Full-automatic intelligent multifunctional high-order nonlinear fluorescence dynamics testing method and device

Abstract

The invention discloses a full-automatic intelligent multifunctional high-order nonlinear fluorescence dynamics testing method and device, and relates to the field of nonlinear optics and nano photonics. An optical testing device composed of an exciting light generation module, an automatic power regulation module, an exciting light modulation module, a sample scanning module, a data acquisition and analysis module and an intelligent control module is established, and under the excitation of incident laser, based on the overall coordination of the intelligent control module, the optical testing device is established. Other modules are driven to complete the dynamic response test of a nonlinear fluorescence sample, and full-automatic intelligent, multifunctional, high-precision, simple, efficient, low-cost and universal fluorescence dynamic test analysis is realized. The problems that traditional manual operation wastes time and labor and the testing precision is low are solved. In addition, the multifunctional integration of the fluorescence response curve, the fluorescence rise time, the fluorescence lifetime and the fluorescence imaging effectively improves the test efficiency and the application scene, and greatly reduces the system cost.

Description

Technical Field

[0001] The invention relates to the fields of nonlinear optics and nanophotonics, and in particular to a fully automatic, intelligent and multifunctional high-order nonlinear fluorescence dynamics testing method and device. Background Art

[0002] Nonlinear fluorescence dynamics testing is a key method for studying nonlinear optical effects, nanophotonics, and their applications in bioimaging, optical sensing, and other fields. Traditional nonlinear fluorescence nanosample testing methods usually rely on manual operations, which are not only time-consuming and labor-intensive, but also extremely susceptible to human errors, resulting in inaccurate data. In addition, due to the complexity of manual testing, especially for some probes with ultra-high nonlinear response characteristics, when the laser power approaches a specific threshold, its fluorescence intensity exhibits significant nonlinear changes. This ultra-high-order nonlinear dependence is difficult to accurately capture in conventional manual measurements, further affecting the scientific explanation and analysis of nonlinear optical phenomena, and hindering the in-depth development of nonlinear optics and nanophotonics.

[0003] At present, in order to meet various testing requirements such as the fluorescence response curve, rise time, decay time and imaging of fluorescent nanoparticles, it is usually necessary to replace different test modules or devices, which not only increases the complexity and time cost of the test process, but also poses challenges to the stability and consistency of the experiment; especially in the study of high-order nonlinear fluorescence dynamics, precise control and automatic adjustment of different powers are particularly critical. However, existing systems are difficult to achieve multi-functional integration and fully automated intelligent testing, which limits their breadth and universality in practical applications. Summary of the invention

[0004] The purpose of the present invention is to provide a fully automatic, intelligent, and multifunctional high-order nonlinear fluorescence dynamics test method and device to overcome the defects of the prior art, realize fully automatic and high-precision measurement of fluorescence response curve, fluorescence rise time, fluorescence lifetime, and fluorescence imaging, and eliminate errors and false information caused by human intervention.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] A fully automatic, intelligent, and multifunctional high-order nonlinear fluorescence dynamics testing device includes: an excitation light generation module, an automatic power adjustment module, an excitation light modulation module, a sample scanning module, a data acquisition and analysis module, and an intelligent control module; under incident laser excitation, based on the overall coordination of the intelligent control module, the remaining modules are driven to complete the dynamic response test of the nonlinear fluorescence nanosample and realize fluorescence dynamics test and analysis.

[0007] The excitation light generation module includes the laser and a filter group placed in sequence along the direction of the laser beam emitted by the laser. The laser is used to generate a steady-state laser beam output, the photon energy of the laser beam matches the excited state absorption of the fluorescent nanoparticles, and the filter is used to purify the laser.

[0008] The automatic power adjustment module includes a gradient attenuation sheet and a micro servo motor connected thereto. The gradient attenuation sheet has different light transmittances in its radial direction or angle, is aligned with the laser output end, and rotates at different angles to correspond to different transmitted light powers; the micro servo motor is used to drive the gradient attenuation sheet to rotate, realize precise control of the angle of the attenuation sheet, and thus adjust the power of the transmitted light.

[0009] The excitation light modulation module includes a focusing lens, a polarization-maintaining fiber, a half-wave plate and a quarter-wave plate. The focusing lens and the polarization-maintaining fiber are used for beam shaping to improve the efficiency of the excitation light beam, and the half-wave plate and the quarter-wave plate are used to change the polarization state of the excitation light to adapt to different excitation modes.

[0010] The sample scanning module includes an infinity correction tube lens system, a dichroic mirror, an imaging objective lens and a three-dimensional displacement stage. The infinity correction tube lens system is used to adjust the beam size to match the entrance pupil of the imaging objective lens, improve the beam utilization rate, and correct the optical aberration under high magnification and high numerical aperture conditions to ensure the clarity of imaging; the dichroic mirror can reflect the excitation light and transmit the sample fluorescence, and is used to separate the excitation light and the fluorescence; the imaging objective lens is used for beam focusing and sample imaging; the three-dimensional displacement stage is used for spatial positioning of the sample, moves in the XYZ direction according to the set resolution, and scans the entire sample area point by point.

[0011] The data acquisition and analysis module is composed of a filter group, a focusing lens and a photodetector placed coaxially in sequence. The data acquisition and analysis module is used to collect fluorescence signals, which are input to the computer end for data processing through the intelligent control module; the sample emits fluorescence in all directions under the excitation of the incident laser, and the imaging objective collects a part of the fluorescence signal. The signal passes through the dichroic mirror, the filter group, and the focusing lens, and is received by the photodetector, and then input to the computer end through the intelligent control module.

[0012] The intelligent control module includes an intelligent control FPGA chip and a computer. The FPGA chip is connected to the computer. The FPGA chip serves as a main control unit, which is used to receive control signals from the computer, convert them into control instructions for the servo motor, monitor the feedback of the servo motor in real time, and record the fluorescence signal. The computer communicates with the FPGA through a USB or Ethernet interface through software such as LabVIEW to complete parameter setting, real-time data transmission, visual display and data analysis, and coordinates various modules of the system.

[0013] The excitation light generation module includes continuous ultraviolet light / visible light / near infrared light or pulsed ultraviolet light / visible light / near infrared light, and filter groups placed in sequence along the direction of the laser beam emitted by the laser. The laser generates a steady-state laser beam output, the photon energy of which matches the excited state absorption of the fluorescent sample, and the filter is used to purify the laser.

[0014] Furthermore, the filter set of the excitation light generating module can be assembled by a rotatable bracket to adjust and match different excitation light wavelengths; the filter set of the data acquisition and analysis module can be assembled by a rotatable bracket to adjust and match different collected fluorescence wavelengths.

[0015] Furthermore, the FPGA chip has a built-in pulse width modulation module for generating a control pulse signal and adjusting the pulse frequency, width and duty cycle; the FPGA chip has a built-in time gating module for accurately controlling the time window of the excitation and acquisition signals for data analysis.

