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

Abstract

The application relates to the field of biological imaging, and particularly discloses a two-photon frequency-domain fluorescence lifetime imaging in-vitro cell microenvironment dynamic monitoring method, which comprises the following steps: S1, two-photon frequency-domain fluorescence lifetime imaging (TP-FD-FLIM) technology is utilized, microenvironment changes are non-destructively characterized through fluorescence lifetime parameters, oxidative stress reaction and neutrophil migration behavior are monitored in real time, and compound effect analysis is combined with an in-vitro cell model; and S2, a frequency-domain fluorescence lifetime signal processing and analysis system based on the physical basis of FD-FLIM is established, dynamic correlation characteristics of phase delay phi and modulation depth M of a fluorescence signal relative to excitation light are analyzed, and quantitative back calculation of a molecular lifetime parameter is carried out. The two-photon frequency-domain fluorescence lifetime imaging in-vitro cell microenvironment dynamic monitoring method can non-destructively and in real time observe neuroinflammatory reaction on a subcellular scale.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of bio-imaging, in particular to a method for dynamic monitoring of in-vitro cell microenvironment by two-photon frequency-domain fluorescence lifetime imaging. BACKGROUND

[0002] Changes in cell microenvironment are important observation objects in biomedical research, and real-time monitoring of 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, fixed tissue imaging based on immunofluorescence cannot obtain dynamic information of inflammation in real time, and although electrophysiological recording can achieve high time resolution monitoring, it is difficult to provide inflammation characteristics at the cellular and molecular levels.

[0003] Fluorescence lifetime imaging (FLIM) is an imaging technique that reflects the characteristics of the microenvironment by measuring the lifetime changes of fluorescent molecules, which can avoid the problem that traditional fluorescence intensity imaging is easily affected by probe concentration and photobleaching. Two-photon fluorescence lifetime imaging (2P-FLIM) uses infrared femtosecond laser excitation to achieve deep imaging of living tissues, and combines frequency modulation technology to improve the efficiency of lifetime signal acquisition. 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.

[0004] Although two-photon fluorescence imaging (TP-FLIM) and fluorescence lifetime imaging (FLIM) techniques have been widely used in biomedical research, existing methods still have the following limitations in real-time monitoring of neuroinflammation: 1. Reliance on fluorescence intensity or fixed samples, which cannot analyze living cell microenvironment in real time and non-destructively; 2. Time-domain FLIM has low data acquisition efficiency and insufficient dynamic resolution. 3. Existing imaging systems have limited ability to analyze weak fluorescence signals.

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

[0006] Patent CN105891170A: In-vivo two-photon excitation delayed detection fluorescence imaging analysis method and device: This patent proposes an imaging method that uses red or near-infrared pulsed laser two-photon excitation to emit fluorescence from in-vivo fluorescent nanoprobes, and detects the fluorescence at the excited site in the body by a delayed detection method. The method aims to improve the imaging depth, reduce the interference of spontaneous fluorescence and background light, and thus improve the reliability of imaging analysis.

[0007] Patent CN104198458A Two-photon fluorescence biological microscopic imaging system and imaging method thereof: This patent introduces a method of using a tunable femtosecond laser source to perform high-resolution imaging of biological tissues through a two-photon fluorescence microscopic imaging system. The system uses specific optical elements and filters to achieve accurate imaging of biological samples.

[0008] Patent CN101254091A Method for realizing high spatial resolution retinal imaging by using second harmonic and two-photon excitation fluorescence: This patent combines second harmonic imaging, polarization-sensitive second harmonic imaging, and two-photon excitation fluorescence imaging to perform high spatial resolution imaging of the retina, thereby achieving 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 prior art solutions mainly focus on general biological tissue imaging or imaging of specific tissues (such as the retina), and have not been specifically targeted at dynamic monitoring of cell microenvironments. In addition, most of the existing two-photon fluorescence lifetime imaging patents use time-domain detection methods rather than frequency-domain detection methods. SUMMARY

[0010] The present application provides a two-photon frequency-domain fluorescence lifetime imaging method for in vitro dynamic monitoring of cell microenvironments, which can non-invasively and real-time observe neuroinflammatory responses at a subcellular scale, and has the advantage of improving the accurate quantitative analysis of fluorescence intensity imaging.

