A fully automatic intelligent and multifunctional high-order nonlinear fluorescence kinetics testing method and device

Abstract

A kind of full-automatic intelligent, multi-functional high-order nonlinear fluorescence kinetics test method and device, it relates to nonlinear optics and nanophotonics field.The optical testing device consisting of excitation light generation module, automatic power regulation module, excitation light modulation module, sample scanning module, data acquisition and analysis module and intelligent control module is built, under the excitation of incident laser, based on the overall coordination of intelligent control module, drive the rest each module to complete the dynamic response test of nonlinear fluorescence sample, realize full-automatic intelligent, multi-functional, high-precision, simple, efficient, low cost, universal fluorescence kinetics test analysis.The problem of traditional manual time-consuming and low test accuracy is solved.In addition, the multi-functional integration of fluorescence response curve, fluorescence rise time, fluorescence lifetime and fluorescence imaging effectively improves the test efficiency and application scenario, greatly reduces the system cost.

Description

Technical Field

[0001] This invention relates to the fields of nonlinear optics and nanophotonics, specifically to a fully automated, intelligent, and multifunctional high-order nonlinear fluorescence dynamics testing method and apparatus. Background Technology

[0002] Nonlinear fluorescence dynamics testing is a crucial tool for studying nonlinear optical effects, nanophotonics, and their applications in bioimaging and optical sensing. Traditional nonlinear fluorescence nanosample testing methods typically rely on manual operation, which is not only time-consuming and labor-intensive but also highly susceptible to human error, leading to inaccurate data. Furthermore, due to the complexity of manual testing, especially for probes with ultra-high nonlinear response characteristics, their fluorescence intensity exhibits significant nonlinear changes when the laser power approaches a specific threshold. This ultra-high-order nonlinear dependence is difficult to accurately capture in conventional manual measurements, further impacting the scientific explanation and analysis of nonlinear optical phenomena and hindering the in-depth development of nonlinear optics and nanophotonics.

[0003] Currently, various testing requirements for fluorescent nanoparticles, such as fluorescence response curves, rise time, decay time, and imaging, typically necessitate the replacement of different testing modules or devices. This not only increases the complexity and time cost of the testing process but also poses challenges to the stability and consistency of the experiments. In particular, in the study of high-order nonlinear fluorescence dynamics, precise control and automatic adjustment of different powers are crucial. However, existing systems struggle to achieve multifunctional integration and fully automated intelligent testing, thus limiting their versatility and applicability in practical applications. Summary of the Invention

[0004] The purpose of this invention is to address the shortcomings of existing technologies by providing a fully automated, intelligent, and multifunctional high-order nonlinear fluorescence dynamics testing method and apparatus. It overcomes the deficiencies of existing technologies, achieving fully automated and high-precision measurement of fluorescence response curves, fluorescence rise time, fluorescence lifetime, and fluorescence imaging, while eliminating errors and false information caused by human intervention.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] A fully automated, 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, the intelligent control module coordinates and drives the other modules to complete the dynamic response test of the nonlinear fluorescent nanosample, thereby realizing fluorescence dynamics testing and analysis.

[0007] The excitation light generation module includes the laser and a group of filters arranged sequentially 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 is matched to the excited-state absorption of the fluorescent nanoparticles, and the filters are used to purify the laser beam.

[0008] The automatic power adjustment module includes a gradient attenuator and a connected miniature servo motor. The gradient attenuator has different transmittance in its radial direction or angle. When aligned with the laser emission end, different rotation angles correspond to different transmitted light power. The miniature servo motor is used to drive the gradient attenuator to rotate, achieving precise control of the attenuator angle, thereby adjusting the power of the transmitted light.

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

[0010] The sample scanning module includes an infinity-corrected lens system, a dichroic mirror, an imaging objective, and a three-dimensional stage. The infinity-corrected lens system adjusts the beam size to match the entrance pupil of the imaging objective, improving beam utilization and correcting optical aberrations under high magnification and high numerical aperture conditions to ensure image clarity. The dichroic mirror reflects excitation light and transmits sample fluorescence, separating the excitation light from the fluorescence. The imaging objective is used for beam focusing and sample imaging. The three-dimensional stage is used for spatial positioning of the sample, moving in the XYZ directions according to a set resolution to scan the entire sample area point by point.

[0011] The data acquisition and analysis module consists of a filter group, a focusing lens, and a photodetector placed coaxially in sequence. The module collects fluorescence signals, which are then input to a computer for data processing via an intelligent control module. When a sample is excited by an incident laser, it emits fluorescence in all directions. The imaging objective collects a portion of the fluorescence signal, which passes through a dichroic mirror, a filter group, and a focusing lens, is received by the photodetector, and then input to the computer via 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 and acts as the main control unit. It receives control signals from the computer, converts them into control commands for the servo motor, and monitors the feedback from the servo motor in real time, recording fluorescence signals. The computer communicates with the FPGA via USB or Ethernet interface through software such as LabVIEW to complete parameter setting, real-time data transmission, visualization, and data analysis, thus coordinating all modules of the system.

[0013] The excitation light generation module includes continuous ultraviolet / visible / near-infrared light or pulsed ultraviolet / visible / near-infrared light, and a filter group arranged sequentially 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 filters are used to purify the laser beam.

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

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

[0016] Furthermore, the micro servo motor can be controlled by pulses to adjust its rotation angle and has an angle feedback system to ensure high-precision positioning. The micro servo motor is controlled by an FPGA chip in the intelligent control module, using pulse width modulation (PWM) signals to control its rotation angle. 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 via a gate, enabling the servo motor to precisely stop at a specific angle.