[0016] Furthermore, the micro servo motor can control the rotation angle through pulses, and has an angle feedback system to ensure high-precision positioning. The micro servo motor is controlled by the FPGA chip of the intelligent control module, and its rotation angle is controlled by the pulse width modulation signal. The frequency and duty cycle of the pulse signal determine the speed and direction of the servo motor. In addition, the FPGA controls the pulse output duration and frequency through the Gate to achieve the precise stop of the servo motor at a specific angle.

[0017] Furthermore, the FPGA chip is connected to the photodetector of the data acquisition and analysis module, and the photodetector collects light signals in the Gate window of the FPGA, converts them into counting pulses, and accumulates them in the counter. After each independent counting is completed, the counter transmits the accumulated results to the FPGA and feeds them back to LabVIEW in real time to realize the fluorescence intensity collection of this time.

[0018] Furthermore, the power attenuation sheet can be replaced by other power regulating devices such as an electrically adjustable optical attenuator, an electro-optic modulator, an acousto-optic modulator, etc., the main function of which is to change the power at the laser output end.

[0019] Furthermore, the micro servo motor can be replaced by a mechanical drive device such as a stepper motor or a piezoelectric drive, and its main function is to accurately control the laser attenuation device.

[0020] Furthermore, the FPGA chip can be replaced by a microcontroller, a single-chip control system, a digital-to-analog converter, a digital signal processor, etc., and its main function is to realize signal conversion and control.

[0021] Furthermore, the photodetector can be replaced by a single photon counter, a photomultiplier tube, a spectrometer, etc., the main function of which is to record photon information.

[0022] Furthermore, the LabVIEW can be replaced by development environments such as Python, Matlab, LabWindows / CVI, etc., and its main function is to realize UI interface design and coordinate various modules of the control system.

[0023] Furthermore, fluorescent samples include quantum dots, organic dyes, traditional upconversion fluorescent nanoparticles, and photon avalanche effect rare earth doped upconversion nanoparticles, etc.

[0024] A fully automatic, intelligent and multifunctional high-order nonlinear fluorescence dynamics test method, comprising the following steps:

[0025] S1: Laser excitation and power modulation: The laser outputs a stable laser beam, which is filtered out by the filter group to remove lasers of other wavelengths. The purified excitation light is quantitatively modulated by the automatic power adjustment module. After being modulated by the focusing objective lens, polarization-maintaining fiber, and polarization wave plate, the aberration is corrected by the tube lens group and the entrance pupil of the imaging objective lens is matched to obtain a Gaussian solid light spot.

[0026] It should be noted that in step S1, the laser determines the required laser wavelength according to the excited state absorption characteristics of the fluorescent sample, including continuous ultraviolet light / visible light / near-infrared light or pulsed ultraviolet light / visible light / near-infrared light, and the photon energy of the light beam matches the excited state absorption of the fluorescent sample.

[0027] It should be noted that in step S1, the excitation power adjustment does not require manual operation, and accurate intelligent control on the computer side is achieved through the automatic power adjustment module.

[0028] It should be noted that in step S1, the automatic power adjustment module is mainly composed of a power attenuation sheet and a micro servo motor. The attenuation sheet has different light transmittances in its radial direction or angle, and is aligned with the laser emission end. Rotating at different angles corresponds to different transmitted light powers. The micro servo motor is used to drive the rotation of the gradient attenuation sheet to achieve precise control of the angle of the attenuation sheet, thereby adjusting the power of the transmitted light. The micro servo motor can control the rotation angle by pulse, and has an angle feedback system to ensure high-precision positioning. The micro motor is controlled by the FPGA chip of the intelligent control module, and its rotation angle is controlled by a pulse width modulation signal. The frequency and duty cycle of the pulse signal determine the speed and direction of the servo motor. In addition, the FPGA controls the pulse output duration and frequency through the Gate to achieve precise stop of the servo motor at a specific angle.

[0029] It should be noted that in step S1, the focusing lens and polarization-maintaining optical fiber are used for beam shaping to improve the efficiency of the excitation light beam, and the half-wave plate and quarter-wave plate are used to change the polarization state of the excitation light to adapt to different excitation modes.

[0030] It should be noted that in step S1, the power attenuation sheet can be replaced by other power regulating devices such as an electric adjustable optical attenuator, an electro-optic modulator, an acousto-optic modulator, etc., and its main function is to change the power of the laser output end; the micro servo motor can be replaced by a mechanical driving device such as a stepper motor and a piezoelectric driver, and its main function is to accurately control the laser attenuation device; the FPGA chip can be replaced by a microcontroller, a single-chip control system, a digital-to-analog converter, a digital signal processor, etc., and its main function is to realize signal conversion and control.

[0031] S2: Fluorescence signal collection and processing: The focused Gaussian solid light spot excites the fluorescent sample at the exit end of the imaging objective lens, and the sample fluorescence enters the collection and analysis module: the fluorescence signal of the sample is collected by the same imaging objective lens, and then focused by the dichroic mirror, filter set and lens and enters the collection optical fiber. The signal is received by the photodetector, stored by the FPGA, and processed in collaboration with LabVIEW on the computer side;

[0032] It should be noted that in step S2, the fluorescent samples include quantum dots, organic dyes, traditional up-conversion fluorescent nanoparticles, and photon avalanche effect rare earth doped up-conversion nanoparticles, etc. The nonlinearity of quantum dots and organic dyes is generally less than 3, the nonlinearity of traditional up-conversion fluorescent nanoparticles is generally less than 6, and the nonlinearity of photon avalanche effect rare earth doped up-conversion nanoparticles is generally greater than 10.

[0033] It should be noted that, in step S2, the filter set is assembled by a rotatable bracket for adjusting and matching different collected fluorescence wavelengths.

[0034] It should be noted that, in step S2, the photoelectric detector includes but is not limited to a photomultiplier tube, a single photon counter, a spectrometer, and the like.

[0035] It should be noted that in step S2, the FPGA chip realizes multiple functions such as pulse signal generation, time gating, fluorescence signal acquisition and processing.

[0036] It should be noted that, in step S2, the LabVIEW implements parameter setting, real-time data transmission and visual display.

[0037] It should be noted that in step S2, the photodetector is connected to the FPGA chip. The photodetector collects light signals in the Gate window of the FPGA to achieve light-to-electrical signal conversion. The ADC channel of the FPGA receives the analog electrical signal of the photodetector, digitizes it into counting pulses, and accumulates it in the counter. After each independent counting is completed, the counter transmits the accumulated result to the FPGA and feeds it back to the LabVIEW program in real time to achieve the fluorescence intensity collection of this time.

[0038] It should be noted that in step S2, the computer end is connected to the FPGA chip. As the main control unit, the FPGA chip is responsible for receiving the control signal of the computer end, converting it into the control instruction of the servo motor, and monitoring the feedback of the servo motor in real time and recording the fluorescence signal. Using the LabVIEW program as the user interface, write the control code to realize the real-time control and adjustment of the laser power, specifically including setting the target power, reading the feedback value and controlling the rotation angle of the servo motor. In addition, the computer end displays the fluorescence intensity signal in real time for subsequent recording and processing.

