In-vitro cell microenvironment dynamic monitoring method for two-photon frequency domain fluorescence lifetime imaging

Abstract

The invention relates to the field of biological imaging, and particularly discloses an in-vitro cell microenvironment dynamic monitoring method for two-photon frequency domain fluorescence lifetime imaging, which comprises the following steps: S1, by using a two-photon frequency domain fluorescence lifetime imaging TP-FD-FLIM technology, losslessly representing microenvironment change through fluorescence lifetime parameters, and monitoring oxidative stress reaction and neutrophil migration behavior in real time, analyzing the effect of the associated compound in combination with an in-vitro cell model; and S2, based on the physical basis of FD-FLIM, establishing a frequency domain fluorescence lifetime signal processing and analysis system under periodic excitation, analyzing the dynamic correlation characteristics of the phase delay phi and the modulation depth M of a fluorescence signal relative to exciting light, and carrying out quantitative backstepping on molecular lifetime parameters. According to the in-vitro cell microenvironment dynamic monitoring method for two-photon frequency domain fluorescence lifetime imaging provided by the invention, neuroinflammatory response can be observed in a lossless and real-time manner on the subcellular scale.

Description

Technical Field

[0001] The present invention relates to the field of bioimaging technology, and specifically to a method for dynamically monitoring the in vitro cell microenvironment by two-photon frequency-domain fluorescence lifetime imaging. Background Art

[0002] Changes in the cell microenvironment are important observation objects in biomedical research. Real-time monitoring of its dynamic changes is of great significance for studying cell microenvironment changes and biological response mechanisms. However, traditional methods for monitoring neuroinflammation have certain limitations. For example, immunofluorescence-based fixed tissue imaging cannot obtain real-time inflammatory dynamic information. Although electrophysiological recording can achieve high-time-resolution monitoring, it is difficult to provide inflammatory characteristics at the cellular and molecular levels.

[0003] Fluorescence lifetime imaging (FLIM) is an imaging technology that reflects microenvironment characteristics by measuring changes in the fluorescence molecule lifetime, and can avoid the problems of traditional fluorescence intensity imaging being easily affected by probe concentration and photobleaching. Two-photon fluorescence lifetime imaging (2P-FLIM) uses infrared femtosecond lasers for excitation to achieve deep imaging of living tissues, and combines frequency-domain modulation technology to improve the acquisition efficiency of lifetime signals. Compared with time-domain FLIM, frequency-domain FLIM has higher signal-to-noise ratio and data acquisition speed, and is particularly suitable for dynamic imaging of living biological samples. In vitro three-dimensional cell models can be used for high-resolution in vitro sample imaging due to their controllability and operability advantages.

[0004] Although two-photon fluorescence imaging (TP-FLIM) and fluorescence lifetime imaging (FLIM) technologies have been widely used in biomedical research, the existing methods still have the following limitations in the real-time monitoring of neuroinflammation: 1. Dependence on fluorescence intensity or fixed samples, unable to analyze the living cell microenvironment in real time and non-destructively; 2. Low data acquisition efficiency of time-domain FLIM and insufficient dynamic resolution; 3. Limited ability of existing imaging systems to resolve weak fluorescence signals.

[0005] In the prior art, there have been various biomedical imaging methods and devices based on two-photon fluorescence imaging technology.

[0006] Patent CN105891170A: A method and device for two-photon excited time-lapse fluorescence imaging analysis of living animals. This patent proposes an imaging method that uses red or near-infrared pulsed lasers to two-photon excite fluorescence nanoprobes in the body and detects the fluorescence of the excited part in the body through time-lapse detection. The purpose of this method is to increase the imaging depth, reduce the interference of autofluorescence and background light, and thus improve the reliability of imaging analysis.

[0007] Patent CN104198458A A femtosecond laser two-photon fluorescence bio-microscopy imaging system and its imaging method: This patent introduces a method for high-resolution imaging of biological tissues using a tunable femtosecond laser source through a two-photon fluorescence microscopy imaging system. The system uses specific optical elements and filters to achieve precise imaging of biological samples.

[0008] Patent CN101254091A Method for high-spatial-resolution retinal imaging using second harmonic generation and two-photon excited fluorescence: This patent combines second harmonic generation imaging, polarization-sensitive second harmonic generation imaging, and two-photon excited fluorescence imaging to perform high-spatial-resolution imaging of the retina for early diagnosis of fundus diseases. This method couples a laser scanning confocal ophthalmoscope with an ultrashort pulse laser to image important functional cell layers of the retina and obtain their functional information.

[0009] However, these existing technical solutions mainly focus on general biological tissue imaging or imaging of specific tissues (such as the retina), and have not specifically targeted the dynamic monitoring of the cellular microenvironment. In addition, most of the patents on two-photon fluorescence lifetime imaging in the prior art use time-domain detection methods rather than frequency-domain detection methods. Summary of the Invention

[0010] In view of the above problems of the prior art, the present invention provides a method for dynamically monitoring the in vitro cellular microenvironment by two-photon frequency-domain fluorescence lifetime imaging, which can non-destructively and real-time observe neuroinflammatory responses at the subcellular scale and has the advantages of improving the precise quantitative analysis of fluorescence intensity imaging.