[0017] Furthermore, the FPGA chip is connected to the photodetector of the data acquisition and analysis module. The photodetector acquires light signals within the FPGA's Gate window, converts them into counting pulses, and accumulates them in a counter. After each independent count is completed, the counter transmits the accumulated result to the FPGA and provides real-time feedback to LabVIEW, thus realizing the fluorescence intensity acquisition for that instance.

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

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

[0020] Furthermore, the FPGA chip can be replaced by a microcontroller, a single-chip microcontroller 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., and its main function is to record photon information.

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

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

[0024] A fully automated, intelligent, and multifunctional high-order nonlinear fluorescence kinetics testing method includes the following steps:

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

[0026] It should be noted that in step S1, the laser determines the required laser wavelength based on the excited-state absorption characteristics of the fluorescent sample, including continuous ultraviolet / visible / near-infrared light or pulsed ultraviolet / visible / near-infrared light, and the photon energy of the 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; precise and intelligent control is achieved on the computer side through the automatic power adjustment module.

[0028] It should be noted that in step S1, the automatic power adjustment module mainly consists of a power attenuator and a micro servo motor. The attenuator has different transmittance in its radial or angular direction. When aligned with the laser emission end, different rotation angles correspond to different transmitted light power. The micro servo motor drives the rotation of the gradient attenuator, achieving precise control of the attenuator angle and thus adjusting the transmitted light power. The micro servo motor can control the rotation angle via 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, using pulse width modulation signals to control its rotation angle. The frequency and duty cycle of the pulse signal determine the speed and direction of the servo motor. Furthermore, the FPGA controls the pulse output duration and frequency via a gate, enabling the servo motor to precisely stop at a specific angle.

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

[0030] It should be noted that in step S1, the power attenuator can be replaced by other power adjustment devices such as an electrically adjustable optical attenuator, an electro-optic modulator, or an acousto-optic modulator, the main function of which is to change the power at the laser emission end; the micro servo motor can be replaced by mechanical drive devices such as a stepper motor or a piezoelectric driver, the main function of which is to precisely control the laser attenuation device; the FPGA chip can be replaced by a microcontroller, a single-chip microcontroller control system, a digital-to-analog converter, or a digital signal processor, the main function of which 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. The sample fluorescence enters the collection and analysis module: The fluorescence signal of the sample is collected by the same imaging objective, focused by the dichroic mirror, filter group and lens and enters the collection fiber. The signal is received by the photodetector, stored by the FPGA and processed in conjunction with the computer LabVIEW.

[0032] It should be noted that in step S2, the fluorescent sample includes quantum dots, organic dyes, conventional upconversion fluorescent nanoparticles, and rare-earth-doped upconversion nanoparticles with photon avalanche effect. The nonlinearity of quantum dots and organic dyes is generally below 3, the nonlinearity of conventional upconversion fluorescent nanoparticles is generally below 6, and the nonlinearity of rare-earth-doped upconversion nanoparticles with photon avalanche effect is generally greater than 10.

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

[0034] It should be noted that in step S2, the photodetector includes, but is not limited to, a photomultiplier tube, a single-photon counter, a spectrometer, etc.

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

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

[0037] It should be noted that in step S2, the photodetector is connected to the FPGA chip. The photodetector acquires light signals within the FPGA's Gate window, realizing photo-to-electrical signal conversion. The FPGA's ADC channel receives the analog electrical signals from the photodetector and digitizes them into counting pulses, which are accumulated in a counter. After each independent count is completed, the counter transmits the accumulated result to the FPGA and feeds it back to the LabVIEW program in real time, realizing the fluorescence intensity acquisition for that instance.

[0038] It should be noted that in step S2, the computer is connected to the FPGA chip. The FPGA chip, acting as the main control unit, receives control signals from the computer, converts them into control commands for the servo motor, and monitors the feedback from the servo motor in real time, recording the fluorescence signal. Using a LabVIEW program as the user interface, control code is written to achieve real-time control and adjustment of the laser power, specifically including setting the target power, reading feedback values, and controlling the rotation angle of the servo motor. Furthermore, the computer displays the fluorescence intensity signal in real time for subsequent recording and processing.

[0039] S3: Nonlinear Power Curve Test: The fluorescent sample under test exhibits a high-order nonlinear response to excitation light intensity. In a typical test, the FPGA chip of the intelligent control module sends commands to a micro servo motor, intelligently controlling the rotation of the attenuator to achieve precise 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 to fit the sample power curve, realizing the fully automatic nonlinear power curve test function for fluorescent samples.

[0040] It should be noted that in step S3, the intelligent control module consists of an intelligent control FPGA chip and a computer. The FPGA has a built-in pulse width modulation module used to generate pulse signals to control other modules and adjust the pulse frequency, width, and duty cycle. In addition, a time gating module precisely controls the time window for excitation and acquisition signals for data analysis. The computer communicates with the FPGA via USB or Ethernet interface using software such as LabVIEW to complete parameter setting, real-time data transmission, visualization, and data analysis, thus comprehensively controlling all modules of the system.

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

[0042] S4: Fluorescence Rise Time and Lifetime Test: The intelligent control module sends pulse generation commands to the excitation light generation module to achieve continuous-pulse mode switching, and adjusts the excitation light input mode and the output mode of the acquisition module in real time to ensure that fluorescence signal acquisition begins immediately at the start and end of the excitation light pulse. The photodetector continuously collects fluorescence intensity at different times and stores it in the FPGA counter. The fluorescence rise curve and decay curve are plotted and fitted via LabVIEW on the computer, realizing fully automated and accurate testing of the in-situ fluorescence intensity, in-situ fluorescence rise curve, and in-situ fluorescence lifetime curve of the sample under test.