[0039] S3: Nonlinear power curve test: The fluorescent sample to be tested has a high-order nonlinear response relationship to the excitation light intensity. In a typical test process, the FPGA chip of the intelligent control module transmits instructions to the micro-servo motor, and intelligently controls the rotation of the attenuation plate to achieve fine adjustment of the excitation light power. As the excitation light power changes, the fluorescence emission intensity of the fluorescent sample changes accordingly; the FPGA chip records the fluorescence emission intensity corresponding to the excitation intensity in real time, and outputs the data to the computer in real time. The data is processed by the LabVIEW program on the computer, and the sample power curve is fitted to realize the fully automatic nonlinear power curve test function of the fluorescent sample;

[0040] It should be noted that in step S3, the intelligent control module is composed of an intelligent control FPGA chip and a computer end. The FPGA has a built-in pulse width modulation module for generating pulse signals to control other modules and adjusting the pulse frequency, width and duty cycle. In addition, the time gating module accurately controls the time window of the excitation and acquisition signals for data analysis. The computer end communicates with the FPGA through a USB or Ethernet interface through software such as LabVIEW to complete parameter setting, real-time data transmission, visual display and data analysis, and coordinates the various modules of the system.

[0041] It should be noted that, in step S3, the LabVIEW program can be written in Python, Matlab, LabWindows / CVI and other development environments to implement UI interface design and coordinate various modules of the control system.

[0042] S4: Fluorescence rise time and lifetime test: The intelligent control module transmits pulse generation instructions to the excitation light generation module to realize the continuous-pulse mode conversion, and adjusts the excitation light input mode and the output mode of the acquisition module in real time to ensure that the fluorescence signal is collected immediately after the excitation light pulse starts and ends. The photodetector continuously collects the fluorescence intensity at different times, stores it in the FPGA counter, and draws and fits the fluorescence rise curve and decay curve through the computer-side LabVIEW, realizing the fully automatic and accurate test of the in-situ fluorescence intensity, in-situ fluorescence rise curve and in-situ fluorescence lifetime curve of the fluorescent sample to be tested;

[0043] It should be noted that in step S4, the intelligent control module realizes the continuous-to-pulse mode conversion by transmitting pulses to the laser module. Specifically, in the pulse mode, the FPGA chip of the intelligent control module accurately controls the power, pulse frequency, width and duty cycle of the excitation light, as well as the collection time window of the fluorescence signal through the built-in pulse width modulation module and time gating module.

[0044] It should be noted that in step S4, the LabVIEW plots a decay curve of fluorescence intensity over time based on the received data, and accurately calculates the rise time and lifetime parameters of the sample by setting an exponential fitting method to measure the fluorescence lifetime or a custom algorithm.

[0045] S5: Fluorescence imaging test: LabVIEW on the computer side drives the three-dimensional translation stage in real time to scan the sample area point by point in the XYZ direction. During each scan, the Gaussian solid light spot is focused on different positions on the imaging plane to excite the fluorescent sample at that position. The photodetector records the fluorescence intensity of each scanning point and stores it in the FPGA counter. By changing the output mode of the intelligent control module and the acquisition module, changing the point scanning size, point scanning time, and point scanning depth, the point-by-point fluorescence intensity of different resolutions and depths can be recorded, and the data can be processed and visualized in real time through LabVIEW on the computer side to achieve fluorescence imaging test.

[0046] It should be noted that in step S5, the three-dimensional translation stage is used for spatial positioning of the sample. With the help of high-precision pulses generated inside the FPGA, the XYZ axis stepping is controlled by setting the stepping interval and frequency, thereby realizing the stepping signal control of the three-dimensional translation stage and the timing management of fluorescence signal acquisition, and scanning the entire sample area point by point.

[0047] It should be noted that in step S5, the LabVIEW program serves as the operating interface of the system, and is used to control the scanning path of the three-dimensional translation stage, the setting of acquisition parameters, the monitoring of the FPGA operating status, and the real-time visualization of data, and to perform spatial mapping of the fluorescence signal intensity value to generate imaging data, support two-dimensional or three-dimensional imaging display, and realize the fully automatic fluorescence imaging test function of fluorescent samples.

[0048] The method of the present invention can realize the excitation power response curve test, fluorescence rise time test, fluorescence decay lifetime test and fluorescence imaging test of the fluorescent sample, and no additional components need to be added during the switching of the test functions, thereby realizing the intelligent and multifunctional integration of fluorescence dynamics testing.

[0049] Compared with the prior art, the technical effects and advantages of the present invention are:

[0050] 1. Improve test accuracy and reduce human errors: Traditional nonlinear fluorescence kinetic test methods usually rely on manual operation and are easily subject to human interference, resulting in inaccurate measurement results. The present invention introduces an intelligent control module to achieve fully automated operation of the system, avoid errors caused by human operation, and improve the accuracy and consistency of the test. This fully automated control method not only ensures the accuracy and repeatability of experimental data, but also avoids false information that may occur during manual intervention, ensuring that the kinetic response of nonlinear fluorescent nanosamples is truly reflected;

[0051] 2. Accurately control power to achieve reliable measurement of high-order nonlinear responses: When the excitation light power of high-order nonlinear fluorescent nanosamples approaches the threshold, the fluorescence intensity will change dramatically, showing an ultra-high-order nonlinear dependence, and the traditional manual power adjustment method cannot accurately capture this complex dynamic behavior. The present invention can finely control the excitation light power through an automatic power adjustment module to achieve effective measurement of ultra-high nonlinear responses, providing high-precision experimental data support for revealing nonlinear physical phenomena. This precise power control makes the measurement of dynamic responses in the power-sensitive area of ​​the probe more reliable, expanding the breadth of application of the test device;

[0052] 3. Realize multi-functional integration to meet various testing needs: Nonlinear fluorescence dynamics testing usually involves the measurement of multiple parameters such as fluorescence response curve, fluorescence rise time, fluorescence decay time and fluorescence imaging, while traditional systems often need to replace different modules to achieve various functions, which is time-consuming and labor-intensive. The present invention designs an integrated and multi-functional testing system, which coordinates multiple modules such as excitation light generation, power regulation, sample scanning and data acquisition through an intelligent control module to meet various needs for nonlinear fluorescence dynamics. It can realize full-process automated testing without any module replacement, greatly improving the convenience of operation and testing efficiency;

[0053] 4. Improve test efficiency and broaden application scenarios: The fully automatic intelligent system design of the present invention simplifies the operation process, significantly improves the test efficiency, and greatly improves the speed and accuracy of multi-parameter fluorescence kinetics measurement. Compared with traditional time-consuming and labor-intensive manual testing, the present invention can complete complex nonlinear kinetic analysis in a short time, and at the same time has universality and is applicable to various types of nonlinear fluorescent nanosamples. This not only meets the high efficiency requirements in scientific research, but is also suitable for batch industrial testing applications and has broad application prospects;

[0054] 5. Reduce system costs and enhance economic applicability: Traditional nonlinear fluorescence dynamics test devices are usually complex in structure, expensive, and difficult to popularize. The present invention efficiently integrates various functional modules into one system through modular design and integrated configuration, simplifies the hardware structure, and reduces manufacturing costs. At the same time, the system can achieve multifunctional testing without replacing any components, further reducing experimental costs. This low-cost and simple test system has important practical significance for researchers and industrial users in the field of nonlinear optics and nanophotonics. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 Schematic diagram of the test optical path device in embodiments 1, 2, 3 and 4 of the present invention.