[0011] To achieve the above-mentioned purposes, the present application provides a two-photon frequency-domain fluorescence lifetime imaging method for in vitro dynamic monitoring of cell microenvironments, which includes the following steps:

[0012] S1, using two-photon frequency-domain fluorescence lifetime imaging (TP-FD-FLIM) technology, and non-invasively representing microenvironment changes through fluorescence lifetime parameters, real-time monitoring oxidative stress response and neutrophil migration behavior, and correlating compound effect analysis with in vitro cell models;

[0013] 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 φ and modulation depth M of the fluorescence signal relative to the excitation light, and quantitatively reversing the molecular lifetime parameters.

[0014] Preferably, the two-photon frequency-domain fluorescence lifetime imaging is performed by using a two-photon fluorescence microscope system, and the imaging process is as follows: a laser is generated by using a tunable femtosecond pulse laser, the power of the laser is regulated by a half-wave plate HWP and a polarization beam splitter PBS, the laser is collimated and expanded by a 4f system, and the laser is controlled to scan in XY directions by a two-dimensional galvanometer mirror; the excitation light is focused on a sample by a high numerical aperture objective lens; and two-photon fluorescence signals are detected by a photomultiplier tube PMT after filtering, so as to realize high spatiotemporal resolution fluorescence lifetime imaging.

[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 microenvironment change is nondestructively characterized by the fluorescence lifetime parameter, the oxidative stress reaction and the neutrophil migration behavior are monitored in real time, and the specific steps of correlating compound effect analysis by combining an in-vitro cell model are as follows:

[0017] S11, monitoring the metabolic state by the fluorescence lifetime: measuring the lifetime change of an endogenous fluorescent molecule, calculating the ratio of NAD / NADH indirectly reflected by a metabolic index, and deducing the redox state and metabolic level of a cell; +

[0018] S12, obtaining the fluorescence lifetime signal of oxidative stress: obtaining the accumulation degree of reactive oxygen species ROS, and the accumulation degree of the reactive oxygen species ROS is represented by a shortened fluorescence lifetime;

[0019] S13, performing inflammation-related cell tracking: marking neutrophils by EGFP, and analyzing the recruitment and migration characteristics of inflammation-related cells in an inflammation area in combination with the lifetime signal.

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

[0021] S211, signal preprocessing: a data acquisition card DAQ collects a synchronous signal, a laser output synchronous reference signal is used, high-order harmonics are suppressed by a low-pass filter LPF, and a low-noise amplifier LNA is used to improve the signal-to-noise ratio;

[0022] S212, signal distribution and phase regulation: four homologous signals are generated from the preprocessed signal by a four-way power distributor, and the phase regulation of 0-2π is performed by a phase shifter Phase Shifter;

[0023] S213, signal amplification and mixing: the adjusted signal is amplified by an LNA and then input into a mixer Mixer, and the fluorescence signal from a photomultiplier tube PMT is mixed to form four quadrature 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 processing is performed to remove noise and interference; the processed PMT signal is mixed with four-way quadrature reference signals for demodulation to extract characteristic information related to the fluorescence lifetime;

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

[0026] Preferably, in S2, the step of quantitatively deducing the molecular lifetime parameter by analyzing the phase delay φ and the modulation depth M of the fluorescence signal relative to the excitation light includes:

[0027] S221, using the frequency domain characteristics of the periodic excitation light to analyze the fluorescence signal, modeling the excitation function, calculating the fluorescence signal and analyzing the fluorescence signal characteristics;

[0028] S222, analyzing the frequency domain signal and the assumed model: the periodic fluorescence signal F(t) is decomposed into a Fourier series, and the frequency domain response is extracted, which is expressed as:

[0029]

[0030] In the formula, the Fourier series q k defined by integration

[0031] S223, phase delay and modulation depth measurement: the fluorescence signal captured by the photomultiplier tube PMT is processed by a three-way bias, the original time domain signal F(t) is decomposed into a direct current DC and a radio frequency RF component, and the RF part contains lifetime information; the fundamental characteristics are extracted by radio frequency mixing technology, and the fluorescence signal and the reference signal are processed in parallel by a four-channel power distribution architecture;

[0032] The specific processing process of the three-way bias signal separation processing of the fluorescence signal captured by the photomultiplier tube PMT is as follows:

[0033] The direct current component represents the time integral intensity of the fluorescence signal, and corresponds to the fundamental frequency coefficient q0 of the frequency domain Fourier series, which is mathematically expressed as:

[0034]

[0035] In the formula, B is the direct current conversion efficiency of the three-way bias, and O BV0 is the system inherent DC offset, q0 is the zeroth Fourier coefficient of the fluorescence signal, representing the steady-state fluorescence intensity, V DC is the biaser DC port component;

[0036] RF component corresponds to the fundamental frequency coefficient q of the frequency domain Fourier series k , which is mathematically expressed as:

[0037]

[0038] or

[0039] In the formula, V RF is the biaser RF port component;

[0040] S224, system calibration and parameter extraction: establish the mapping relationship between the phase shifter bias voltage and the phase shift, calibrate the mixer loss coefficient and the gain coefficient, calculate the key parameter ratio M / B, generate four sets of quadrature reference signals, and correct the channel gain difference;

[0041] S225, phasor analysis and life quantitative backstepping: analyze the four sets of quadrature phase shift signals, eliminate the DC offset O M by matrix operation, and extract the fluorescence lifetime by the proportional relationship between the imaginary part and the real part of the first harmonic Fourier coefficient of the fluorescence; the average lifetime of multi-exponential decay is calculated by the horizontal component g and the vertical component s of the phasor diagram.

[0042] Preferably, S221 utilizes the frequency domain characteristics of the periodic excitation light to analyze the fluorescence signal, and the excitation function is modeled to calculate and obtain the fluorescence signal and analyze the specific steps of the fluorescence signal characteristics. The time-domain excitation function of the femtosecond pulse sequence with a repetition frequency of 80MHz and a pulse width of less than 100fs is modeled as a Dirac comb distribution, and the expression is:

[0043] I(t)=Pδ T (t);

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

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

[0046]

[0047] In the formula, c is the concentration of the fluorophore, For the single-exponential decay fluorophore, the pulse response function f(t) is:

[0048] For the single-exponential decay fluorophore, the pulse response function f(t) is:

[0049]

[0050] where τ represents the fluorescence lifetime, the average time for a molecule to return from an excited state to a ground state, reflecting the physicochemical properties of its microenvironment.

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

[0052]

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

[0054] 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 a voltage-controlled phase shifter in each independent channel to generate a four-way quadrature reference signal input to the local oscillator port LO of the mixer, and perform four-channel synchronous demodulation to realize real-time phase analysis of the fluorescence signal, which is expressed as:

[0056] v LO (t, O) = sin(ωt + φ);

[0057] where v LO is the signal of the LO port.

[0058] S2232, four mixers respectively input the PMT signal and the phase-shifted reference signal to the RF and LO ports, and obtain the inherent DC offset O M , which is expressed as:

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

[0060] where O(P RF ) is a function of RF power, and d is a system constant offset.

[0061] S2233. Correct the mixer output by passing the mixer output signal through a low-pass filter to eliminate all higher harmonic components and converting the filtered signal into a pure DC voltage. The expression for the corrected mixer output is as follows:

[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, and ν IF This is the output function of the mixer;

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

[0065]

[0066] S2234. The four demodulated signals required for fluorescence lifetime calculation are synchronously sampled and digitized by the data acquisition card DAQ.

[0067] Preferably, in S225, the formula for calculating the average lifetime of multi-exponential decay is as follows:

[0068] Therefore, this invention proposes a method for dynamic monitoring of the in vitro cellular microenvironment using two-photon frequency-domain fluorescence lifetime imaging, with the following beneficial effects:

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

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

[0071] (3) This invention constructs a quantitative assessment model to analyze the oxidative stress and metabolic characteristics of the cellular microenvironment.

[0072] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0073] Figure 1 This is the overall design flowchart of the in vitro cell microenvironment dynamic monitoring method for two-photon frequency domain fluorescence lifetime imaging according to the present invention;

[0074] Figure 2 is the imaging process diagram of the in-vitro cell microenvironment dynamic monitoring method of the two-photon frequency domain fluorescence lifetime imaging of the application;

[0075] Figure 3 is the frequency domain signal processing flowchart of the in-vitro cell microenvironment dynamic monitoring method of the two-photon frequency domain fluorescence lifetime imaging of the application;

[0076] Figure 4 is the zebrafish neurogenic inflammation model detection principle diagram of the in-vitro cell microenvironment dynamic monitoring method of the two-photon frequency domain fluorescence lifetime imaging of the application. DETAILED DESCRIPTION

[0077] In order to make the technical solutions, advantages and purposes of the application clearer, the technical solutions of the embodiments of the application will be clearly and completely described below. The described embodiments are part of the embodiments of the application, rather than all the embodiments. Based on the described embodiments of the application, all other embodiments obtained by those skilled in the art without creative labor belong to the protection scope of the present application.