[0043] It should be noted that in step S4, the intelligent control module achieves continuous-pulse mode switching by sending pulses to the laser module. Specifically, in pulse mode, the FPGA chip of the intelligent control module precisely controls the power, pulse frequency, pulse width and duty cycle of the excitation light, as well as the acquisition 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, LabVIEW plots a fluorescence intensity decay curve over time based on the received data. The rise time and lifetime parameters of the sample are accurately calculated by setting an exponential fitting method to measure fluorescence lifetime or using a custom algorithm.

[0045] S5: Fluorescence Imaging Test: The computer-based LabVIEW drives a 3D displacement stage in real time to scan the sample area point by point in the XYZ direction. During each scan, a Gaussian solid spot is focused on different positions on the imaging plane, exciting the fluorescent sample at that position. The photodetector records the fluorescence intensity of each scan point and stores it in the FPGA counter. By changing the output mode of the intelligent control module and the acquisition module, the point scanning size, point scanning time, and point scanning depth can be changed to achieve the recording of point-by-point fluorescence intensity at different resolutions and depths. The data is output in real time and processed and visualized by the computer-based LabVIEW to realize fluorescence imaging test.

[0046] It should be noted that in step S5, the three-dimensional displacement 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 step interval and frequency, thereby realizing the step signal control of the three-dimensional displacement 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 system's operating interface, used to control the scanning path of the three-dimensional displacement stage, set the acquisition parameters, monitor the FPGA's operating status, and visualize the data in real time. It also spatially maps the fluorescence signal intensity values ​​to generate imaging data, supports two-dimensional or three-dimensional imaging display, and realizes the fully automated fluorescence imaging test function for fluorescent samples.

[0048] The method of this invention can perform excitation power response curve testing, fluorescence rise time testing, fluorescence decay lifetime testing, and fluorescence imaging testing of fluorescent samples. Furthermore, no additional components are required when switching between testing functions, thus achieving intelligent and multifunctional integration of fluorescence dynamics testing.

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

[0050] 1. Improved testing accuracy and reduced human error: Traditional nonlinear fluorescence kinetic testing methods typically rely on manual operation, which is susceptible to human interference, leading to inaccurate measurement results. This invention introduces an intelligent control module to achieve fully automated operation of the system, avoiding human error and improving testing accuracy and consistency. This fully automated control method not only ensures the accuracy and repeatability of experimental data but also avoids false information that may arise during manual intervention, ensuring that the kinetic response of the nonlinear fluorescent nanosample is truly reflected.

[0051] 2. Precise power control enables reliable measurement of high-order nonlinear responses: When the excitation power of a high-order nonlinear fluorescent nanosample approaches the threshold, the fluorescence intensity undergoes drastic changes, exhibiting an ultra-high-order nonlinear dependence. Traditional manual power adjustment methods cannot accurately capture this complex kinetic behavior. This invention, through an automatic power adjustment module, can precisely control the excitation power, achieving effective measurement of ultra-high nonlinear responses and providing high-precision experimental data support for revealing nonlinear physical phenomena. This precise power control makes kinetic response measurements more reliable within the probe's power-sensitive region, expanding the application scope of the testing device.

[0052] 3. Achieve multifunctional integration to meet diverse testing needs: Nonlinear fluorescence kinetics testing typically involves measuring multiple parameters such as fluorescence response curves, fluorescence rise time, fluorescence decay time, and fluorescence imaging. Traditional systems often require replacing different modules to achieve these functions, which is time-consuming and labor-intensive. This invention designs an integrated, multifunctional testing system that uses an intelligent control module to coordinate multiple modules, including excitation light generation, power adjustment, sample scanning, and data acquisition. This meets various needs for nonlinear fluorescence kinetics and enables fully automated testing without replacing any modules, greatly improving operational convenience and testing efficiency.

[0053] 4. Improved Testing Efficiency and Expanded Application Scenarios: The fully automated intelligent system design of this invention simplifies the operation process and significantly improves testing efficiency, resulting in a substantial increase in the speed and accuracy of multi-parameter fluorescence kinetic measurements. Compared to traditional time-consuming and labor-intensive manual testing, this invention can complete complex nonlinear kinetic analyses in a short time, while also possessing universality, applicable to various types of nonlinear fluorescent nanosamples. This not only meets the high-efficiency requirements of scientific research but is also suitable for batch industrial testing applications, demonstrating broad application prospects.

[0054] 5. Reduced System Costs and Enhanced Economic Applicability: Traditional nonlinear fluorescence dynamics testing devices are typically complex in structure and expensive, hindering widespread adoption. This invention, through modular design and integrated configuration, efficiently integrates various functional modules into a single system, simplifying the hardware structure and reducing manufacturing costs. Furthermore, the system enables multifunctional testing without replacing any components, further reducing experimental costs. This low-cost, simple testing system has significant practical implications for researchers and industrial users in the fields of nonlinear optics and nanophotonics. Attached Figure Description

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

[0056] Figure 2 The diagram shows the user interface of the testing software in embodiments 1, 2, 3 and 4 of this invention.

[0057] Figure 3 The design flow and logic diagram of the test software in embodiments 1, 2, 3 and 4 of the present invention are shown.

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

[0059] Figure 5 The figures show the photon avalanche dynamics power curve test and nonlinear fitting results for four types of upconversion fluorescent nanoparticles in Example 2 of this invention. Figure a shows the test system as NaYF4:Tm(0.5%), Figure b shows the test system as NaYF4:Tm(4%), Figure c shows the test system as NaYF4:Tm(8%), and Figure d shows the test system as NaYF4:Tm(8%)@NaYF4.

[0060] Figure 6 The figures show the fluorescence rise time and fluorescence lifetime test and fitting results of NaYF4:Tm(8%) in Example 3 of this invention. Figure a shows the fluorescence rise curve and fitting results, and Figure b shows the fluorescence lifetime curve and fitting results.