[0056] Figure 2 1 is an operation interface diagram of the test software in Examples 1, 2, 3 and 4 of the present invention.

[0057] Figure 3 The following is a design flow and logic diagram of the test software in Embodiments 1, 2, 3 and 4 of the present invention.

[0058] Figure 4 Schematic diagram of the energy transfer and photon avalanche upconversion process of the luminescence energy level in the typical upconversion fluorescent nanoparticle NaYF4:Tm system in Examples 2, 3 and 4 of the present invention.

[0059] Figure 5 The photon avalanche kinetic power curve test and nonlinear fitting results of four upconversion fluorescent nanoparticles in Example 2 of the present invention are shown in Figure a. The test system is NaYF4:Tm (0.5%), the test system is NaYF4:Tm (4%), the test system is NaYF4:Tm (8%), and the test system is NaYF4:Tm (8%)@NaYF4 in Figure d.

[0060] Figure 6 The fluorescence rise time and fluorescence lifetime test and fitting results of NaYF4:Tm(8%) in Example 3 of the present invention are shown in Figure a. The fluorescence rise curve and fitting results are shown in Figure b. The fluorescence lifetime curve and fitting results are shown in Figure 4.

[0061] Figure 7 The super-resolution fluorescence imaging and optical resolution analysis of NaYF4:Tm(8%)@NaYF4 in Example 4 of the present invention are shown in Figure a. Figure a is a super-resolution fluorescence imaging, and Figure b is a Gaussian fitting analysis of the optical resolution of the two nanoparticles in Figure a. DETAILED DESCRIPTION

[0062] The present invention will be further described below. It should be noted that the following examples are based on the technical solution and provide detailed implementation methods and specific operating processes, but the protection scope of the present invention is not limited to the following examples.

[0063] Example 1

[0064] like Figure 1 As shown, a fully automatic, intelligent, and multifunctional high-order nonlinear fluorescence dynamics testing device described in an embodiment of the present invention includes an excitation light generating module, an automatic power adjustment module, an excitation light modulation module, a sample scanning module, a data acquisition and analysis module, and an intelligent control module. Under the excitation of the incident laser, based on the overall coordination of the intelligent control module, the remaining modules are driven to complete the dynamic response test of the nonlinear fluorescence nanosample, thereby realizing fully automatic, intelligent, multifunctional, high-precision, simple, efficient, low-cost, and universal fluorescence dynamics testing and analysis.

[0065] The excitation light generation module includes a continuous near-infrared laser 1 and a first filter 2 placed in sequence along the direction of the laser beam emitted by the laser. The automatic power adjustment module includes a gradient attenuation plate 3 and a micro servo motor 4 connected thereto. The excitation light modulation module includes a focusing objective lens 5, a polarization-maintaining single-mode optical fiber 6, a half-wave plate 7 and a quarter-wave plate 8. The infinity correction tube lens system 9 is used to adjust the light beam size to match the entrance pupil of the imaging objective lens 11 to improve the light beam utilization rate, and to correct optical aberrations under high magnification and high numerical aperture conditions to ensure imaging clarity. The dichroic mirror 10 can reflect the excitation light and transmit the sample fluorescence, and is used to separate the excitation light from the fluorescence. The three-dimensional translation stage 12 is used for spatial positioning of the sample, moves in the XYZ direction according to the set step size, and scans the entire sample area point by point.

[0066] The data acquisition and analysis module is composed of a second filter 14, a focusing lens 15 and a single photon counter 17 placed coaxially in sequence. The sample emits fluorescence in all directions under the excitation of the incident laser, and the imaging objective 11 collects part of the fluorescence signal. The signal passes through the dichroic mirror 10, the reflector 13, the second filter 14, the focusing lens 15, and is connected to the multi-mode polarization-maintaining fiber 16, and is finally received by the single photon counter 17. The intelligent control module is composed of an intelligent control FPGA chip 18 and a computer terminal 19. The computer terminal completes parameter setting, real-time data transmission, visual display and data analysis through software such as LabVIEW, and coordinates various modules of the system.

[0067] The operation interface of the above device written based on FPGA-LabVIEW is shown in Figure 2 The operation interface includes four typical parts. Area 1 is used to visualize the laser power and the number of fluorescence emission photons, and to perform axial focus positioning based on the number of emission photons; Area 2 is used to visualize the spectral information of the sample and to select a specific fluorescence emission band; Area 3 is used to adjust the laser power and start the automatic test cycle; Area 4 is used to visualize and fit the power curve test results.

[0068] The logic block diagram of the above device written based on FPGA-LabVIEW can be found in Figure 3 A typical workflow is: start the Labview program, move the axial position of the piezoelectric stage through the operation interface, and locate the optimal axial position according to the recorded number of photons; adjust the PWM pulse to control the servo motor to change the rotation angle to adjust the incident laser power, and record the number of emitted photons of the sample under different incident powers for plotting and fitting to achieve fully automatic testing of the power curve; set the delay time and width of the Gate window to turn on / off the laser to achieve sample fluorescence rise time and decay time testing; adjust the PWM pulse to achieve the timing management of the stage step signal control and fluorescence signal acquisition, and the photoelectric detection module digitizes the fluorescence intensity of each scanning point and stores it in the buffer area in real time to achieve two-dimensional or three-dimensional imaging display.

[0069] The fully automatic, intelligent, and multifunctional high-order nonlinear fluorescence dynamics testing method described in the embodiment of the present invention comprises the following steps:

[0070] S1: Laser excitation and power modulation: The laser 1 outputs a stable laser beam, which is filtered out by the filter group 2 to remove laser beams of other wavelengths. The purified excitation light is quantitatively modulated by the automatic power adjustment module, and modulated by the focusing objective lens 5, polarization-maintaining fiber 6, half-wave plate 7 and quarter-wave plate 8. The aberration is corrected by the tube lens group 9 and the entrance pupil of the imaging objective lens 11 is matched to obtain a Gaussian solid light spot.

[0071] It should be noted that in step S1, the excitation wavelength output by the laser is 1064 nm, which is used to match the excited state absorption of the lanthanide-doped fluorescent nanoparticles, and the continuous / pulse mode of the laser is adjustable.

[0072] It should be noted that in step S1, the automatic power adjustment module is mainly composed of a power attenuation sheet 3 and a micro servo motor 4. The attenuation sheet 3 has different light transmittances in its radial direction or angle, and is aligned with the output end of the laser 1. Rotating at different angles corresponds to different transmitted light powers. The micro servo motor 4 is used to drive the rotation of the gradual attenuation sheet 3 to achieve precise control of the angle of the attenuation sheet 3, thereby adjusting the power of the transmitted light.

