such as LM741) and two resistors (R1, R2) to become an inverting amplifier. The 1 MHz synchronization signal is input to resistor R1, connected to the inverting input (-) of the operational amplifier, and the non-inverting input (+) is grounded. The inverting output of the signal is obtained through resistor R2.

[0061] The frequency multiplication circuit relies on a non-linear element, such as a diode, to generate harmonics. The initial 1MHz signal is first shaped into an approximate square wave by a limiter, and then passed through a diode to generate a waveform containing multiple integer harmonic components. By using a precisely designed bandpass filter, only the 2MHz component is selected to obtain the desired frequency multiplication signal, which is used as the trigger signal of the acquisition card to achieve the synchronous acquisition of two-photon and three-photon signals.

[0062] The analog frequency multiplication circuit structure is relatively simple, easy to implement and debug; the required components are cheap and the cost is low.

[0063] 2. Digital signal processor (DSP)

[0064] First, the 1MHz analog signal is digitized by a high-speed analog-to-digital converter (ADC). After the DSP receives these digital samples, it multiplies all the signal samples by -1 to achieve inversion. Then, using algorithms such as Fast Fourier Transform (FFT), the non-fundamental frequency components are removed by mathematical means, the fundamental frequency components are copied, and their amplitudes are increased as needed. After that, the digital signal is converted back to an analog signal by a digital-to-analog converter (DAC), i.e. a 2MHz output signal is obtained. This signal is used as the trigger signal of the acquisition card to achieve the synchronous acquisition of two-photon and three-photon signals. The sampling rate of the ADC and DAC must be at least twice the target frequency, which should be 4MHz or higher. The real-time processing capability of the DSP needs to be sufficient to perform complex operations such as FFT. The DSP can accurately control the frequency and phase of the signal, and allows the parameters to be adjusted by programming.

[0065] 3. Field programmable gate array (FPGA) and digital phase-locked loop (DPLL)

[0066] In the FPGA, first design a simple digital logic circuit that receives a 1MHz signal as input, then inverts each input data bit (e.g. changes 1 to 0, 0 to 1). This can achieve the inversion of the signal.

[0067] The input 1MHz synchronization signal is sent to the DPLL module, which includes a phase frequency detector, a loop filter and a numerically controlled oscillator (NCO). When configuring the DPLL, the parameters of the loop filter and the control word of the NCO must be accurately set to match the 2MHz output. The DPLL will continuously adjust the output of the NCO to keep the same phase and integer multiple of the frequency of the input signal. Finally, an output pin of the FPGA will generate the required 2MHz signal. This scheme requires accurate programming of the electronic components inside the FPGA and modulation of the control word of the NCO to achieve a stable frequency multiplication output. This signal is used as the trigger signal for the acquisition card to achieve synchronous acquisition of two-photon and three-photon signals. The digital phase-locked loop can provide a high-stability frequency multiplication signal, and the FPGA provides high flexibility and programmability, allowing complex digital logic to be implemented.

[0068] In addition, as Figure 2 shown, the embodiment provides a multi-photon imaging system combining two-photon and three-photon imaging technology, which can realize real-time, multi-plane imaging of different regions of the brain and synchronous data collection, specifically including:

[0069] The three-photon excitation light is emitted by a three-photon laser 1 at a pulse frequency of 1MHz, and the pulse width is usually in the range of tens of femtoseconds; after adjustment by a pair of amplification lenses 2, 3, its path is turned by a mirror 4 and enters the main light path of the system; at the same time, in the two-photon incident light path, the two-photon excitation light generated by a two-photon laser 5 is emitted at a pulse frequency of 80MHz and a pulse width of about one hundred femtoseconds, and is modulated by an acousto-optic modulator 6; then, the light beam is precisely adjusted by a pair of lens groups 7, 8 with a magnification of one, where the second lens is mounted on a precision adjustable displacement stage, allowing the lens spacing to be finely adjusted, providing high flexibility and precision for focusing on different brain regions; after being processed by another pair of amplification lenses 9, 10, the two-photon excitation light and the three-photon excitation light are coupled at a dichroic mirror 11 and continue to transmit along the main light path of the system.

[0070] In the main light path, the two excitation lights are guided by a scanning galvanometer 12 and a scanning lens 13, reflected by a mirror 14 and a dichroic mirror 16, introduced into an objective lens 18 by a tube lens 15, and finally focused on different depths of the sample.

[0071] In the collection light path, the objective lens 18 collects the fluorescence signal from the sample, which is separated by the dichroic mirror 16 and focused by the lens 19; the optical filter 20 is used to remove the leakage of the excitation light, and the band-pass filter 21 further screens out the target fluorescence waveband, and finally detected by the photomultiplier tube 22. Figure 3 Signal result graph for the embodiment.