[0011] To achieve the above object, the present invention proposes a method for dynamically monitoring the in vitro cellular microenvironment by two-photon frequency-domain fluorescence lifetime imaging, including the following steps:

[0012] S1. Using two-photon frequency-domain fluorescence lifetime imaging (TP-FD-FLIM) technology, non-destructively characterizing microenvironment changes through fluorescence lifetime parameters, real-time monitoring oxidative stress responses and neutrophil migration behaviors, and combining in vitro cell model-related compound effect analysis;

[0013] S2. Establishing a frequency-domain fluorescence lifetime signal processing and analysis system based on the physical basis of FD-FLIM under periodic excitation, analyzing the dynamic correlation characteristics of the phase delay φ and modulation depth M of the fluorescence signal relative to the excitation light, and quantitatively inferring the molecular lifetime parameters.

[0014] Preferably, a two-photon fluorescence microscope system is used for two-photon frequency-domain fluorescence lifetime imaging. The imaging process is as follows: a tunable femtosecond pulsed laser is used to generate a laser. After the power is regulated by a half-wave plate (HWP) and a polarization beam splitter (PBS), it is collimated and expanded through a 4f system, and the XY scanning is controlled by a two-dimensional galvanometer scanner. The excitation light is focused on the sample through a high numerical aperture objective lens, and the two-photon fluorescence signal is detected by a photomultiplier tube (PMT) after filtering to achieve fluorescence lifetime imaging with high spatio-temporal resolution.

[0015] Preferably, the frequency-domain fluorescence lifetime signal processing and analysis system established under periodic excitation includes a four-phase quadrature mixer, a phase shift compensation module, and a phasor analysis algorithm.

[0016] Preferably, in S1, the changes in the microenvironment are characterized by fluorescence lifetime parameters without damage, and the oxidative stress response and neutrophil migration behavior are monitored in real time. The specific steps for analyzing the compound effect in combination with an in vitro cell model are as follows:

[0017] S11. Monitoring the fluorescence lifetime to characterize the metabolic state: Measuring the lifetime changes of endogenous fluorescent molecules, calculating the ratio of NAD + / NADH, and deducing the redox state and metabolic level of the cells;

[0018] S12. Obtaining the fluorescence lifetime signal of oxidative stress: Obtaining the accumulation degree of reactive oxygen species (ROS). The accumulation degree of ROS is manifested as a shortening of the fluorescence lifetime;

[0019] S13. Tracking inflammation-related cells: Labeling neutrophils with EGFP, and analyzing the recruitment and migration characteristics of inflammation-related cells in the inflammation area by combining the lifetime signal.

[0020] Preferably, in S2, for the frequency-domain fluorescence lifetime signal processing and analysis system established under periodic excitation, the steps of frequency-domain fluorescence lifetime signal processing include:

[0021] S211. Signal preprocessing: The data acquisition card (DAQ) collects the synchronization signal, and the laser outputs a synchronization reference signal. After suppressing the high-order harmonics by a low-pass filter (LPF), the signal-to-noise ratio is improved by a low-noise amplifier (LNA);

[0022] S212. Signal distribution and phase regulation: The preprocessed signal generates four homologous signals by a four-way power divider, and the phase is regulated from 0 to 2π through a phase shifter;

[0023] S213. Signal amplification and mixing: The adjusted signal is amplified by an LNA and then input into a mixer, where it is mixed with the fluorescence signal from the photomultiplier tube (PMT) to form four orthogonal reference signals;

[0024] S214. PMT Signal Processing and Demodulation: The weak current signal output by the PMT is first converted into a voltage signal by a transimpedance amplifier, and current separation and filtering are performed to remove noise and interference; the processed PMT signal is mixed and demodulated with four-channel quadrature reference signals to extract the characteristic information related to the fluorescence lifetime;

[0025] S215. Signal Analysis and Calibration: The fluorescence signal is analyzed using four-phase matrix operations to calculate the key parameters of the fluorescence lifetime, phasor coordinates, and intensity image; a full 50Ω impedance matching architecture is combined with electromagnetic shielding measures for impedance matching and electromagnetic shielding to optimize the signal-to-noise ratio; a dynamic calibration algorithm is used to correct the channel deviation in real time to ensure the accurate analysis of weak fluorescence signals.

[0026] Preferably, in S2, the steps for quantitatively inferring the molecular lifetime parameters by analyzing the dynamic correlation characteristics of the phase delay φ and modulation depth M of the fluorescence signal relative to the excitation light include:

[0027] S221. Analyze the fluorescence signal using the frequency domain characteristics of the periodic excitation light, model the excitation function, calculate the obtained fluorescence signal, and analyze the fluorescence signal characteristics;

[0028] S222. Analyze the frequency domain signal and the assumed model: Decompose the periodic fluorescence signal F(t) into Fourier series, extract the frequency domain response, and its expression is:

[0029]

[0030] In the formula, the Fourier series q k Is defined by the integral as

[0031] S223. Phase Delay and Modulation Depth Measurement: Perform signal separation processing on the bias tee of the fluorescence signal captured by the photomultiplier tube PMT, decompose the original time domain signal F(t) into direct current DC and radio frequency RF components, and the RF part contains lifetime information; use radio frequency mixing technology to extract the fundamental wave characteristics, and parallelly transmit and process the fluorescence signal and the reference signal through a four-channel power distribution architecture;

[0032] The specific processing process for performing signal separation processing on the bias tee of the fluorescence signal captured by the photomultiplier tube PMT is:

[0033] The direct current component characterizes the time integral intensity of the fluorescence signal, corresponding to the fundamental frequency coefficient q0 of the frequency domain Fourier series, and the mathematical expression is:

[0034]