[0061] Figure 7 The images show the super-resolution fluorescence imaging and optical resolution analysis of NaYF4:Tm(8%)@NaYF4 in Example 4 of this invention. Figure a is the super-resolution fluorescence imaging, and Figure b is the Gaussian fitting analysis of the optical resolution of the two nanoparticles in Figure a. Detailed Implementation

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

[0063] Example 1

[0064] like Figure 1 As shown in the embodiment of the present invention, a fully automated, intelligent, and multifunctional high-order nonlinear fluorescence dynamics testing device comprises 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 fluorescent nanosample, realizing fully automated, intelligent, multifunctional, high-precision, simple, efficient, low-cost, and universal fluorescence dynamics testing and analysis.

[0065] The excitation light generation module includes a continuous-wave near-infrared laser 1 and a first filter 2 placed sequentially along the direction of the laser beam emitted by the laser. The automatic power adjustment module includes a graded attenuator 3 and a connected miniature servo motor 4. The excitation light modulation module includes a focusing objective 5, a polarization-maintaining single-mode fiber 6, a half-wave plate 7, and a quarter-wave plate 8. The infinity correction tube system 9 is used to adjust the beam size to match the entrance pupil of the imaging objective 11, improving beam utilization and correcting optical aberrations under high magnification and high numerical aperture conditions to ensure image clarity. The dichroic mirror 10 reflects the excitation light and transmits sample fluorescence, used to separate the excitation light from the fluorescence. The three-dimensional displacement stage 12 is used for spatial positioning of the sample, moving in the XYZ directions according to a set step size to scan the entire sample area point by point.

[0066] The data acquisition and analysis module consists of a second filter 14, a focusing lens 15, and a single-photon counter 17, all placed coaxially in sequence. When the sample is excited by the incident laser, it emits fluorescence in all directions. The imaging objective 11 collects a portion of the fluorescence signal, which then passes through a dichroic mirror 10, a reflecting mirror 13, the second filter 14, and the focusing lens 15 before being fed into a multimode polarization-maintaining fiber 16 and finally received by the single-photon counter 17. The intelligent control module consists of an intelligent control FPGA chip 18 and a computer terminal 19. The computer terminal uses software such as LabVIEW to complete parameter settings, real-time data transmission, visualization, and data analysis, comprehensively controlling all modules of the system.

[0067] The user interface for the above-mentioned device, developed using FPGA-LabVIEW, can be found here. Figure 2 The user interface includes four typical sections: Section 1 is used to visualize the laser power and the number of fluorescence emitted photons, and to perform axial focusing based on the number of emitted photons; Section 2 is used to visualize the spectral information of the sample and to select specific fluorescence emission bands; Section 3 is used to adjust the laser power and start the automatic test cycle; and Section 4 is used to visualize and fit the power curve test results.

[0068] The logic block diagram of the above device, written using FPGA-LabVIEW, can be found here. Figure 3 A typical workflow is as follows: Start the LabVIEW program, move the piezoelectric displacement stage axially through the interface, and determine the optimal axial position based on the recorded photon count; control the servo motor with PWM pulses to change the rotation angle and adjust the incident laser power, and record the emitted photon count of the sample under different incident powers for plotting and fitting to achieve fully automated power curve testing; set the delay time and width of the Gate window to turn the laser on / off, enabling testing of sample fluorescence rise time and decay time; control the PWM pulses to achieve timing management of the displacement stage step signal control and fluorescence signal acquisition; the photodetector module digitizes the fluorescence intensity of each scanning point and stores it in the buffer in real time, enabling two-dimensional or three-dimensional imaging display.

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

[0070] S1: Laser excitation and power modulation: Laser 1 outputs a stable laser beam. The laser beam is filtered out by the filter group 2 to remove other wavelengths of laser light. The purified excitation light is quantitatively modulated by the automatic power adjustment module. After being modulated by the focusing objective 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 matched with the entrance pupil of the imaging objective 11 to obtain a Gaussian solid 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 laser's continuous / pulse mode is adjustable.

[0072] It should be noted that in step S1, the automatic power adjustment module mainly consists of a power attenuator 3 and a micro servo motor 4. The attenuator 3 has different transmittance in its radial or angular direction. Aligned with the emission end of the laser 1, different rotation angles correspond to different transmitted light power. The micro servo motor 4 is used to drive the rotation of the gradient attenuator 3, realizing precise control of the angle of the attenuator 3, thereby adjusting the power of the transmitted light.

[0073] It should be noted that in step S1, the micro servo motor 3 can be controlled by pulses to adjust its rotation angle, and it 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 pulse width modulation signals. The frequency and duty cycle of the pulse signals determine the speed and direction of the micro servo motor 4. In addition, the FPGA chip 18 can implement a pulse counting function to accurately record the rotation angle of the micro servo motor 4.

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

[0075] It should be noted that in step S1, the power attenuator 3 can be replaced by other power adjustment devices such as an electrically adjustable optical attenuator, an electro-optic modulator, or an acousto-optic modulator, the main function of which is to change the power of the laser emission end; the micro servo motor 4 can be replaced by mechanical drive devices such as a stepper motor or a piezoelectric driver, the main function of which is to precisely control the laser attenuation device; the FPGA chip 18 can be replaced by a microcontroller, a single-chip microcontroller control system, a digital-to-analog converter, or a digital signal processor, the main function of which 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. 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 group 13 and the focusing lens 15 and then enters the multimode polarization-maintaining fiber 16. The signal is received by the single photon counter 17 (Excelitas, SPCM20328-107), stored by the FPGA chip 18, and processed by the LabVIEW program compiled on the computer 19.