[0078] Unless otherwise defined, the technical terms or scientific terms used in the application shall have the usual meanings understood by those skilled in the art to which the application belongs.

[0079] As shown in Figures 1-3 The application proposes an in-vitro cell microenvironment dynamic monitoring method of two-photon frequency domain fluorescence lifetime imaging, which comprises the following steps:

[0080] S1, using two-photon frequency domain fluorescence lifetime imaging (TP-FD-FLIM) technology, and non-destructively characterizing microenvironment changes through fluorescence lifetime parameters, real-time monitoring oxidative stress response and neutrophil migration behavior, and combining in-vitro cell model correlation compound effect analysis, the specific steps are as follows:

[0081] S11, monitoring fluorescence lifetime to characterize metabolic state: measuring the lifetime change of endogenous fluorescent molecules, calculating the ratio of NAD + / NADH indirectly reflected by metabolic indicators, deducing the redox state and metabolic level of cells;

[0082] S12, obtaining fluorescence lifetime signal of oxidative stress: obtaining the accumulation degree of reactive oxygen species (ROS), and the accumulation degree of reactive oxygen species (ROS) is manifested as the shortening of fluorescence lifetime;

[0083] S13, performing inflammation-related cell tracking: marking neutrophils through EGFP, and combining lifetime signal to analyze the recruitment and migration characteristics of inflammation-related cells in the inflammation area.

[0084] S2, the physical basis of the FD-FLIM is established on a frequency domain fluorescence lifetime signal processing and analysis system under periodic excitation, and the phase delay φ and the modulation depth M of the fluorescence signal relative to the excitation light are dynamically correlated, and the quantitative inverse of the molecular lifetime parameter is calculated.

[0085] In the formula, the frequency domain fluorescence lifetime signal processing and analysis system is established under periodic excitation, and the steps of the frequency domain fluorescence lifetime signal processing include:

[0086] S211, signal preprocessing: the data acquisition card DAQ collects the synchronization signal, adopts the laser output synchronization reference signal, suppresses the high-order harmonic after low-pass filtering LPF, and improves the signal-to-noise ratio by low-noise amplifier LNA;

[0087] S212, signal distribution and phase control: the preprocessed signal is generated by four-way power distributor to generate four-way homologous signals, and the phase is adjusted by 0-2π through phase shifter Phase Shifter;

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

[0089] S214, PMT signal processing and demodulation: the weak current signal output by PMT is first converted into voltage signal through transimpedance amplifier, and current separation and filtering processing are performed to remove noise and interference; the processed PMT signal is mixed with the four-way quadrature reference signal for demodulation, and the characteristic information related to the fluorescence lifetime is extracted;

[0090] S215, signal analysis and calibration: four-phase matrix operation is used to analyze the fluorescence signal, and the key parameters of the fluorescence lifetime, the phasor coordinate and the intensity image are calculated; 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; dynamic calibration algorithm is used to correct the channel deviation in real time, and the accurate analysis of the weak fluorescence signal is ensured.

[0091] In S2, the steps of analyzing the phase delay φ and the modulation depth M of the fluorescence signal relative to the excitation light and the quantitative inverse of the molecular lifetime parameter include:

[0092] S221, the frequency domain characteristics of the periodic excitation light are used to analyze the fluorescence signal, the excitation function is modeled, the fluorescence signal is calculated and analyzed, and the specific steps are as follows: the time-domain excitation function of the femtosecond pulse sequence excitation fluorescence molecule with a pulse width less than 100 fs is modeled as Dirac comb distribution, and the expression is:

[0093] I(t)=Pδ T (t).

[0094] 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, δ T (t) is a Dirac comb function with period T, representing a periodic pulse sequence;

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

[0096]

[0097] where c is the concentration of the fluorophore, is the pulse response function of a single-exponential decay fluorophore, and the instrument response function IRF is much lower than the lifetime value of the fluorophore, so IRF is ignored.