[0035] In the formula, B is the direct current conversion efficiency of the bias tee, O Bis the inherent DC offset of the system, q0 is the zero-order Fourier coefficient of the fluorescence signal, representing the steady-state fluorescence intensity, V DC is the DC port component of the bias;

[0036] The RF component corresponds to the fundamental frequency coefficient q of the Fourier series in the frequency domain k , and the mathematical expression is:

[0037]

[0038] or

[0039] where V RF is the RF port component of the bias;

[0040] S224. System calibration and parameter extraction: Establish the mapping relationship between the bias voltage of the phase shifter and the phase offset, calibrate the loss coefficient and gain coefficient of the mixer, calculate the ratio M / B of the key parameters, generate four groups of orthogonal reference signals, and correct the channel gain difference;

[0041] S225. Phasor analysis and life quantification backtracking: Analyze the four groups of orthogonal phase shift signals, use matrix operations to eliminate the DC offset O M , and extract the fluorescence lifetime through the proportional relationship with the imaginary and real parts of the first harmonic Fourier coefficient of the fluorescence; calculate the average lifetime of the multi-exponential decay through the horizontal component g and vertical component s of the phasor diagram.

[0042] Preferably, the specific steps of S221 for analyzing the fluorescence signal by using the frequency domain characteristics of the periodic excitation light, modeling the excitation function, calculating the obtained fluorescence signal and analyzing the characteristics of the fluorescence signal are to model the time-domain excitation function of the femtosecond pulse sequence with a repetition frequency of 80 MHz and a pulse width less than 100 fs for exciting the fluorescent molecules as a Dirac comb distribution, and the expression is:

[0043] I(t) = Pδ T (t);

[0044] where P is a coefficient related to the average power of the laser beam, I(t) is the time distribution function of the excitation light, and δ T (t) is the Dirac comb function with a period of T, representing the periodic pulse sequence;

[0045] In the two-photon excitation process, the fluorescence signal F(t) is proportional to the convolution of the square of the excitation light intensity and the impulse response function f(t) of the fluorophore, and the fluorescence signal F(t) is expressed as:

[0046]

[0047] where c is the concentration of the fluorophore, It is the impulse response function of a single-exponential decay fluorophore. Here, the instrument response function IRF is much lower than the lifetime value of the fluorophore, and the IRF can be ignored.

[0048] Preferably, in S222, assuming the model is divided into a single-component model and a multi-component model, for the impulse response function f(t) of a single-exponential decay fluorophore:

[0049]

[0050] In the formula, τ represents the fluorescence lifetime, which is the average time for the molecule to return from the excited state to the ground state and reflects the physicochemical properties of its microenvironment;

[0051] For multi-component fluorescent samples, the lifetimes of each component are analyzed by measuring the phase difference and modulation ratio, and the impulse response function is extended to:

[0052]

[0053] In the formula, a i is the intensity-weighted fractional contribution of the fluorophore with lifetime τ i , τ i are different lifetimes in the multi-component, and the Fourier coefficient q of the multi-component fluorescent sample k is:

[0054] Preferably, in S223, the specific steps for parallel transmission and processing of the fluorescence signal and the reference signal through a four-channel power distribution architecture are as follows:

[0055] S2231. Set the preset phase offset of the voltage-controlled phase shifter in each independent channel to generate four-way quadrature reference signals and input them into the local oscillator port LO of the mixer for four-channel synchronous demodulation to achieve real-time phase analysis of the fluorescence signal. Its expression is:

[0056] v LO (t, O) = sin(ωt + φ);

[0057] In the formula, ν LO is the signal of the LO port.

[0058] S2232. The four mixers respectively connect the PMT signal and the phase-shifted reference signal to their respective radio frequency RF and local oscillator LO ports, and obtain the inherent DC offset in the output signal. The inherent DC offset O M The expression is:

[0059] O M = O(P RF ) + d;

[0060] In the formula, O(P RF ) is a function of the radio frequency power, and d is the inherent constant offset of the system;

[0061] S2233. Correct the output of the mixer. Process the output signal of the mixer through low-pass filtering to eliminate all high-order harmonic components, and convert the filtered signal to represent a pure DC voltage. Among them, the corrected expression of the mixer output is:

[0062] v IF (t,φ) = MV LO (t,φ)v RF (t) + O M ;

[0063] In the formula, M is the conversion loss coefficient of the mixer, IF is the intermediate frequency, ν IF is the output function of the mixer;

[0064] The mathematical expression of the pure DC voltage converted from the filtered signal is:

[0065]

[0066] S2234. Synchronously sample and digitally convert the four demodulation signals required for fluorescence lifetime calculation through the data acquisition card DAQ.

[0067] Preferably, in S225, the calculation formula for the average lifetime of multi-exponential decay is

[0068] Therefore, the present invention proposes a method for dynamically monitoring the in vitro cell microenvironment by two-photon frequency-domain fluorescence lifetime imaging, and its beneficial effects are as follows:

[0069] (1) The method for dynamically monitoring the in vitro cell microenvironment by two-photon frequency-domain fluorescence lifetime imaging proposed by the present invention uses the change of fluorescence lifetime to characterize the redox state and metabolic characteristics of the inflammatory microenvironment, and can provide more accurate quantitative analysis than traditional fluorescence intensity imaging. It provides a new tool for dynamically monitoring the cell microenvironment and analyzing biological responses, and realizes real-time and non-destructive dynamic analysis of in vitro cell microenvironment samples, avoiding the invasiveness and lag of traditional methods.