[0072] The high regulation and fine operation of the system ensure that it can flexibly adapt to changing experimental requirements and maintain high accuracy and repeatability of data acquisition. Through the signal acquisition scheme designed in this embodiment, the imaging system not only supports two-photon and three-photon imaging on a single plane, but also realizes the synchronous collection of interlayer imaging data, providing a powerful new tool for in-depth analysis of complex biological structures and functional networks. In this embodiment, simultaneous imaging of two planes is achieved. In addition, more planes can be simultaneously realized through time multiplexing decomposition strategy, and an electric focusing lens can be introduced into the optical path to obtain the ability of rapid scanning along the z-axis direction, which is not described here.

[0073] The above merely illustrates the specific embodiments of the present application, but the protection scope of the present application is not limited thereto, and any person skilled in the art can easily think of various equivalent modifications or replacements within the technical range disclosed by the present application, which should be covered within the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. A method for multi-photon imaging simultaneous multi-region data acquisition, characterized in that, The application relates to a multi-photon imaging system and a method for synchronously collecting multi-region data. The multi-photon imaging system is built by using a two-photon laser and a three-photon laser with set parameters, and the focusing depth of the two-photon and three-photon is adjusted; The synchronous signal generated by the three-photon laser is reversely processed to activate the acousto-optic modulator of the synchronous two-photon laser, and the excitation light is alternately outputted; The synchronous signal generated by the three-photon laser is multiplied to drive the photomultiplier tube through a collection card, and the multi-region two-photon and three-photon imaging signals are synchronously collected.

2. The method of claim 1, wherein, The focusing depth of the two-photon and three-photon is adjusted by using a wavefront modulation technology, specifically, the wavefront curvature of the incident laser is shaped by using a spatial light modulator, the propagation path of the light beam in the sample is controlled, the three-photon or two-photon light path is added to adjust the focusing depth of the light beam, and the excitation light of the two-photon and three-photon is focused on different depth layers of the sample.

3. The method of claim 1, wherein, The focusing depth of the two-photon and three-photon is adjusted by using a zoom lens.

4. The method of claim 1, wherein, The focusing depth of the two-photon and three-photon is adjusted by using a refractive index gradient lens.

5. The method of claim 4, wherein, The axial position of the single refractive index gradient lens is combined or adjusted to obtain the focus points of different depths.

6. The method of claim 1, wherein, The focusing depth of the two-photon and three-photon is adjusted by using an optical focusing adjustment device, specifically, a lens group with a magnification of 1 is installed on a displacement table, the distance between the lenses is changed, the focus point position of the light beam is adjusted, and the excitation light of the two-photon and three-photon is focused on different depth layers of the sample.

7. The method of claim 1, wherein, The synchronous signal generated by the three-photon laser is reversely processed by using a reverse circuit and an analog frequency multiplication circuit, the frequency multiplication signal is used as the trigger signal of the collection card to drive the photomultiplier tube, and the multi-region two-photon and three-photon imaging signals are synchronously collected.

8. The method of claim 1, wherein, The synchronous signal generated by the three-photon laser is reversely processed by using a digital signal processor, specifically, The synchronous signal generated by the three-photon laser is digitized through an analog-to-digital converter and sent to the digital signal processor, the digital signal processor receives the signals, multiplies all the signal samples by-1 to realize the reverse, removes the non-base frequency components of the synchronous signal generated by the three-photon laser through a mathematical method, copies the base frequency components, increases the amplitude of the base frequency components as required, and then converts the digital signal into an analog signal through a digital-to-analog converter, the converted analog signal is used as the trigger signal of the collection card to drive the photomultiplier tube, and the multi-region two-photon and three-photon imaging signals are synchronously collected. The sampling rate of the analog-to-digital converter and the digital-to-analog converter is more than twice the target frequency. A digital logic circuit is arranged in a field programmable logic gate array, receives the synchronous signal generated by the three-photon laser, and reverses each input data bit to realize signal reversal.

9. The method of claim 1, wherein, A digital phase-locked loop module is arranged in the field programmable logic gate array, the synchronous signal generated by the three-photon laser is frequency-multiplied, and the frequency-multiplied signal is outputted from the field programmable logic gate array pin to be used as the trigger signal of the collection card to drive the photomultiplier tube, and the multi-region two-photon and three-photon imaging signals are synchronously collected. The multi-photon imaging system adopts the method in any one of claims 1-9 to synchronously collect multi-region data.

10. A multi-photon imaging system, characterized by, ​

Images