[0073] It should be noted that in step S1, the micro servo motor 3 can control the rotation angle by pulses, and has an angle feedback system to ensure high-precision positioning. The micro servo motor 4 is controlled by the FPGA chip 18 of the intelligent control module, and its rotation angle is controlled by a pulse width modulation signal. The frequency and duty cycle of the pulse signal determine the speed and direction of the micro servo motor 4. In addition, the FPGA chip 18 can realize a pulse counting function for accurately recording the rotation angle of the micro servo motor 4.

[0074] It should be noted that in step S1, the focusing lens 5 and the polarization-maintaining optical fiber 6 are used for beam shaping to improve the efficiency of the excitation light beam, and the half-wave plate 7 and the quarter-wave plate 8 are used to change the polarization state of the excitation light to adapt to different excitation modes.

[0075] It should be noted that in step S1, the power attenuation sheet 3 can be replaced by other power regulating devices such as an electric adjustable optical attenuator, an electro-optic modulator, an acousto-optic modulator, etc., and its main function is to change the power of the laser output end; the micro servo motor 4 can be replaced by a mechanical driving device such as a stepper motor and a piezoelectric driver, and its main function is to accurately control the laser attenuation device; the FPGA chip 18 can be replaced by a microcontroller, a single-chip control system, a digital-to-analog converter, a digital signal processor, etc., and its main function is to realize signal conversion and control.

[0076] S2: Fluorescence signal collection and processing: The focused Gaussian solid light spot excites the fluorescent sample at the exit end of the imaging objective 11, and the sample fluorescence enters the collection and analysis module: the fluorescence signal of the sample is collected by the same imaging objective, passes through the dichroic mirror 10, the filter set 13 and the focusing lens 15, enters the multi-mode polarization-maintaining fiber 16, and is received by the single-photon counter 17 (Excelitas, SPCM20328-107), stored by the FPGA chip 18, and processed by the LabVIEW program compiled by the computer end 19;

[0077] It should be noted that in step S2, the fluorescent sample is a photon avalanche effect rare earth doped up-conversion nanoparticle, and its nonlinearity is generally greater than 10.

[0078] It should be noted that in step S2, the filter group is assembled by a rotatable bracket, and has multiple optical filters in the 400 nm to 1000 nm band built in.

[0079] It should be noted that, in step S2, the FPGA chip 18 realizes multiple functions such as pulse signal generation, time gating, fluorescence signal acquisition and processing.

[0080] It should be noted that, in step S2, the LabVIEW program compiled by the computer end 19 realizes parameter setting, real-time data transmission and visual display.

[0081] It should be noted that in step S2, the single photon counter 17 is connected to the FPGA chip 18. The single photon counter 17 collects light signals in the Gate window of the FPGA chip 18 to achieve light-to-electrical signal conversion. The ADC channel of the FPGA chip 18 receives the analog electrical signal of the single photon counter 17, digitizes it into counting pulses, and accumulates it in the counter. After each independent counting is completed, the counter transmits the accumulated result to the FPGA chip 18 and feeds it back to the LabVIEW program compiled by the computer end 19 in real time to achieve the fluorescence intensity collection of this time.

[0082] It should be noted that in step S2, the computer terminal 19 is connected to the FPGA chip 18. As the main control unit, the FPGA chip 18 is responsible for receiving the control signal of the LabVIEW program in the computer terminal 19, converting it into the control instruction of the micro servo motor 4, and monitoring the feedback of the micro servo motor 4 in real time and recording the fluorescence signal. The LabVIEW program compiled by the computer terminal 19 is used as a user interface to write control codes to achieve real-time control and adjustment of the laser power, including setting the target power, reading the feedback value, and controlling the rotation angle of the micro servo motor 4 and displaying the fluorescence intensity signal in real time for subsequent recording and processing.

[0083] S3: Nonlinear power curve test: The fluorescent sample to be tested has a high-order nonlinear response relationship to the excitation light intensity. The FPGA chip 18 transmits instructions to the micro-servo motor 4, and intelligently controls the rotation of the attenuation plate 3 to achieve fine adjustment of the excitation light power. As the excitation light power changes, the fluorescence emission intensity of the fluorescent sample changes accordingly; the FPGA chip 18 records the fluorescence emission intensity corresponding to the excitation intensity in real time, and outputs the data to the computer terminal 19 in real time, and processes the data through the LabVIEW program to fit the sample power curve, thereby realizing the fully automatic nonlinear power curve test function of the fluorescent sample;

[0084] It should be noted that in step S3, the FPGA chip 18 has a built-in pulse width modulation module for generating pulse signals for controlling other modules and adjusting the pulse frequency, width and duty cycle. In addition, the time gating module accurately controls the time window of the excitation and acquisition signals for data analysis. The computer terminal 19 communicates with the FPGA chip 18 via USB through the LabVIEW program to complete parameter setting, real-time data transmission, visual display and data analysis, and coordinates the various modules of the system.

[0085] S4: Fluorescence rise time and lifetime test: FPGA chip 18 transmits pulse generation instructions to laser 1 to realize continuous-pulse mode conversion, and adjusts the excitation light input mode and the output mode of the acquisition module in real time to ensure that the fluorescence signal is collected immediately after the excitation light pulse starts and ends. The single-photon counter 17 continuously collects the fluorescence intensity at different times, stores it in the counter of FPGA chip 18, and draws and fits the fluorescence rise curve and decay curve through the LabVIEW program compiled by the computer end 19, so as to realize the fully automatic and accurate test of the in-situ fluorescence intensity, in-situ fluorescence rise curve and in-situ fluorescence lifetime curve of the fluorescent sample to be tested;

[0086] It should be noted that in step S4, the FPGA chip 18 accurately controls the power, pulse frequency, width and duty cycle of the excitation light, as well as the collection time window of the fluorescence signal through the built-in pulse width modulation module and time gating module.

[0087] It should be noted that in step S4, the LabVIEW program compiled by the computer terminal 19 plots the decay curve of fluorescence intensity over time based on the received data. The rise time and lifetime parameters of the sample are accurately calculated by setting the exponential fitting method to measure the fluorescence lifetime or a custom algorithm.

[0088] S5: Fluorescence imaging test: The LabVIEW program compiled by the computer end 19 drives the three-dimensional translation stage 12 in real time to scan the sample area point by point in the XYZ direction. During each scanning process, the Gaussian solid light spot is focused on different positions on the imaging plane to excite the fluorescent sample at that position. The single photon counter 17 records the fluorescence intensity of each scanning point and stores it in the FPGA chip 18 counter; by changing the output mode of the intelligent control module and the acquisition module, changing the point scanning size, point scanning time, and point scanning depth, the point-by-point fluorescence intensity of different resolutions and depths can be recorded, and the real-time output is performed through the LabVIEW program compiled by the computer end 19 for data processing and visualization, thereby realizing fluorescence imaging test.