[0070] (2) The present invention improves the fluorescence lifetime detection efficiency and signal-to-noise ratio through frequency-domain signal processing.

[0071] (3) The present invention constructs a quantitative evaluation model to analyze the oxidative stress and metabolic characteristics of the cell microenvironment.

[0072] Next, through the drawings and embodiments, the technical solutions of the present invention will be further described in detail. Description of the Drawings

[0073] Figure 1 is the overall design flow chart of the method for dynamically monitoring the in vitro cell microenvironment by two-photon frequency-domain fluorescence lifetime imaging of the present invention;

[0074] Figure 2 It is the imaging process diagram of the method for dynamically monitoring the in vitro cell microenvironment by two-photon frequency-domain fluorescence lifetime imaging of the present invention;

[0075] Figure 3 It is the flowchart of frequency-domain signal processing of the method for dynamically monitoring the in vitro cell microenvironment by two-photon frequency-domain fluorescence lifetime imaging of the present invention;

[0076] Figure 4 It is the schematic diagram of the detection principle of the zebrafish neuroinflammation model in the embodiment of the method for dynamically monitoring the in vitro cell microenvironment by two-photon frequency-domain fluorescence lifetime imaging of the present invention. Detailed implementation manners

[0077] To make the technical solutions, advantages and objectives of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the described embodiments of the present invention without creative efforts fall within the protection scope of this application.

[0078] Unless otherwise defined, the technical terms or scientific terms used in the present invention shall have the ordinary meanings understood by those of ordinary skill in the art to which the present invention belongs.

[0079] As Figures 1-3 shown, the present invention proposes a method for dynamically monitoring the in vitro cell microenvironment by two-photon frequency-domain fluorescence lifetime imaging, including the following steps:

[0080] S1. Utilize the two-photon frequency-domain fluorescence lifetime imaging TP-FD-FLIM technology, and non-destructively characterize the microenvironment changes through fluorescence lifetime parameters, and real-time monitor the oxidative stress response and neutrophil migration behavior, combined with the analysis of the effects of associated compounds in the in vitro cell model. The specific steps are as follows:

[0081] S11. Monitor the fluorescence lifetime to characterize the metabolic state: Measure the lifetime changes of endogenous fluorescent molecules, calculate the ratio of NAD + / NADH indirectly reflected by the metabolic index, and deduce the redox state and metabolic level of the cells;

[0082] S12. Obtain the fluorescence lifetime signal of oxidative stress: Obtain the accumulation degree of reactive oxygen species ROS. The accumulation degree of reactive oxygen species ROS is manifested as a shortening of the fluorescence lifetime;

[0083] S13. Perform tracking of inflammation-related cells: Label neutrophils with EGFP, and analyze the recruitment and migration characteristics of inflammation-related cells in the inflammation area in combination with the lifetime signal.

[0084] S2. Based on the physical basis of FD-FLIM, a frequency-domain fluorescence lifetime signal processing and analysis system is established under periodic excitation to analyze the dynamic correlation characteristics of the phase delay φ and modulation depth M of the fluorescence signal relative to the excitation light, and to quantitatively infer the molecular lifetime parameters.

[0085] Among them, for the frequency-domain fluorescence lifetime signal processing and analysis system established under periodic excitation, the steps of frequency-domain fluorescence lifetime signal processing include:

[0086] S211. Signal preprocessing: The data acquisition card DAQ collects the synchronization signal, the laser outputs the synchronization reference signal, after suppressing the high-order harmonics by the low-pass filter LPF, and the signal-to-noise ratio is improved by the low-noise amplifier LNA;

[0087] S212. Signal distribution and phase regulation: The preprocessed signal generates four homologous signals by a four-way power divider, and the phase regulation of 0 to 2π is carried out through the phase shifter Phase Shifter;

[0088] S213. Signal amplification and mixing: The adjusted signal is amplified by the LNA and then input into the mixer Mixer, and is mixed with the fluorescence signal from the photomultiplier tube PMT to form four-way quadrature reference signals;

[0089] S214. PMT signal processing and demodulation: The weak current signal output by the PMT is first converted into a voltage signal by a transimpedance amplifier, and current separation and filtering processing are carried out to remove noise and interference; the processed PMT signal is mixed and demodulated with the four-way quadrature reference signals to extract the characteristic information related to the fluorescence lifetime;

[0090] S215. Signal analysis and calibration: The fluorescence signal is analyzed by four-phase matrix operation, and the key parameters of the fluorescence lifetime, phasor coordinates and intensity image are calculated; the impedance matching and electromagnetic shielding are carried out by adopting a full 50Ω impedance matching architecture and combining electromagnetic shielding measures to optimize the signal-to-noise ratio; the channel deviation is corrected in real time by adopting a dynamic calibration algorithm to ensure the accurate analysis of the weak fluorescence signal.