[0077] It should be noted that in step S2, the fluorescent sample is a rare earth-doped upconversion nanoparticle with photon avalanche effect, 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 contains multiple optical filters in the 400 nm to 1000 nm wavelength band.

[0079] It should be noted that in step S2, the FPGA chip 18 performs 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 terminal 19 implements parameter setting, real-time data transmission and visualization 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 acquires optical signals within the Gate window of the FPGA chip 18, realizing photo-to-electrical signal conversion. The ADC channel of the FPGA chip 18 receives the analog electrical signals from the single-photon counter 17, digitizes them into counting pulses, and accumulates them in the counter. After each independent count is completed, the counter transmits the accumulated result to the FPGA chip 18 and feeds it back in real time to the LabVIEW program compiled on the computer 19, realizing the fluorescence intensity acquisition for that instance.

[0082] It should be noted that in step S2, the computer terminal 19 is connected to the FPGA chip 18. The FPGA chip 18, as the main control unit, is responsible for receiving control signals from the LabVIEW program within the computer terminal 19, converting them into control commands for the micro servo motor 4, and monitoring the feedback from the micro servo motor 4 in real time and recording the fluorescence signal. The LabVIEW program compiled on the computer terminal 19 serves as the user interface, containing control code to achieve real-time control and adjustment of the laser power. Specifically, this includes setting the target power, reading feedback values, 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 under test exhibits a high-order nonlinear response to the excitation light intensity. The FPGA chip 18 sends commands to the micro servo motor 4, intelligently controlling the rotation of the attenuator 3 to achieve precise 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 19 in real time. The data is processed by a LabVIEW program to fit the sample power curve, realizing a fully automated nonlinear power curve test function for the fluorescent sample.

[0084] It should be noted that in step S3, the FPGA chip 18 has a built-in pulse width modulation module used to generate pulse signals to control other modules and adjust the pulse frequency, width, and duty cycle. In addition, the time gating module precisely controls the time window for excitation and acquisition signals for data analysis. The computer 19 communicates with the FPGA chip 18 via USB through a LabVIEW program to complete parameter setting, real-time data transmission, visualization, and data analysis, thus comprehensively controlling all modules of the system.

[0085] S4: Fluorescence Rise Time and Lifetime Test: FPGA chip 18 sends pulse generation commands to laser 1 to achieve continuous-pulse mode switching, and adjusts the excitation light input mode and the output mode of the acquisition module in real time to ensure that fluorescence signal acquisition begins immediately at the start and end of the excitation light pulse. Single-photon counter 17 continuously acquires fluorescence intensity at different times and stores it in the counter of FPGA chip 18. The fluorescence rise curve and decay curve are plotted and fitted by LabVIEW program compiled by computer 19, realizing fully automatic and accurate testing of in-situ fluorescence intensity, in-situ fluorescence rise curve and in-situ fluorescence lifetime curve of the sample under test.

[0086] It should be noted that in step S4, the FPGA chip 18 precisely controls the power, pulse frequency, width and duty cycle of the excitation light, as well as the acquisition 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 on the computer terminal 19 plots a fluorescence intensity decay curve over time based on the received data. The rise time and lifetime parameters of the sample are accurately calculated by setting an exponential fitting method to measure fluorescence lifetime or a custom algorithm.

[0088] S5: Fluorescence Imaging Test: The LabVIEW program compiled on the computer 19 drives the three-dimensional displacement stage 12 in real time to scan the sample area point by point in the XYZ direction. During each scan, a Gaussian solid 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 scan point and stores it in the counter of the FPGA chip 18. By changing the output mode of the intelligent control module and the acquisition module, the point scanning size, point scanning time, and point scanning depth are changed to achieve the recording of the fluorescence intensity at different resolutions and depths. The data is output in real time and processed and visualized by the LabVIEW program compiled on the computer 19 to realize the fluorescence imaging test.

[0089] It should be noted that in step S5, the three-dimensional displacement 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 step interval and frequency, thereby realizing the step signal control of the three-dimensional displacement 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 on the computer terminal 19 serves as the system's operating interface. It is used to control the scanning path of the three-dimensional displacement stage 12, set the acquisition parameters, monitor the operating status of the FPGA chip 18, and visualize the data in real time. It also spatially maps the fluorescence signal intensity values ​​to generate imaging data, supports two-dimensional or three-dimensional imaging display, and realizes the fully automatic fluorescence imaging test function of fluorescent samples.

[0091] Example 2

[0092] Photon avalanche upconversion is a highly nonlinear optical process that typically occurs in certain rare-earth-doped materials, such as Tm. 3+ Plasmon ionization. In this phenomenon, low-intensity incident light can induce rapid excitation of electrons within the material, forming a positive feedback loop that triggers a series of multi-step excitation processes, ultimately producing fluorescence emission far exceeding the initial excitation. This process exhibits highly nonlinear characteristics because even under low-power excitation, the avalanche-like feedback significantly amplifies the fluorescence output, causing the intensity of the emitted light to exhibit an exponentially nonlinear relationship with the excitation light power.

[0093] The photon avalanche upconversion effect is highly dependent on the concentration of dopant ions in the material. At lower doping concentrations, the inter-ion spacing is larger, resulting in relatively low cross-relaxation energy transfer efficiency, which limits the effectiveness of positive feedback loops during photon avalanche and leads to insignificant nonlinearity. Conversely, at higher doping concentrations, the inter-ion spacing shortens, cross-relaxation energy transfer efficiency increases, and multi-step excitation and positive feedback loops between ions are more easily achieved, thus enhancing the nonlinear response. Therefore, different doping concentrations directly affect the optical nonlinearity and threshold of photon avalanche upconversion, which are measured by the power curves of the fluorescence excitation-emission intensity of the test samples.