[0089] It should be noted that in step S5, the three-dimensional translation stage 12 is used for spatial positioning of the sample. With the help of high-precision pulses generated inside the FPGA chip 18, the XYZ axis stepping is controlled by setting the stepping interval and frequency, thereby realizing the stepping signal control of the three-dimensional translation stage 12 and the timing management of fluorescence signal acquisition, and scanning the entire sample area point by point.

[0090] It should be noted that in step S5, the LabVIEW program compiled by the computer end 19 is used as the operating interface of the system to control the scanning path of the three-dimensional translation stage 12, the setting of acquisition parameters, the monitoring of the operating status of the FPGA chip 18 and the real-time visualization of data, and spatial mapping of the fluorescence signal intensity value to generate imaging data, support two-dimensional or three-dimensional imaging display, and realize the fully automatic fluorescence imaging test function of fluorescent samples.

[0091] Example 2

[0092] Photon avalanche upconversion is a highly nonlinear optical process that usually occurs in certain rare earth doped materials, such as Tm 3+ In this phenomenon, relatively low-intensity incident light can trigger rapid excitation of electrons in the material, forming a positive feedback loop, which triggers a series of multi-step excitation processes and ultimately produces fluorescence emission far higher than the initial excitation. This process has a highly nonlinear characteristic, because even under low-power excitation, the avalanche feedback will greatly amplify the fluorescence output, making the intensity of the emitted light show an exponentially nonlinear relationship with the excitation light power.

[0093] The photon avalanche upconversion effect is highly dependent on the doping ion concentration in the material. At lower doping concentrations, the distance between ions is larger and the cross-relaxation energy transfer efficiency is relatively low, which limits the effectiveness of the positive feedback loop in the photon avalanche process and leads to a less obvious degree of nonlinearity. At higher doping concentrations, the distance between ions is shortened, the cross-relaxation energy transfer efficiency is improved, and multi-step excitation and positive feedback loops between ions are easier to achieve, thereby enhancing the nonlinear response. Therefore, different doping concentrations will directly affect the optical nonlinearity and threshold size of photon avalanche upconversion, which is measured by the power curve of the fluorescence excitation-emission intensity of the test sample.

[0094] Synthesize NaYF4:Tm systems with different concentrations and structures, see Figure 4 , showing their common energy transfer pathway. At high doping concentrations, the photon avalanche process is as follows: in the ground state 3 Tm of H6 level 3+ The ions undergo a non-resonant 1064 nm ground state absorption and a small number of them transition to 3 The F4 level is then re-excited to the3 H4 energy level. 3 Tm at H6 level 3+ Ions and a 3 Tm at H4 level 3+ The ions undergo cross relaxation, producing two 3 Tm of F4 level 3+ ions, again through the resonant 1064 nm excited state absorption to produce two 3 Tm at H4 level 3+ ions. As this positive feedback cycle of ground state absorption-cross relaxation-excited state absorption continues to accumulate, it eventually leads to 3 Tm of F4 level 3+ The number of ions increases dramatically, and finally the excited state transitions to the luminescent state via the resonant 1064 nm absorption. 3 H4 energy level, produces 800 nm photon avalanche fluorescence.

[0095] Rare earth doped upconversion nanoparticles with the compositions of NaYF4:Tm(0.5%), NaYF4:Tm(4%), NaYF4:Tm(8%) and NaYF4:Tm(8%)@NaYF4 were prepared for testing. Under 1064 nm laser excitation, the designed fully automatic intelligent device was used to perform fully automatic testing and fitting of the power curve. Figure 5 The test and fitting results of the power curves of photon avalanche nanoparticles with different doping concentrations are shown. The test system in Figure a is NaYF4:Tm (0.5%), the test system in Figure b is NaYF4:Tm (4%), the test system in Figure c is NaYF4:Tm (8%), and the test system in Figure d is NaYF4:Tm (8%)@NaYF4. Their optical nonlinear fitting results are 2, 20, 25, and 21, respectively. The test results show that low Tm 3+ The ion-doped (0.5%) system shows almost no photon avalanche upconversion process, while the high Tm 3+ The ion-doped (4%, 8%) systems all produce typical photon avalanche phenomena. In addition, compared with the monomer core-structured NaYF4:Tm(8%) system, the core-shell structured NaYF4:Tm(8%)@NaYF4 system exhibits lower photon avalanche threshold power and lower optical nonlinearity.

[0096] Example 3

[0097] Rare earth doped upconversion nanoparticles with the composition of NaYF4:Tm (8%) were tested and prepared. Under 1064 nm laser excitation, the fluorescence rise curve and fluorescence decay curve were fully automatically tested and fitted using the designed fully automatic intelligent device. Figure 6The fluorescence rise curve and lifetime decay curve of NaYF4:Tm(8%) nanoparticles are shown. Figure 6 A in the figure shows a slow fluorescence rise curve, and the fitted fluorescence rise time is 5 ms; Figure 6 Figure b shows a fast fluorescence decay curve with a fluorescence lifetime of 28 μs after fitting, demonstrating the fluorescence dynamics of NaYF4:Tm(8%) rare earth doped upconversion nanoparticles.

[0098] Example 4

[0099] Rare earth-doped upconversion nanoparticles with the composition of NaYF4:Tm(8%)@NaYF4 were prepared for monodisperse test. Under 1064 nm laser excitation, fluorescence super-resolution imaging was performed using the designed fully automatic intelligent device, and the half-width at half maximum of the imaging resolution was fitted. Figure 7 The fluorescence super-resolution imaging and optical resolution analysis results of NaYF4:Tm(8%)@NaYF4 nanoparticles are shown. Figure 7 a is the result of optical scanning super-resolution imaging of nanoparticles by the invented intelligent testing method, where serial numbers 1 and 2 are two single nanoparticles in the imaging field of view; Figure 7 Figures b and c are the optical resolution analysis results of nanoparticles 1 and 2, respectively. Using the full width at half maximum analysis method, Gaussian fitting was performed on the line profiles of nanoparticles 1 and 2 and their full width at half maximum was solved as the resolution index, showing imaging resolutions of 77 nm and 66 nm, respectively.

[0100] The multifunctional integrated design and automated operation of the present invention not only significantly improve the test efficiency and accuracy, but also greatly reduce the overall cost of the system, making the study of high-order nonlinear fluorescence dynamics more efficient, simple and economical, laying a solid foundation for the further research and application of nonlinear optics and nanophotonics, and will be of great significance for enhancing my country's international influence in the field of nonlinear optics and nanophotonics.

[0101] For those skilled in the art, various corresponding changes and modifications can be made according to the above technical solutions and concepts, and all of these changes and modifications should be included in the protection scope of the claims of the present invention.