[0091] In S2, the steps of analyzing the dynamic correlation characteristics of the phase delay φ and modulation depth M of the fluorescence signal relative to the excitation light and quantitatively inferring the molecular lifetime parameters include:

[0092] S221. Analyze the fluorescence signal by using the frequency-domain characteristics of the periodic excitation light, model the excitation function, calculate and obtain the fluorescence signal and analyze the fluorescence signal characteristics. The specific steps are to model the time-domain excitation function of the femtosecond pulse sequence with a repetition frequency of 80 MHz and a pulse width less than 100 fs for exciting the fluorescence molecules as a Dirac comb distribution, and the expression is:

[0093] I(t) = Pδ T (t);

[0094] Wherein, P is a coefficient related to the average power of the laser beam, I(t) is the time distribution function of the excitation light, and δ T (t) is a Dirac comb function with a period of T, representing a periodic pulse sequence;

[0095] In the two-photon excitation process, the fluorescence signal F(t) is proportional to the convolution of the square of the excitation light intensity and the impulse response function f(t) of the fluorophore, and the fluorescence signal F(t) is expressed as:

[0096]

[0097] Wherein, c is the concentration of the fluorophore, is the impulse response function of the single-exponential decay fluorophore. Here, the instrument response function IRF is much lower than the lifetime value of the fluorophore, and IRF is negligible.

[0098] S222. Analyzing the frequency-domain signal and the assumed model: Decompose the periodic fluorescence signal F(t) into Fourier series, and extract the frequency-domain response. Its expression is:

[0099]

[0100] Wherein, the Fourier series q k is defined by the integral as

[0101] The assumed model is divided into a single-component model and a multi-component model. For the impulse response function f(t) of the single-exponential decay fluorophore:

[0102]

[0103] Wherein, τ represents the fluorescence lifetime, which is the average time for the molecule to return from the excited state to the ground state, reflecting the physical and chemical properties of its microenvironment;

[0104] For multi-component fluorescence samples, the lifetimes of each component are analyzed by measuring the phase difference and the modulation ratio, and the impulse response function is extended to:

[0105]

[0106] Wherein, a i is the intensity-weighted fractional contribution of the fluorophore with a lifetime of τ i , τ i are different lifetimes in the multi-component. The Fourier coefficient qk of the multi-component fluorescence sample is:

[0107] S223. Phase Delay and Modulation Depth Measurement: Signal separation processing is performed on the bias tee of the fluorescence signal captured by the photomultiplier tube PMT. The original time-domain signal F(t) is decomposed into a DC and an RF component. The RF part contains lifetime information. The fundamental wave characteristics are extracted using RF mixing technology, and the fluorescence signal and the reference signal are processed in parallel through a four-channel power distribution architecture;

[0108] The specific processing process of signal separation processing on the bias tee of the fluorescence signal captured by the photomultiplier tube PMT is as follows:

[0109] S224. System Calibration and Parameter Extraction: Establish the mapping relationship between the bias voltage of the phase shifter and the phase offset, calibrate the loss coefficient and gain coefficient of the mixer, calculate the ratio M / B of the key parameters, generate four sets of orthogonal reference signals, and correct the channel gain difference;

[0110] S225. Phasor Analysis and Inverse Lifespan Quantification: Analyze the four sets of orthogonal phase-shifted signals, and use matrix operations to eliminate the DC offset O M , and extract the fluorescence lifetime by its proportional relationship with the imaginary and real parts of the first harmonic Fourier coefficient of the fluorescence. Calculate the average lifetime of the multi-exponential decay through the horizontal component g and vertical component s of the phasor diagram.

[0111] The DC component characterizes the time-integrated intensity of the fluorescence signal, corresponding to the fundamental frequency coefficient q0 of the Fourier series in the frequency domain. The mathematical expression is:

[0112]

[0113] In the formula, B is the DC conversion efficiency of the bias tee, O B is the inherent DC offset of the system, q0 is the zero-order Fourier coefficient of the fluorescence signal, characterizing the steady-state fluorescence intensity, V DC is a function of the DC port of the mixer;

[0114] The RF component corresponds to the fundamental frequency coefficient q k in the Fourier series of the frequency domain, and the mathematical expression is:

[0115]

[0116] Or

[0117] In the formula, V RF is a function of the RF port of the mixer

[0118] In S223, the specific steps of parallel transmission and processing of the fluorescence signal and the reference signal through the four-channel power distribution architecture are as follows:

[0119] S2231. Set the preset phase offset of the voltage-controlled phase shifter in each independent channel to generate four-channel quadrature reference signals and input them into the local oscillator port LO of the mixer for four-channel synchronous demodulation to achieve real-time phase analysis of the fluorescence signal. The expression is as follows:

[0120] v LO (t, O) = sin(ωt + φ);

[0121] In the formula, ν LO is the signal at the LO port;

[0122] Among them, for each reference signal path, a phase shifter is used to introduce a voltage-controlled phase shift of φ to the signal, and the reference signal is sent to the local oscillator LO port of the mixer.

[0123] S2232. Four mixers respectively connect the PMT signal and the phase-shifted reference signal to their respective radio frequency RF and local oscillator LO ports, and obtain the inherent DC offset in the output signal. The inherent DC offset O M The expression is:

[0124] O M = O(P RF ) + d;

[0125] In the formula, O(PRF) is a function of the radio frequency power, and d is the inherent constant offset of the system;

[0126] S2233. Correct the output of the mixer. The output signal of the mixer is processed by low-pass filtering to eliminate all high-order harmonic components, and the filtered signal is converted to a pure DC voltage. Among them, the corrected expression of the mixer output is:

[0127] v IF (t, φ) = MV LO (t, φ)v RF (t) + O M ;

[0128] In the formula, M is the conversion loss coefficient of the mixer, IF is the intermediate frequency, and ν IF is the output function of the mixer;

[0129] The mathematical expression of the pure DC voltage converted from the filtered signal is:

[0130]

[0131] S2234. Synchronously sample and digitally convert the four-channel demodulation signals required for fluorescence lifetime calculation through the data acquisition card DAQ.