[0094] For the synthesis of NaYF4:Tm systems with different concentrations and structures, see [link to documentation]. Figure 4 This demonstrates their common energy transfer pathway. At high doping concentrations, the photon avalanche process is as follows: [In the ground state...] 3 Tm at the H6 level 3+ Ions undergo a small-scale transition to the phonon-assisted state via non-resonant absorption at the 1064 nm ground state. 3 The F4 energy level is subsequently re-excited to the 1064 nm excited state via resonant absorption.3 H4 energy level. A level at which... 3 Tm at the H6 level 3+ Ions and a state 3 Tm at H4 level 3+ Ions undergo cross-relaxation, producing two states of cross-relaxation. 3 Tm at F4 level 3+ The ions, again through resonance at the 1064 nm excited state, generate two states... 3 Tm at H4 level 3+ Ions. As this positive feedback loop of ground-state absorption-cross-relaxation-excited-state absorption accumulates, it eventually leads to... 3 Tm at F4 level 3+ The number of ions surges, eventually transitioning to luminescence via a resonant absorption transition at the 1064 nm excited state. 3 The H4 energy level produces 800 nm photonic avalanche fluorescence.

[0095] Rare-earth-doped upconversion nanoparticles with compositions of NaYF4:Tm(0.5%), NaYF4:Tm(4%), NaYF4:Tm(8%), and NaYF4:Tm(8%)@NaYF4 were prepared for testing. Power curves were automatically measured and fitted using a designed fully automated intelligent device under 1064 nm laser excitation. Figure 5 The test and fitting results of power curves for photon avalanche nanoparticles with different doping concentrations are presented. Figure a shows the test system of NaYF4:Tm(0.5%), Figure b shows the test system of NaYF4:Tm(4%), Figure c shows the test system of NaYF4:Tm(8%), and Figure d shows the test system of 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 hardly exhibits the photon avalanche upconversion process, while the high Tm system... 3+ Both ion-doped (4%) and 8% systems exhibited typical photon avalanche phenomena. Furthermore, compared to the monolithic NaYF4:Tm(8%) system, the core-shell NaYF4:Tm(8%)@NaYF4 system exhibited lower photon avalanche threshold power and lower optical nonlinearity.

[0096] Example 3

[0097] Rare earth-doped upconversion nanoparticles with a composition of NaYF4:Tm(8%) were prepared for testing. Under 1064 nm laser excitation, the fluorescence rise curve and fluorescence decay curve were automatically tested and fitted using the designed fully automated intelligent device. Figure 6The fluorescence rise curve and lifetime decay curve of NaYF4:Tm(8%) nanoparticles are shown. Figure 6 Figure 'a' shows a slow fluorescence rise curve, with a fitted fluorescence rise time of 5 ms; Figure 6 Yes, b shows a rapid fluorescence decay curve with a fitted fluorescence lifetime of 28 μs, 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 NaYF4:Tm(8%)@NaYF4 were prepared for monodisperse testing. Fluorescence super-resolution imaging was performed using a designed fully automated intelligent device under 1064 nm laser excitation, and the full width at half maximum (FWHM) of the imaging resolution was fitted. Figure 7 The results of fluorescence super-resolution imaging and optical resolution analysis of NaYF4:Tm(8%)@NaYF4 nanoparticles are presented. Figure 7 a is the result of optical scanning super-resolution imaging of nanoparticles using the invented intelligent testing method, where serial number 1 and serial number 2 are two single nanoparticles in the imaging field of view. Figure 7 b and c in the figure represent the optical resolution analysis results of nanoparticles 1 and 2, respectively. Using the full width at half maximum (FWHM) analysis method, Gaussian fitting was performed on the line profiles of nanoparticles 1 and 2, and their FWHM was calculated as the resolution index, showing the imaging resolutions of 77 nm and 66 nm, respectively.

[0100] This invention, with its multifunctional integrated design and automated operation, not only significantly improves testing efficiency and accuracy but also greatly reduces the overall cost of the system. This makes the study of high-order nonlinear fluorescence dynamics more efficient, convenient, and economical, laying a solid foundation for further research and application in nonlinear optics and nanophotonics. It will also be of great significance in 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 based on the above technical solutions and concepts, and all such changes and modifications should be included within the protection scope of the claims of this invention.

Claims

1. A fully automated, intelligent, and multifunctional high-order nonlinear fluorescence dynamics testing 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 incident laser excitation, the intelligent control module coordinates and drives the other modules to complete the dynamic response test of the nonlinear fluorescent sample, thereby realizing fluorescence dynamic test and analysis. The excitation light generation module includes a continuous ultraviolet / visible / near-infrared laser or a pulsed ultraviolet / visible / near-infrared laser, and a filter group arranged sequentially 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; the filter group is used to purify the laser. The automatic power adjustment module includes a gradient attenuator and a connected miniature servo motor; the gradient attenuator has different transmittance in its radial direction or angle, and when aligned with the laser emission end, different rotation angles correspond to different transmitted light power; the miniature servo motor is used to drive the gradient attenuator to rotate, thereby achieving precise control of the attenuator angle and adjusting the power of the transmitted light. The excitation light modulation module includes a focusing objective lens, a polarization-maintaining fiber, a half-wave plate, and a quarter-wave plate. Focusing objectives and polarization-maintaining fibers are used for beam shaping to improve the efficiency of the excitation beam, while half-wave plates and quarter-wave plates are used to change the polarization state of the excitation beam to adapt to different excitation modes. The sample scanning module includes an infinity-corrected lens system, a dichroic mirror, an imaging objective, and a three-dimensional displacement stage. The infinity-corrected lens system is used to adjust the beam size to match the entrance pupil of the imaging objective, improving beam utilization and correcting optical aberrations under high magnification and high numerical aperture conditions to ensure image clarity. The dichroic mirror reflects the excitation light and transmits sample fluorescence, used to separate the excitation light and fluorescence. The imaging objective is used for beam focusing and sample imaging. The three-dimensional displacement stage is used for spatial positioning of the fluorescent sample, moving in the XYZ directions according to the set resolution to scan the entire sample area point by point. The data acquisition and analysis module consists 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 then input to the computer for data processing via the intelligent control module. The sample emits fluorescence in all directions under the excitation of the incident laser. The imaging objective lens collects a portion of the fluorescence signal, which passes through a dichroic mirror, a filter group, and a focusing lens, and is received by the photodetector. The signal is then input to the computer via the intelligent control module. The intelligent control module includes an intelligent control FPGA chip and a computer. The FPGA chip is connected to the computer and acts as the main control unit. It receives control signals from the computer, converts them into control commands for the servo motor, and monitors the feedback of the servo motor in real time and records fluorescence signals. The computer communicates with the FPGA via USB or Ethernet through LabVIEW software to complete parameter setting, real-time data transmission, visualization, and data analysis, and coordinates the various modules of the system.