Claims

1. A fully automatic, intelligent, multifunctional high-order nonlinear fluorescence dynamics test device, characterized in that include: Excitation light generation module, automatic power adjustment module, excitation light modulation module, sample scanning module, data acquisition and analysis module, intelligent control module; Under the excitation of incident laser, based on the overall coordination of the intelligent control module, the remaining modules are driven to complete the dynamic response test of nonlinear fluorescence samples and realize fluorescence dynamic test analysis; The excitation light generation module includes continuous ultraviolet light / visible light / near infrared light or pulsed ultraviolet light / visible light / near infrared light, and a filter group placed in sequence along the direction of the laser beam emitted by the laser; the laser generates a steady-state laser beam output, the photon energy of the beam matches the excited state absorption of the fluorescent sample; the filter group is used to purify the laser; The automatic power adjustment module includes a gradient attenuation sheet and a micro servo motor connected thereto; the gradient attenuation sheet has different light transmittances in its radial direction or angle, is aligned with the laser emission end, and rotates at different angles to correspond to different transmitted light powers; the micro servo motor is used to drive the gradient attenuation sheet to rotate, thereby achieving precise control of the angle of the attenuation sheet, thereby adjusting the power of the transmitted light; The excitation light modulation module includes a focusing objective lens, a polarization-maintaining optical fiber, a half-wave plate and a quarter-wave plate; The focusing objective lens and polarization-maintaining optical fiber are used for beam shaping to improve the efficiency of the excitation light beam, and the half-wave plate and quarter-wave plate are used to change the polarization state of the excitation light to adapt to different excitation modes; The sample scanning module includes an infinity correction tube lens system, a dichroic mirror, an imaging objective lens and a three-dimensional translation stage; the infinity correction tube lens system is used to adjust the light beam size to match the entrance pupil of the imaging objective lens, improve the light beam utilization rate, and correct the optical aberration under high magnification and high numerical aperture conditions to ensure the clarity of imaging; the dichroic mirror can reflect the excitation light and transmit the sample fluorescence, and is used to separate the excitation light and the fluorescence; the imaging objective lens is used for light beam focusing and sample imaging; the three-dimensional translation stage is used for spatial positioning of the fluorescent sample, moves in the XYZ direction according to the set resolution, and scans the entire sample area point by point; The data acquisition and analysis module is composed of a filter group, a focusing lens and a photodetector which are coaxially placed in sequence; the data acquisition and analysis module is used to collect fluorescence signals, which are input to the computer end for data processing via the intelligent control module; the sample emits fluorescence in all directions under the excitation of the incident laser, and the imaging objective lens collects a part of the fluorescence signal, which passes through the dichroic mirror, the filter group and the focusing lens, is received by the photodetector, and is input to the computer end via the intelligent control module; The intelligent control module includes an intelligent control FPGA chip and a computer end; the FPGA chip is connected to the computer end, and the FPGA chip serves as a main control unit, which is used to receive control signals from the computer end, convert them into control instructions for the servo motor, and monitor the feedback of the servo motor in real time and record the fluorescence signal; the computer end communicates with the FPGA through the USB or Ethernet interface through LabVIEW software to complete parameter setting, real-time data transmission, visual display and data analysis, and coordinates various modules of the system.

2. A fully automatic, intelligent, multifunctional high-order nonlinear fluorescence dynamics testing device as claimed in claim 1, characterized in that The filter set of the excitation light generation module is assembled by a rotatable bracket, and is used to adjust and match different excitation light wavelengths; the filter set of the data acquisition and analysis module can be assembled by a rotatable bracket, and is used to adjust and match different acquisition fluorescence wavelengths; The FPGA chip can have a built-in pulse width modulation module to generate control pulse signals and adjust the pulse frequency, width and duty cycle; the FPGA chip can have a built-in time gating module to accurately control the time window of excitation and acquisition signals for data analysis.

3. A fully automatic, intelligent, multifunctional high-order nonlinear fluorescence dynamics testing device as claimed in claim 1, characterized in that The micro servo motor can control the rotation angle through pulses, and has an angle feedback system to ensure high-precision positioning; the micro servo motor is controlled by the FPGA chip of the intelligent control module, and its rotation angle is controlled by a pulse width modulation (PWM) signal. The frequency and duty cycle of the pulse signal determine the speed and direction of the servo motor; in addition, the FPGA controls the pulse output duration and frequency through the Gate to achieve precise stop of the servo motor at a specific angle; The FPGA chip is connected to the photodetector of the data acquisition and analysis module. The photodetector collects light signals in the Gate window of the FPGA, converts them into counting pulses, and accumulates them in the counter. After each independent counting is completed, the counter transmits the accumulated results to the FPGA and feeds them back to LabVIEW in real time to realize the fluorescence intensity collection of this time.

4. A fully automatic, intelligent, multifunctional high-order nonlinear fluorescence dynamics testing device as claimed in claim 1, characterized in that The gradient attenuation sheet is replaced by an electric adjustable optical attenuator, an electro-optic modulator, and an acousto-optic modulator to change the power of the laser output end; the micro servo motor can be replaced by a stepper motor and a piezoelectric driver to accurately control the laser attenuation device; the FPGA chip is replaced by a microcontroller, a single-chip control system, a digital analog converter, and a digital signal processor to achieve signal conversion and control; the photodetector is replaced by a photomultiplier tube and a spectrometer to record photon information; the LabVIEW can be replaced by Python, Matlab, and LabWindows / CVI development environments to achieve UI interface design and coordinate various modules of the control system; The fluorescent samples may include quantum dots, organic dyes, conventional up-conversion fluorescent nanoparticles, and photon avalanche effect rare earth doped up-conversion nanoparticles.

5. A fully automatic, intelligent, and multifunctional high-order nonlinear fluorescence dynamics test method, characterized in that The following steps are involved: S1: Laser excitation and power modulation: The laser outputs a stable laser beam, which is filtered out by the filter group to remove lasers of other wavelengths. The purified excitation light is quantitatively modulated by the automatic power adjustment module. After being modulated by the focusing objective lens, polarization-maintaining fiber, and polarization wave plate, the aberration is corrected by the tube lens group and the entrance pupil of the imaging objective lens is matched to obtain a Gaussian solid light spot. S2: Fluorescence signal collection and processing: The focused Gaussian solid light spot excites the fluorescent sample at the exit end of the imaging objective lens, and the sample fluorescence enters the collection and analysis module: the fluorescence signal of the sample is collected by the same imaging objective lens, and then focused by the dichroic mirror, filter set and lens and enters the collection optical fiber. The signal is received by the photodetector, stored by the FPGA, and processed in collaboration with LabVIEW on the computer side; S3: Nonlinear power curve test: The fluorescent sample to be tested has a high-order nonlinear response relationship to the excitation light intensity. In a typical test process, the FPGA chip of the intelligent control module transmits instructions to the micro-servo motor, intelligently controls the rotation of the attenuation plate to achieve fine adjustment of the excitation light power. As the excitation light power changes, the fluorescence emission intensity of the fluorescent sample changes accordingly. The FPGA chip records the fluorescence emission intensity corresponding to the excitation intensity in real time, and outputs the data to the computer in real time. The LabVIEW program on the computer performs data processing, fits the sample power curve, and realizes the fully automatic nonlinear power curve test function of the fluorescent sample. S4: Fluorescence rise time and life test: The intelligent control module transmits pulse generation instructions to the excitation light generation module to realize the continuous-pulse mode conversion, and adjusts the excitation light input mode and the output mode of the acquisition module in real time to ensure that the fluorescence signal is collected immediately after the excitation light pulse starts and ends; The photoelectric detector continuously collects the fluorescence intensity at different times and stores it in the counter of FPGA. The fluorescence rise curve and decay curve are drawn and fitted by LabVIEW on the computer, so as to realize the fully automatic and accurate test of the in-situ fluorescence intensity, in-situ fluorescence rise curve and in-situ fluorescence lifetime curve of the fluorescent sample to be tested. S5: Fluorescence imaging test: Calculate the different positions focused on the imaging plane, excite the fluorescent sample at that position, and the photodetector records the fluorescence intensity of each scanning point. The computer-side LabVIEW drives the three-dimensional translation stage in real time to scan the sample area point by point in the XYZ direction. During each scan, the Gaussian solid spot is stored in the FPGA counter. By changing the output mode of the intelligent control module and the acquisition module, changing the point scanning size, point scanning time, and point scanning depth, the point-by-point fluorescence intensity of different resolutions and depths can be recorded, and the data is processed and visualized in real time through the computer-side LabVIEW to achieve fluorescence imaging test.