[0132] The frequency-domain fluorescence lifetime signal processing and analysis system based on periodic excitation of the present invention includes a four-phase quadrature mixer, a phase shift compensation module, and a phasor analysis algorithm.

[0133] In the present invention, a two-photon fluorescence microscope system is used for two-photon frequency-domain fluorescence lifetime imaging. The imaging process is as follows: a tunable femtosecond pulsed laser is used to generate a laser with a wavelength range of 680 - 1300 nm, a pulse width of 100 fs, and a repetition frequency of 80 MHz. However, the specific repetition frequency can be adjusted according to the required imaging speed. After the power is regulated by a half-wave plate (HWP) and a polarization beam splitter (PBS), it is collimated and expanded by a 4f system, and the XY scanning is controlled by a two-dimensional galvanometer scanner. The excitation light is focused on the sample through a high numerical aperture objective lens, and the two-photon fluorescence signal is detected by a photomultiplier tube (PMT) after filtering to achieve fluorescence lifetime imaging with high spatio-temporal resolution.

[0134] As Figure 4 shown, the present invention provides an application of a method for dynamically monitoring the in vitro cell microenvironment of two-photon frequency-domain fluorescence lifetime imaging in the detection of a zebrafish neuroinflammation model. The specific processing process is as follows:

[0135] 1. Construct a zebrafish neuroinflammation model

[0136] Embryo culture and pretreatment: Healthy zebrafish embryos within 4 days post-fertilization (4 dpf) are selected to ensure the consistency of individual development and improve the repeatability of the experiment. The embryo culture medium is E3 medium (containing 0.003% N-phenylthiourea, PTU), which can effectively inhibit melanogenesis, reduce PMT signal interference, and the culture medium is replaced regularly to maintain a stable metabolic environment.

[0137] Copper sulfate-induced neuroinflammation: At 4 dpf, zebrafish larvae are incubated in solutions of different concentrations (5 - 50 μM) of CuSO4 (dissolved in E3 medium) for 2 hours to induce oxidative stress and sterile inflammation. Preliminary experiments determined that 25 μM CuSO4 is the optimal concentration, which can effectively induce the production of ROS, H2O2, and OH - , activate inflammatory factors TNF-α and IL-1, thereby recruiting EGFP-labeled neutrophils to the injury site, while ensuring the survival rate of zebrafish. Observe the necrotic apoptosis of lateral line hair cells to confirm the successful establishment of the inflammation model, and use the change in fluorescence lifetime as a quantitative index of the degree of inflammation.

[0138] Anesthesia and fixation: Soak zebrafish embryos in 60 μM Tricaine solution (Sigma, #E10521) for 1 - 2 minutes to inhibit motor activity and ensure imaging stability. After anesthesia, rinse three times with fresh E3 medium to remove residual anesthetic. Take 1.5% low melting point agarose gel pre-warmed to 37 °C, gently wrap and fix the anesthetized embryos at the bottom of the imaging dish, ensuring unobstructed respiration and stable positioning for subsequent imaging.

[0139] 2. Perform imaging with the TP-FD-FLIM system

[0140] Imaging system parameter settings: Use a 920 nm femtosecond laser (80 MHz repetition rate) as the fluorescence excitation light source, and perform high-resolution imaging through an objective lens with a numerical aperture (NA) of 0.75.

[0141] FLIM imaging parameters: The single-pixel dwell time is set to 10 μs, the imaging resolution is 512 × 512 pixels, and the imaging rate is 25 frames per second (0.04 s / frame)

[0142] Data acquisition and processing: Continuously acquire fluorescence lifetime and fluorescence intensity data of EGFP-labeled neutrophils through TP-FD-FLIM technology to monitor cell migration trajectories. Analyze the changes in fluorescence lifetime to quantitatively evaluate the accumulation of ROS and the severity of inflammation non-invasively. Record the migration dynamics of neutrophils at the inflammation site, and combine with the fluorescence lifetime change curve to confirm the characteristics of the inflammatory response induced by CuSO4.

[0143] 3. Ibuprofen anti-inflammatory experiment

[0144] After inducing inflammation with CuSO4, transfer the zebrafish embryos to E3 culture medium containing Ibuprofen and incubate for 2 hours to observe the anti-inflammatory effect. The TP-FD-FLIM system monitors the effects of Ibuprofen on inflammatory signals, including reduced ROS accumulation, prolonged fluorescence lifetime, and reduced neutrophil recruitment. Through quantitative analysis of fluorescence lifetime, verify that Ibuprofen reduces the accumulation of PGE-2 by inhibiting COX-2 activity, thereby reducing inflammatory factors TNF-α and IL-1 and promoting cell repair.

[0145] It can be seen from the experimental model that the present invention analyzes oxidative stress response and neutrophil migration in real time through changes in fluorescence lifetime, establishes an optical evaluation system for the severity of inflammation based on fluorescence lifetime, and provides a quantitative index for the screening of bioactive substances.

[0146] Therefore, the present invention provides a method for dynamically monitoring the in vitro cell microenvironment of two-photon frequency-domain fluorescence lifetime imaging. By using the TP-FD-FLIM technology, it realizes the non-destructive dynamic monitoring of the in vitro cell microenvironment model for the first time, breaking through the traditional invasive method that relies on the detection of inflammatory factors. The present invention uses the FD-FLIM frequency-domain fluorescence lifetime imaging technology, breaking through the limitations of the traditional TCSPC time-domain detection, improving the imaging speed and signal-to-noise ratio, and having significant advantages in neuroinflammation monitoring, imaging quality and signal processing optimization, providing a quantitative optical evaluation means for the screening of bioactive substances and the study of cell responses.