2. The fully automated, intelligent, and multifunctional high-order nonlinear fluorescence dynamics testing device as described in claim 1, characterized in that... The filter group of the excitation light generation module is assembled with a rotatable bracket for adjusting and matching different excitation light wavelengths; the filter group of the data acquisition and analysis module can be assembled with a rotatable bracket for adjusting and matching 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 also have a built-in time gating module to precisely control the time window for excitation and acquisition signals for data analysis.

3. The fully automated, intelligent, and multifunctional high-order nonlinear fluorescence dynamics testing device as described in claim 1, characterized in that... The micro servo motor can be controlled by pulses to rotate 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 pulse width modulation (PWM) signals. The frequency and duty cycle of the pulse signals determine the speed and direction of the servo motor. In addition, the FPGA controls the pulse output duration and frequency through the gate to enable the servo motor to stop precisely at a specific angle. The FPGA chip is connected to the photodetector of the data acquisition and analysis module. The photodetector acquires light signals within the Gate window of the FPGA, converts them into counting pulses, and accumulates them in the counter. After each independent count is completed, the counter transmits the accumulated result to the FPGA and feeds it back to LabVIEW in real time, realizing the fluorescence intensity acquisition for that time.

4. The fully automated, intelligent, and multifunctional high-order nonlinear fluorescence dynamics testing device as described in claim 1, characterized in that... The gradient attenuator is replaced by an electrically adjustable optical attenuator, an electro-optic modulator, or an acousto-optic modulator to change the power at the laser emission end; the micro servo motor can be replaced by a stepper motor or a piezoelectric driver for precise control of the laser attenuation device; the FPGA chip is replaced by a microcontroller, a single-chip microcontroller control system, a digital-to-analog converter, or a digital signal processor for signal conversion and control; the photodetector is replaced by a photomultiplier tube or a spectrometer for recording photon information; and LabVIEW can be replaced by Python, Matlab, or LabWindows / CVI development environments for UI design and overall control of various modules of the system. The fluorescent sample may include quantum dots, organic dyes, conventional upconversion fluorescent nanoparticles, and rare earth-doped upconversion nanoparticles with photon avalanche effect.

5. A fully automated, intelligent, and multifunctional high-order nonlinear fluorescence kinetic testing method, characterized in that... The method using the fully automated, intelligent, and multifunctional high-order nonlinear fluorescence dynamics testing device as described in any one of claims 1 to 4 includes the following steps: S1: Laser excitation and power modulation: The laser outputs a stable laser beam, which is filtered out by a filter group to remove other wavelengths of laser light. The purified excitation light is quantitatively modulated by an automatic power adjustment module. After being modulated by a focusing objective, a polarization-maintaining fiber, and a polarization plate, the aberration is corrected by a tube lens group and matched with the entrance pupil of the imaging objective to obtain a Gaussian solid 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. The sample fluorescence enters the collection and analysis module: The fluorescence signal of the sample is collected by the same imaging objective, focused by the dichroic mirror, filter group and lens and enters the collection fiber. The signal is received by the photodetector, stored by the FPGA and processed in conjunction with the computer LabVIEW. S3: Nonlinear Power Curve Test: The fluorescent sample under test exhibits a high-order nonlinear response to the excitation light intensity. In a typical test, the FPGA chip of the intelligent control module sends commands to the micro servo motor, intelligently controlling the rotation of the attenuator 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 then processed by the LabVIEW program on the computer to fit the sample power curve, realizing the fully automatic nonlinear power curve test function for the fluorescent sample. S4: Fluorescence rise time and lifetime test: The intelligent control module sends pulse generation commands to the excitation light generation module to realize continuous-pulse mode switching, and adjusts the excitation light input mode and the output mode of the acquisition module in real time to ensure that fluorescence signal acquisition begins immediately at the start and end of the excitation light pulse; the photodetector continuously acquires fluorescence intensity at different times and stores it in the FPGA counter. The fluorescence rise curve and decay curve are plotted and fitted by LabVIEW on the computer to realize fully automatic and accurate testing of the in-situ fluorescence intensity, in-situ fluorescence rise curve and in-situ fluorescence lifetime curve of the fluorescent sample under test; S5: Fluorescence Imaging Test: Calculates the fluorescence intensity of the sample at different positions focused on the imaging plane, excites the sample at those positions, and records the fluorescence intensity of each scanning point using a photodetector. The computer-side LabVIEW drives a 3D displacement stage in real time to scan the sample area point by point in the XYZ directions. During each scan, a Gaussian solid spot is stored in an FPGA counter. By changing the output modes of the intelligent control module and the acquisition module, the point scanning size, scanning time, and scanning depth can be altered to record the fluorescence intensity at different resolutions and depths. The data is output in real time and processed and visualized via LabVIEW on the computer, thus achieving fluorescence imaging testing.