[0098] S222, analyze the frequency domain signal and the assumed model: the periodic fluorescence signal F(t) is decomposed into a Fourier series, and the frequency domain response is extracted, which is expressed as:

[0099]

[0100] where the Fourier series q k is defined by integration

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

[0102]

[0103] where τ represents the fluorescence lifetime, representing the average time of the molecule returning from the excited state to the ground state, reflecting the physicochemical properties of its microenvironment;

[0104] For a multi-component fluorescent sample, the lifetime of each component is analyzed by measuring the phase difference and the modulation ratio, and the pulse response function is expanded as:

[0105]

[0106] where a i is the intensity-weighted fractional contribution of the fluorophore with a lifetime τ i , τ i is the different lifetime in the multi-component, and the Fourier coefficient qk of the multi-component fluorescent sample is:

[0107] S223, phase delay and modulation depth measurement: the signal separation processing is performed on the fluorescence signal captured by the photomultiplier PMT biased three-way, the original time domain signal F(t) is decomposed into direct current DC and radio frequency RF components, the RF part contains lifetime information; the radio frequency mixing technology is used to extract the fundamental characteristic, 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 of the fluorescence signal captured by the photomultiplier PMT biased three-way is as follows:

[0109] S224, system calibration and parameter extraction: the mapping relationship between the phase shifter bias voltage and the phase shift is established, the mixer loss coefficient and the gain coefficient are calibrated, the key parameter ratio M / B is calculated, four sets of orthogonal reference signals are generated, and the channel gain difference is corrected;

[0110] S225, phasor analysis and lifetime quantitative backstepping: analyzing four sets of orthogonal phase shift signals, eliminating direct current offset O M by matrix operation, and extracting the fluorescence lifetime by the proportional relationship between the imaginary part and the real part of the first harmonic Fourier coefficient of the fluorescence; the average lifetime of multi-exponential decay is calculated by the horizontal component g and the vertical component s of the phasor diagram.

[0111] The direct current component represents 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:

[0112]

[0113] In the formula, B is the direct current conversion efficiency of the biased three-way, O B is the system inherent direct current offset, q0 is the zero-order Fourier coefficient of the fluorescence signal, representing the steady-state fluorescence intensity, V DC is the function of the direct current port of the mixer;

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

[0115]

[0116] Or

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

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

[0119] S2231, preset phase offset in each independent channel, generate four-way quadrature reference signal input mixer local oscillator port LO, four-channel synchronous demodulation to realize real-time phase analysis of fluorescence signal, its expression is:

[0120] v LO (t, O) = sin (ωt + φ);

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

[0122] Wherein, 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 access the RF and LO ports of each reference signal with the phase-shifted reference signal, 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 RF power, and d is a system constant offset;

[0126] S2233, the mixer output is corrected, the mixer output signal is processed by low-pass filter to eliminate all high harmonic components, and the filtered signal is converted to pure DC voltage, wherein the expression of the corrected 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 filtered signal converted to pure DC voltage is:

[0130]

[0131] S2234, the four demodulation signals required for fluorescence lifetime calculation are sampled and digitized by the data acquisition card DAQ.

[0132] The application is established on a frequency domain fluorescence lifetime signal processing and analysis system under periodic excitation, which comprises a four-phase quadrature mixer, a phase shift compensation module and a phasor analysis algorithm.

[0133] In the application, a two-photon fluorescence microscope system is used for two-photon frequency domain fluorescence lifetime imaging, and the imaging process is to use a tunable femtosecond pulse laser to generate laser with a wavelength range of 680-1300 nm, a pulse width of 100 fs and a repetition frequency of 80 MHz, but 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, the laser is collimated and expanded by a 4f system, and the XY scanning is controlled by a two-dimensional galvanometer mirror, the excitation light is focused to the sample by a high numerical aperture objective, and the two-photon fluorescence signal is detected by a photomultiplier tube PMT after filtering, so as to realize high spatiotemporal resolution fluorescence lifetime imaging.