6. A fully automatic, intelligent, multifunctional high-order nonlinear fluorescence dynamics test method as claimed in claim 5, characterized in that In step S1, the laser determines the required laser wavelength according to the excited state absorption characteristics of the fluorescent probe, including continuous ultraviolet light / visible light / near infrared light or pulsed ultraviolet light / visible light / near infrared light; In step S1, the polarization wave plate may be a half wave plate or a quarter wave plate, which is used to change the polarization state of the excitation light to adapt to different excitation modes; In step S1, the excitation power does not need to be adjusted manually, and precise intelligent control of the computer end is achieved through the automatic power adjustment module; the automatic power adjustment module is mainly composed of a power attenuation sheet and a micro servo motor; the attenuation sheet has different transmittances in its radial direction or angle, and is aligned with the laser output end. Different rotation angles correspond to different transmitted light powers; the micro servo motor is used to drive the rotation of the gradient attenuation sheet to achieve precise control of the angle of the attenuation sheet, thereby adjusting the power of the transmitted light; the micro servo motor can control the rotation angle through pulses, and has an angle feedback system to ensure high-precision positioning; the micro motor is controlled by the FPGA chip of the intelligent control module, and its rotation angle is controlled by a pulse width modulation signal. The frequency and duty cycle of the pulse signal determine the speed and direction of the servo motor; in addition, the FPGA controls the pulse output duration and frequency through the Gate to achieve precise stop of the servo motor at a specific angle; the power attenuation sheet can be replaced by an electric adjustable optical attenuator, an electro-optic modulator, or an acousto-optic modulator to change the power of the laser output end; the micro servo motor can be replaced by a stepper motor or a piezoelectric driver to accurately control the laser attenuation device.

7. A fully automatic, intelligent, and multifunctional high-order nonlinear fluorescence dynamics test method as claimed in claim 5, characterized in that In step S2, the fluorescent sample includes quantum dots, organic dyes, conventional upconversion fluorescent nanoparticles, and photon avalanche effect rare earth doped upconversion nanoparticles; the nonlinearity of quantum dots and organic dyes is generally less than 3, the nonlinearity of conventional upconversion fluorescent nanoparticles is generally less than 6, and the nonlinearity of photon avalanche effect rare earth doped upconversion nanoparticles is generally greater than 10; In step S2, the filter set is assembled by a rotatable bracket to adjust and match different collected fluorescence wavelengths; The photoelectric detector includes a photomultiplier tube, a single photon counter, and a spectrometer; the FPGA chip realizes the generation of pulse signals, time gating, fluorescence signal acquisition and processing functions; the LabVIEW realizes parameter setting, real-time data transmission and visual display; In step S2, the photodetector is connected to the FPGA chip; the photodetector collects light signals in the Gate window of the FPGA to achieve light-to-electrical signal conversion; the ADC channel of the FPGA receives the analog electrical signal of the photodetector, digitizes it into counting pulses, and accumulates it in the counter; after each independent counting is completed, the counter transmits the accumulated result to the FPGA and feeds it back to the LabVIEW program in real time to achieve the fluorescence intensity collection of this time; In step S2, the computer end is connected to the FPGA chip; the FPGA chip serves as a main control unit, which is responsible for receiving the control signal from the computer end, converting it into a control instruction of the servo motor, and monitoring the feedback of the servo motor in real time and recording the fluorescence signal; using the LabVIEW program as a user interface, writing the control code to achieve real-time control and adjustment of the laser power, specifically including setting the target power, reading the feedback value, and controlling the rotation angle of the servo motor; in addition, the computer end displays the fluorescence intensity signal in real time for subsequent recording and processing.

8. A fully automatic, intelligent, multifunctional high-order nonlinear fluorescence dynamics test method as claimed in claim 5, characterized in that In step S3, the intelligent control module is composed of an intelligent control FPGA chip and a computer end; the FPGA has a built-in pulse width modulation module for generating pulse signals for controlling other modules and adjusting the pulse frequency, width and duty cycle; in addition, the time gating module accurately controls the time window of the excitation and acquisition signals for data analysis; the computer end communicates with the FPGA through the USB or Ethernet interface through the LabVIEW software to complete parameter setting, real-time data transmission, visual display and data analysis, and coordinates the various modules of the system; In step S3, the LabVIEW program can be replaced by Python, Matlab, LabWindows / CVI development environment to implement UI interface design and coordinate various modules of the control system.

9. A fully automatic, intelligent, and multifunctional high-order nonlinear fluorescence dynamics test method as claimed in claim 5, characterized in that In step S4, the intelligent control module realizes the continuous-to-pulse mode conversion by transmitting pulses to the laser module; specifically, in the pulse mode, the FPGA chip of the intelligent control module accurately controls the power, pulse frequency, width and duty cycle of the excitation light, as well as the collection time window of the fluorescence signal through the built-in pulse width modulation module and time gating module; In step S4, the LabVIEW plots a decay curve of the fluorescence intensity over time based on the received data; and accurately calculates the rise time and lifetime parameters of the sample by setting an exponential fitting method to measure the fluorescence lifetime or a custom algorithm.

10. A fully automatic, intelligent, and multifunctional high-order nonlinear fluorescence dynamics test method as claimed in claim 5, characterized in that In step S5, the three-dimensional translation stage is used for spatial positioning of the sample. With the help of high-precision pulses generated inside the FPGA, the XYZ axis stepping is controlled by setting the stepping interval and frequency, so as to realize the timing management of the stepping signal control of the three-dimensional translation stage and the fluorescence signal collection, and scan the entire sample area point by point; In step S5, the LabVIEW program is used as the operating interface of the system to control the scanning path of the three-dimensional translation stage, the setting of acquisition parameters, the monitoring of the FPGA operating status and the real-time visualization of data, and to spatially map the fluorescence signal intensity value to generate imaging data, support two-dimensional or three-dimensional imaging display, and realize the fully automatic fluorescence imaging test function of fluorescent samples.

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