6. The fully automated, intelligent, and multifunctional high-order nonlinear fluorescence dynamics testing method as described in claim 5, characterized in that... In step S1, the laser determines the required laser wavelength based on the excited-state absorption characteristics of the fluorescent probe, including continuous ultraviolet / visible / near-infrared light or pulsed ultraviolet / visible / near-infrared light; In step S1, the polarization waveplate can be a half-waveplate or a quarter-waveplate, used to change the polarization state of the excitation light to adapt to different excitation modes; In step S1, the excitation power adjustment does not require manual operation; precise intelligent control is achieved on the computer side through an automatic power adjustment module. The automatic power adjustment module mainly consists of a power attenuator and a micro servo motor. The attenuator has different transmittance in its radial or angular direction. Aligned with the laser emission end, different rotation angles correspond to different transmitted light power. The micro servo motor drives the rotation of the gradient attenuator, achieving precise control of the attenuator angle and thus adjusting the transmitted light power. The micro servo motor can control the rotation angle via 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, using pulse width modulation signals to control its rotation angle. The frequency and duty cycle of the pulse signal determine the speed and direction of the servo motor. Furthermore, the FPGA controls the pulse output duration and frequency via a gate, enabling the servo motor to precisely stop at a specific angle. The power attenuator can be replaced by an electrically adjustable optical attenuator, an electro-optic modulator, or an acousto-optic modulator to change the power at the laser emission end. The micro servo motor can be replaced by a stepper motor or a piezoelectric driver for precise control of the laser attenuation device.

7. The fully automated, intelligent, and multifunctional high-order nonlinear fluorescence dynamics testing method as described in claim 5, characterized in that... In step S2, the fluorescent sample includes quantum dots, organic dyes, conventional upconversion fluorescent nanoparticles, and rare earth-doped upconversion nanoparticles with photon avalanche effect; 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 rare earth-doped upconversion nanoparticles with photon avalanche effect is generally greater than 10. In step S2, the filter group is assembled by a rotatable bracket for adjusting and matching different fluorescence collection wavelengths; The photodetector includes a photomultiplier tube, a single-photon counter, and a spectrometer; the FPGA chip realizes the functions of pulse signal generation, time gating, fluorescence signal acquisition and processing; the LabVIEW realizes parameter setting, real-time data transmission and visualization display. In step S2, the photodetector is connected to the FPGA chip; the photodetector collects light signals within the FPGA's Gate window to achieve photo-to-electric signal conversion; the FPGA's ADC channel receives the analog electrical signals from the photodetector and digitizes them into counting pulses, which are accumulated in the counter; after each independent count is completed, the counter transmits the accumulated result to the FPGA and feeds it back to the LabVIEW program in real time to realize the fluorescence intensity acquisition for that time. In step S2, the computer is connected to the FPGA chip. The FPGA chip acts as the main control unit, responsible for receiving control signals from the computer, converting them into control commands for the servo motor, and monitoring the feedback from the servo motor and recording the fluorescence signal in real time. A LabVIEW program is used as the user interface to write control code to achieve real-time control and adjustment of the laser power, specifically including setting the target power, reading feedback values, and controlling the rotation angle of the servo motor. In addition, the computer displays the fluorescence intensity signal in real time for subsequent recording and processing.

8. The fully automated, intelligent, and multifunctional high-order nonlinear fluorescence dynamics testing method as described in claim 5, characterized in that... In step S3, the intelligent control module consists of an intelligent control FPGA chip and a computer. The FPGA has a built-in pulse width modulation module, which generates pulse signals to control other modules and adjusts the pulse frequency, width, and duty cycle. In addition, the time gating module precisely controls the time window for excitation and acquisition signals for data analysis. The computer communicates with the FPGA via USB or Ethernet interface through LabVIEW software to complete parameter setting, real-time data transmission, visualization, and data analysis, and to coordinate the control of all modules of the system. In step S3, the LabVIEW program can be written using Python, Matlab, or LabWindows / CVI development environments, and is used to implement the UI interface design and to coordinate the various modules of the control system.

9. The fully automated, intelligent, and multifunctional high-order nonlinear fluorescence dynamics testing method as described in claim 5, characterized in that... In step S4, the intelligent control module achieves continuous-pulse mode switching by sending pulse generators to the laser module; specifically, in pulse mode, the FPGA chip of the intelligent control module precisely controls the power, pulse frequency, pulse width and duty cycle of the excitation light, as well as the acquisition time window of the fluorescence signal through the built-in pulse width modulation module and time gating module. In step S4, LabVIEW plots a fluorescence intensity decay curve over time based on the received data; and accurately calculates the rise time and lifetime parameters of the sample by setting the application of exponential fitting method to measure fluorescence lifetime or a custom algorithm.

10. The fully automated, intelligent, and multifunctional high-order nonlinear fluorescence dynamics testing method as described in claim 5, characterized in that... In step S5, the three-dimensional displacement 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 step interval and frequency, thereby realizing the step signal control of the three-dimensional displacement stage and the timing management of fluorescence signal acquisition, and scanning the entire sample area point by point. In step S5, the LabVIEW program serves as the system's operating interface, used to control the scanning path of the three-dimensional displacement stage, set the acquisition parameters, monitor the FPGA's operating status, and visualize the data in real time. It also spatially maps the fluorescence signal intensity values ​​to generate imaging data, supports two-dimensional or three-dimensional imaging display, and realizes the fully automated fluorescence imaging test function for fluorescent samples.

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