[0134] As shown in Figure 4 , the application provides an application of a two-photon frequency domain fluorescence lifetime imaging in vitro cell microenvironment dynamic monitoring method in zebrafish neurogenic inflammation model detection, and the specific processing process is as follows:

[0135] 1. Constructing a zebrafish neurogenic inflammation model

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

[0137] Copper sulfate induced neurogenic inflammation: at 4dpf, the zebrafish larvae are incubated in different concentrations (5-50 μM) of CuSO4 (dissolved in E3 culture medium) solution for 2 hours to induce oxidative stress and sterile inflammation. The pre-experiment determines 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. The necrotic apoptosis of lateral line hair cells is observed to confirm the successful establishment of the inflammation model, and the fluorescence lifetime change is used as a quantitative indicator of the degree of inflammation.

[0138] Anesthesia and immobilization: Zebrafish embryos were immersed in a 60 mM Tricaine solution (Sigma, #E10521) for 1-2 minutes to suppress motor activity and ensure imaging stability. After anesthesia, the embryos were rinsed three times with fresh E3 medium to remove residual anesthetics. A 1.5% low-melting agarose gel pre-warmed to 37°C was used to gently wrap and immobilize the anesthetized embryos on the bottom of the imaging dish, ensuring unobstructed respiration and stable positioning for subsequent imaging.

[0139] 2. TP-FD-FLIM system imaging

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

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

[0142] Data acquisition and processing: The fluorescence lifetime and fluorescence intensity data of EGFP-labeled neutrophils were continuously collected using TP-FD-FLIM technology to monitor cell migration trajectories. Changes in fluorescence lifetime were analyzed to non-invasively and quantitatively assess ROS accumulation and inflammation severity. The migration dynamics of neutrophils at the inflammation site were recorded, and the CuSO4-induced inflammation characteristics were confirmed by combining the fluorescence lifetime change curve.

[0143] 3. Ibuprofen anti-inflammatory experiment

[0144] After CuSO4-induced inflammation, zebrafish embryos were transferred to E3 culture solution containing Ibuprofen for 2 hours to observe the anti-inflammatory effect. The TP-FD-FLIM system monitored the effects of Ibuprofen on inflammation signals, including reduced ROS accumulation, prolonged fluorescence lifetime, and reduced neutrophil recruitment. Through quantitative analysis of fluorescence lifetime, it was verified that Ibuprofen reduced the accumulation of PGE-2 by inhibiting COX-2 activity, thereby reducing inflammation factors TNF-α and IL-1 and promoting cell repair.

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

[0146] Therefore, the application provides an in-vitro cell microenvironment dynamic monitoring method of two-photon frequency domain fluorescence lifetime imaging, adopts the TP-FD-FLIM technology, and first realizes non-invasive dynamic monitoring of an in-vitro cell microenvironment model, and breaks through the invasive method of the traditional dependence on inflammatory factor detection. The application adopts the FD-FLIM frequency domain fluorescence lifetime imaging technology, breaks through the limitation of the traditional TCSPC time domain detection, improves the imaging speed and the signal-to-noise ratio, has significant advantages in the aspects of neural inflammation monitoring, imaging quality and signal processing optimization, and provides a quantitative optical evaluation means for biological active substance screening and cell response research.

[0147] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application rather than limit them, although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that: the technical solutions of the present application can still be modified or replaced by equivalents, 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 application.