[0147] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that they can still modify or equivalently replace the technical solutions of the present invention, and these modifications or equivalent replacements cannot make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for dynamic monitoring of in vitro cell microenvironment by two-photon frequency-domain fluorescence lifetime imaging, characterized in that: The following steps are involved: S1. Using two-photon frequency-domain fluorescence lifetime imaging (TP-FD-FLIM) technology, and non-destructively characterizing microenvironmental changes through fluorescence lifetime parameters, real-time monitoring of oxidative stress response and neutrophil migration behavior, combined with in vitro cell model-associated compound effect analysis; S2. The physical basis of FD-FLIM is based on a frequency-domain fluorescence lifetime signal processing and analysis system under periodic excitation. It analyzes the dynamic correlation characteristics between the phase delay φ of the fluorescence signal relative to the excitation light and the modulation depth M, and quantitatively infers the molecular lifetime parameters.

2. The in vitro cell microenvironment dynamic monitoring method using two-photon frequency domain fluorescence lifetime imaging according to claim 1, characterized in that: A two-photon fluorescence microscope system was used for two-photon frequency-domain fluorescence lifetime imaging. The imaging process was to use a tunable femtosecond pulse laser to generate laser light, adjust the power through a half-wave plate HWP and a polarization beam splitter PBS, collimate and expand the beam through a 4f system, and control the XY scanning through a two-dimensional galvanometer galvanometer. The excitation light was focused onto the sample through a high numerical aperture objective lens, and the two-photon fluorescence signal was detected by a photomultiplier tube PMT after filtering to achieve fluorescence lifetime imaging with high temporal and spatial resolution.

3. The in vitro cell microenvironment dynamic monitoring method using two-photon frequency domain fluorescence lifetime imaging according to claim 1, characterized in that: The frequency domain fluorescence lifetime signal processing and analysis system based on periodic excitation includes a four-phase orthogonal mixer, a phase shift compensation module and a phase analysis algorithm.

4. The in vitro cell microenvironment dynamic monitoring method using two-photon frequency domain fluorescence lifetime imaging according to claim 1, characterized in that: In S1, the fluorescence lifetime parameters were used to non-destructively characterize microenvironment changes, monitor oxidative stress response and neutrophil migration behavior in real time, and the specific steps for analyzing the effects of associated compounds in an in vitro cell model were as follows: S11. Monitor fluorescence lifetime to characterize metabolic status: measure the lifetime changes of endogenous fluorescent molecules and calculate NAD indirectly reflected by metabolic indicators + / NADH ratio, deriving the redox state and metabolic level of cells; S12. Obtaining the fluorescence lifetime signal of oxidative stress: Obtaining the accumulation degree of reactive oxygen species ROS, which is manifested as a shortened fluorescence lifetime; S13. Track inflammation-related cells: Use EGFP to mark neutrophils, and combine lifespan signals to analyze the recruitment and migration characteristics of inflammation-related cells in the inflammatory area.

5. The in vitro cell microenvironment dynamic monitoring method using two-photon frequency domain fluorescence lifetime imaging according to claim 1, characterized in that: In S2, a frequency domain fluorescence lifetime signal processing and analysis system is established under periodic excitation, and the steps of frequency domain fluorescence lifetime signal processing include: S211, signal preprocessing: the data acquisition card DAQ collects the synchronization signal, uses the laser to output the synchronization reference signal, suppresses the high-order harmonics by the low-pass filter LPF, and improves the signal-to-noise ratio by the low-noise amplifier LNA; S212, signal distribution and phase control: the pre-processed signal is converted into four homologous signals by a four-way power divider, and the phase is controlled by a phase shifter in the range of 0 to 2π; S213, signal amplification and mixing: the adjusted signal is amplified by LNA and input into the mixer, and mixed with the fluorescence signal from the photomultiplier tube PMT to form four orthogonal reference signals; S214, PMT signal processing and demodulation: The weak current signal output by the PMT is first converted into a voltage signal by a transimpedance amplifier, and then current separation and filtering are performed to remove noise and interference; the processed PMT signal is mixed and demodulated with four orthogonal reference signals to extract characteristic information related to the fluorescence lifetime; S215, signal analysis and calibration: Four-phase matrix operation is used to analyze fluorescence signals and calculate key parameters of fluorescence lifetime, phasor coordinates and intensity image. A full 50Ω impedance matching architecture is used in combination with electromagnetic shielding measures for impedance matching and electromagnetic shielding to optimize the signal-to-noise ratio. A dynamic calibration algorithm is used to correct channel deviations in real time to ensure accurate analysis of weak fluorescence signals.