Claims

1. An in vitro cell microenvironment dynamic monitoring method of two-photon frequency domain fluorescence lifetime imaging, characterized in that, Comprise the following steps: S1, using two-photon frequency domain fluorescence lifetime imaging TP-FD-FLIM technology, and through the fluorescence lifetime parameter nondestructive characterization of microenvironment changes, real-time monitoring of oxidative stress and neutrophil migration behavior, combined with in vitro cell model correlation compound effect analysis; S2, the physical basis of FD-FLIM is established on the frequency domain fluorescence lifetime signal processing and analysis system under periodic excitation, and the phase delay of fluorescence signal relative to excitation light is analyzed Dynamic correlation with modulation depth M characteristics, quantitative inverse of molecular lifetime parameters; In S2, the phase delay of the fluorescence signal relative to the excitation light is analyzed The step of quantitatively inferring the molecular lifetime parameter in dynamic correlation with the modulation depth M comprises: S221, using the frequency domain characteristics of periodic excitation light to analyze the fluorescence signal, excitation function modeling, calculation of fluorescence signal and analysis of fluorescence signal characteristics; S222, analysis of frequency domain signal and hypothetical model: the periodic fluorescence signal F(t) is decomposed into Fourier series, and the frequency domain response is extracted, and its expression is: ； where the Fourier series q k is defined by the integral , k is the harmonic number (k = 0, ±1, ±2,...), is the complex exponential function, is the fundamental angular frequency; S223, phase delay and modulation depth measurement: the fluorescence signal captured by the photomultiplier PMT is biased three-way signal separation processing, the original time domain signal F(t) is decomposed into DC and RF components, and the RF part contains lifetime information; using RF mixing technology to extract the fundamental characteristics, and through the four-channel power distribution architecture parallel transmission processing fluorescence signal and reference signal; The specific processing process of the fluorescence signal captured by the photomultiplier PMT biased three-way signal separation processing is: The DC component represents 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: ； In the formula, B is the direct current conversion efficiency of the bias tee, O B is the system inherent direct current offset, q0 is the zero-order Fourier coefficient of the fluorescent signal, representing the steady-state fluorescent intensity, V DC is the bias tee direct current port component, T is the acquisition time period of the fluorescent signal, P is a coefficient related to the average power of the excitation laser, is the concentration of the fluorescent dye; A radio frequency (RF) component corresponds to a base frequency coefficient of a frequency domain Fourier series , which is mathematically expressed as: ； ； where V RF is the biaser RF port component, is the harmonic number, (k = 0, ±1, ±2,...), is the complex exponential function, is the fundamental angular frequency; S224, system calibration and parameter extraction: establish the mapping relationship between the bias voltage of the phase shifter and the phase shift, calibrate the loss coefficient and gain coefficient of the mixer, calculate the key parameter ratio M / B, generate four sets of orthogonal reference signals, and correct the channel gain difference; S225、Phase analysis and life quantitative backstepping: analyze four groups of orthogonal phase shift signals, and eliminate direct current offset O by matrix operation M And the imaginary part and the real part of the first harmonic Fourier coefficient of the fluorescence are proportional to the fluorescence lifetime; the average lifetime of multi-exponential decay is calculated by the horizontal component g and the vertical component s of the phase diagram.

2. The in-vitro cell microenvironment dynamic monitoring method of two-photon frequency-domain fluorescence lifetime imaging according to claim 1, characterized in that, A two-photon fluorescence microscope system is used for two-photon frequency domain fluorescence lifetime imaging, and the imaging process is to use a tunable femtosecond pulse laser to generate laser, which is controlled by a half-wave plate HWP and a polarization beam splitter PBS after power regulation, collimated and expanded by a 4f system, and scanned in XY by a two-dimensional galvanometer mirror. The excitation light is focused to the sample by a high numerical aperture objective lens, and the two-photon fluorescence signal is detected by a photomultiplier PMT after filtering, so as to realize high spatiotemporal resolution fluorescence lifetime imaging.

3. The in-vitro cell microenvironment dynamic monitoring method of 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 quadrature mixer, a phase shift compensation module and a phasor analysis algorithm.

4. The in-vitro cell microenvironment dynamic monitoring method of two-photon frequency-domain fluorescence lifetime imaging according to claim 1, characterized in that, In S1, the fluorescence lifetime parameter is used to nondestructively characterize the microenvironment changes, to real-time monitor the oxidative stress and neutrophil migration behavior, and to combine the in vitro cell model correlation compound effect analysis. The specific steps are: S11, monitoring fluorescence lifetime to characterize metabolic state: measuring the lifetime change of endogenous fluorescent molecules, calculating the ratio of metabolic indicators indirectly reflected by NAD + / NADH, deducing the redox state and metabolic level of cells; S12, obtain the fluorescence lifetime signal of oxidative stress: obtain the accumulation degree of reactive oxygen species ROS, and the accumulation degree of reactive oxygen species ROS is manifested as the shortening of fluorescence lifetime; S13, perform inflammation-related cell tracking: mark neutrophils by EGFP, and analyze the recruitment and migration characteristics of inflammation-related cells in the inflammation area by combining the lifetime signal.

5. The in-vitro cell microenvironment dynamic monitoring method of two-photon frequency-domain fluorescence lifetime imaging according to claim 1, characterized in that, In S2, the frequency domain fluorescence lifetime signal processing and analysis system based on periodic excitation is established, and the steps of frequency domain fluorescence lifetime signal processing include: S211, signal preprocessing: the data acquisition card DAQ collects the synchronization signal, adopts the laser output synchronization reference signal, suppresses the high-order harmonic after low-pass filtering LPF, and improves the signal-to-noise ratio by low-noise amplifier LNA; S212, signal distribution and phase control: the preprocessed signal is generated by a four-way power distributor to generate four homologous signals, and a phase shifter is used for phase control of 0~2π; S213, signal amplification and mixing: the adjusted signal is amplified by LNA and then input into the mixer to mix with the fluorescent signal from the photomultiplier tube (PMT) to form four-way 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 current separation and filtering processing are performed to remove noise and interference; the processed PMT signal is mixed with the four-way orthogonal reference signal for demodulation to extract the characteristic information related to the fluorescence lifetime; S215, signal analysis and calibration: four-phase matrix operation is used to analyze the fluorescent signal, and the key parameters of the fluorescence lifetime, phasor coordinates and intensity image are calculated; 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; dynamic calibration algorithm is used to correct the channel deviation in real time to ensure accurate analysis of the weak fluorescent signal.

6. The in-vitro cell microenvironment dynamic monitoring method of two-photon frequency-domain fluorescence lifetime imaging according to claim 1, characterized in that, S221, the frequency domain characteristics of the periodic excitation light are used to analyze the fluorescent signal, and the excitation function model is built to calculate and analyze the specific steps of the fluorescent signal characteristics. The time-domain excitation function of the femtosecond pulse sequence with a repetition frequency of 80MHz and a pulse width of less than 100fs is modeled as a Dirac comb distribution, and the expression is: ； 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, T (t) is a Dirac comb function with period T, representing a periodic sequence of pulses; In the two-photon excitation process, the fluorescent signal F(t) is proportional to the convolution of the excitation light intensity square and the pulse response function f(t) of the fluorophore, and the fluorescent signal F(t) is expressed as: ； where c is the concentration of the fluorophore, is the impulse response function of the single-exponential decaying fluorophore, where the instrument response function, IRF, is much lower than the lifetime value of the fluorophore, and IRF is negligible.

7. The in-vitro cell microenvironment dynamic monitoring method of two-photon frequency-domain fluorescence lifetime imaging according to claim 1, characterized in that, In S222, the model is divided into single-component model and multi-component model, and the pulse response function f(t) of the single exponential decay fluorophore is: ； In the formula, denotes the fluorescence lifetime, the average time for a molecule to return from an excited state to the ground state, reflecting the physicochemical properties of its microenvironment; For multi-component fluorescent samples, the phase difference and modulation ratio are measured to analyze the lifetime of each component, and the pulse response function is extended as: ； wherein is the intensity weighted fractional contribution of the lifetime of the fluorophore, is the different lifetimes in the multi-component.

8. The in-vitro cell microenvironment dynamic monitoring method of two-photon frequency-domain fluorescence lifetime imaging according to claim 1, characterized in that, In S223, the specific steps of parallel transmission and processing of fluorescent signal and reference signal through four-channel power distribution architecture are as follows: S2231, a voltage-controlled phase shifter is set in each independent channel to generate a four-way orthogonal reference signal input into the local oscillator port (LO) of the mixer for four-channel synchronous demodulation to realize real-time phase analysis of the fluorescent signal, and the expression is: ； In the formula, is a signal of the LO port; S2232、The four mixers respectively input the PMT signal and the phase-shifted reference signal into the RF and LO ports, and obtain the inherent DC offset O in the output signal. M The expression is: ； where O(P RF ) is a function of the radio frequency power and d is a constant offset inherent to the system. S2233, the mixer output is corrected, and the mixer output signal is processed by low-pass filtering to eliminate all high harmonic components, and the filtered signal is converted into a pure direct current voltage, wherein the expression of the corrected mixer output is: ； where M is the conversion loss coefficient of the mixer, IF is the intermediate frequency, is the output function of the mixer; The mathematical expression of the filtered signal converted into a pure direct current voltage is: ； S2234, the four-way demodulation signals required for fluorescence lifetime calculation are synchronously sampled and digitized by a data acquisition card (DAQ).

9. The in-vitro cell microenvironment dynamic monitoring method of two-photon frequency-domain fluorescence lifetime imaging according to claim 1, characterized in that, In S225, the calculation formula of the average lifetime of the polyexponential decay is .

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