6. The in vitro cell microenvironment dynamic monitoring method using two-photon frequency domain fluorescence lifetime imaging according to claim 1, characterized in that: In S2, the steps of analyzing the dynamic correlation characteristics of the phase delay φ of the fluorescence signal relative to the excitation light and the modulation depth M and quantitatively inferring the molecular lifetime parameters include: S221, analyzing the fluorescence signal using the frequency domain characteristics of the periodic excitation light, modeling the excitation function, calculating and obtaining the fluorescence signal, and analyzing the characteristics of the fluorescence signal; S222. Analyze frequency domain signal and hypothesized model: Decompose the periodic fluorescence signal F(t) into Fourier series and extract the frequency domain response, which is expressed as: In the formula, the Fourier series q k The integral is defined as S223, Phase delay and modulation depth measurement: The fluorescence signal captured by the photomultiplier tube PMT is subjected to signal separation processing by biasing the tee, and the original time domain signal F(t) is decomposed into DC and RF components, and the RF part contains lifetime information; the fundamental wave characteristics are extracted by RF mixing technology, and the fluorescence signal and reference signal are processed in parallel through a four-channel power distribution architecture; The specific process of signal separation processing of the fluorescence signal captured by the photomultiplier tube PMT is as follows: The DC component represents the time-integrated intensity of the fluorescence signal, which corresponds to the fundamental frequency coefficient q0 of the frequency domain Fourier series. The mathematical expression is: Where B is the DC conversion efficiency of the bias tee, O B is the inherent DC offset of the system, q0 is the zero-order Fourier coefficient of the fluorescence signal, representing the steady-state fluorescence intensity, V DC is the DC port component of the bias device; The fundamental frequency coefficient q of the frequency domain Fourier series corresponding to the RF component k , mathematically expressed as: or Where V RF is the bias tee RF port component S224, system calibration and parameter extraction: establish a mapping relationship between the phase shifter bias voltage and the phase offset, calibrate the mixer loss coefficient and gain coefficient, calculate the key parameter ratio M / B, generate four sets of orthogonal reference signals, and correct the channel gain difference; S225, phase analysis and quantitative life inversion: Analyze four sets of orthogonal phase shift signals and use matrix operations to eliminate DC offset O M The fluorescence lifetime is extracted through the proportional relationship between the imaginary and real parts of the first harmonic Fourier coefficient of the fluorescence; the average lifetime of the multi-exponential decay is calculated through the horizontal component g and the vertical component s of the phasor diagram.

7. The in vitro cell microenvironment dynamic monitoring method using two-photon frequency domain fluorescence lifetime imaging according to claim 6, characterized in that: S221 uses the frequency domain characteristics of periodic excitation light to analyze fluorescence signals, model excitation functions, calculate fluorescence signals, and analyze the characteristics of fluorescence signals. The specific steps are to model the time domain excitation function of the excited fluorescent molecules with a pulse width of less than 100fs using a femtosecond pulse sequence with a repetition frequency of 80MHz as a Dirac comb distribution, and the expression is: I(t)=Pδ T (t); Where P is a coefficient related to the average power of the laser beam, I(t) is the time distribution function of the excitation light, and δ T (t) is a Dirac comb function with a period of T, representing a periodic pulse train; In the two-photon excitation process, the fluorescence signal F(t) is proportional to the convolution of the square of the excitation light intensity and the impulse response function f(t) of the fluorophore. The fluorescence signal F(t) is expressed as: Where c is the concentration of the fluorophore, is the impulse response function of the single exponential decay fluorophore. Here, the instrument response function IRF is much lower than the lifetime value of the fluorophore, and IRF can be ignored.

8. The in vitro cell microenvironment dynamic monitoring method using two-photon frequency domain fluorescence lifetime imaging according to claim 6, characterized in that: In S222, it is assumed that the model is divided into a single-component model and a multi-component model, and the impulse response function f(t) of the single exponential decay fluorophore is: In the formula, τ represents the fluorescence lifetime, which indicates the average time for a molecule to return from an excited state to a ground state, reflecting the physical and chemical properties of its microenvironment; For multi-component fluorescent samples, the lifetime of each component is analyzed by measuring the phase difference and modulation ratio, and the impulse response function is expanded to: In the formula, a i is the life span τ i The intensity-weighted fractional contribution of the fluorophore, τ i is the different lifetimes of the multi-components, and the Fourier coefficient q of the multi-component fluorescence sample k for:

9. The in vitro cell microenvironment dynamic monitoring method using two-photon frequency domain fluorescence lifetime imaging according to claim 6, characterized in that: In S223, the specific steps of parallel transmission and processing of the fluorescence signal and the reference signal through the four-channel power distribution architecture are as follows: S2231, setting a preset phase offset of a voltage-controlled phase shifter in each independent channel, generating four-way orthogonal reference signals and inputting them into the mixer local oscillator port LO, performing four-channel synchronous demodulation to realize real-time phase resolution of the fluorescence signal, and the expression is: v LO (t, O)=sin(ωt+φ); Where, ν LO is the signal of the LO port. S2232, the four mixers respectively connect the PMT signal and the phase-shifted reference signal to their respective RF and LO ports, and obtain the inherent DC offset in the output signal. The inherent DC offset is O M The expression is: O M =O(P RF )+d; In the formula, O(P RF ) is a function of RF power, and d is the system inherent constant offset; S2233, correcting the mixer output, subjecting the mixer output signal to low-pass filtering to eliminate all high-order harmonic components, and converting the filtered signal into a pure DC voltage, wherein the corrected mixer output expression is: in IF (t,φ)=MV LO (t,φ)v RF (t)+O M ; Where M is the conversion loss coefficient of the mixer, IF is the intermediate frequency, and ν IF is the output function of the mixer; The mathematical expression of the pure DC voltage converted from the filtered signal is: S2234. The four-channel demodulation signals required for fluorescence lifetime calculation are synchronously sampled and digitally converted through a data acquisition card DAQ.

10. The in vitro cell microenvironment dynamic monitoring method using two-photon frequency domain fluorescence lifetime imaging according to claim 6, characterized in that: In S225, the calculation formula for the average life of multi-exponential decay is:

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