Implementation and application of FRET–FLIM technology

With the development of the new detection methods and the function of °uorescent molecule, researchers hope to further explore the internal mechanisms of organisms, monitor changes in the intracellular microenvironment, and dynamic processes of molecular interactions in cells. Fluorescence resonance energy transfer (FRET) describes the energy transfer process between donor °uorescent molecules and acceptor °uorescent molecules. It is an important means to detect protein–protein interactions and protein conformation changes in cells. Fluorescence lifetime imaging microscopy (FLIM) enables noninvasive measurement of the °uorescence lifetime of °uorescent particles in vivo. The FRET–FLIM technology, which is use FLIM to quantify and analyze FRET, enables real-time monitoring of dynamic changes of proteins in biological cells and analysis of protein interaction mechanisms. The distance between donor and acceptor and their respective °uorescent lifetime, which are of great importance for studying the mechanism of intracellular activity can be obtained by data analysis and algorithm ̄tting.


Introduction
With the continuous developments in optical technologies, particularly the introduction of°uorescence microscopy, biological images can be obtained at the cellular and subcellular levels. This raises concerns about changes in cells and their internal environments. Researchers are beginning to focus on exploring the dynamic processes of proteins, DNA and RNA.
Fluorescence resonance energy transfer (FRET) is a process via which excited donor°uorescent molecules excite nearby acceptor°uorescent molecules through energy transfer. There are some requirements for FRET:¯rst, the distance between donor and acceptor should be less than 10 nm; second, the emission spectrum of donor overlaps with the excitation spectrum of acceptor; third, there is no overlap between the emission spectrum of donor and acceptor; fourth, there is no overlap between the excitation spectrum of donor and acceptor;¯fth, the dipole of donor and acceptor should have the same direction. Moreover, FRET is also a®ected by the nature of the proteins. It is only possible when the distance between the donor and acceptor molecules is less than 10 nm; therefore, FRET can be used to study protein-protein interactions. Yeast two-hybrid system, Western blot and Co-Immunoprecipitation are traditional means in detecting the interaction between biomoleculars. However, these methods destroy the natural state of cells, fail to conduct protein subcellular localization and re°ect the spatio-temporal dynamic information of protein-protein interactions under the physiological conditions of living cells. Hence, FRET is unique and indispensable to measure small distances between biomolecules. There are two conventional FRET measurement technologies:°uorescence intensitybased method (FIBM) and°uorescence lifetimebased method (FLBM). FIBM is the most straightforward; however, it is strongly dependent on the concentration of the sample and requires calibration for reliable data analysis. In contrast, FLBM provides absolute light measurements. Fluorescence-lifetime imaging microscopy (FLIM) belongs to the method of FIBM. FLIM can detect the lifetime of°uorescent molecules in a single cell. Besides,°uorescence lifetime imaging of°uorescent proteins is an e®ective quantitative tool for noninvasive study of intracellular processes. The proportion of donor-receptor interaction can be determined by using FLIM to quantify FRET, which is important for biologically relevant research.
FRET-FLIM technology uses the°uorescence lifetime change of a sample to detect the dynamic process of the reaction between the donor and ac-ceptor°uorescent molecules. It can obtain lots of information, such as intracellular protein-protein interactions and conformational changes to proteins in the cells. [1][2][3][4][5][6] In the past three or four decades, FRET-FLIM technology has been used to evaluate various intracellular processes such as analysis of kinase and phosphatase activity, monitoring of messenger RNA kinetics, and assessment of metabolic processes. [7][8][9] Nowadays, researchers gradually realized that FRET-FLIM technology can solve more biophysical and biochemical issues. Using FRET-FLIM technology, Ahmed et al. 10 observed direct interaction between S6K1 and raptor; Venditti et al. 11 searched for molecular determinants of the ER-TGN contact sites (ERTGoCS) and found that both structural tethers and a proper lipid composition are needed for ERTGoCS integrity; Dikovskaya et al. 12 improved the FLIM/FRET methodology to visualize and measure the interaction between Nrf2 and Keap1 in single cells.
Here, we have introduced the principle and implementation of FRET-FLIM technology and then focused on its applications in the biological eld relative to four primary aspects: the internal mechanism of cellular action, causes of diseases, evaluation of drug treatment e®ects, and biosensors. This paper provides common means of information collection and data processing. The speci¯c information is shown in Tables 1 and 2.

FRET-FLIM Principle, Detection
Method and Data Processing 2.1. FRET-FLIM principle As indicated in Fig. 1(a), the electrons of a°uorescent molecule absorb energy, transitioning from the ground state to a high energy level of the excited state. After vibrational relaxation, the molecule falls to a low energy level of the excited state. These electrons then return to the ground state from the excited state while releasing photons that generate°u orescence. When FRET occurs, energy is transferred from the donor to the acceptor molecule. Then, the°uorescence of the donor becomes gradually weakened, and the acceptor°uorescent. The°u orescence lifetime of the sample gradually changes with the occurrence of FRET. As indicated in Fig. 1(b), FRET pairs can link in either a singlestranded or double-stranded form. In the singlestranded form, the FRET pairs link the same molecule, and FRET occurs when changes in the molecular structure alter the distance between the donor and acceptor molecules. In the doublestranded form, two independent targets are labeled with di®erent°uorescent molecules, and FRET occurs when the two objects interact and the distance between the°uorescent molecules is reduced su±ciently. 13-18

Detection method
Similar to the simple FLIM information acquisition technology, both the frequency and timedomain methods can be used for FRET-FLIM.
The frequency-domain method uses a continuous light source with sinusoidal (Cosine) modulation to excite the sample. This method obtains°uorescence lifetime information from the delayed phase and the decreasing modulation amplitude of light. In contrast, the time-domain method uses highly repetitive pulsed laser excitation. The°u orescence lifetime information is obtained from the attenuation curve of the sample's°uorescence intensity. The frequency and time domains can be converted using Fourier transform; therefore, in principle, both the frequency-and time-domain methods yield the same information.
In the frequency-domain method, a high-frequency (typically 10-100 MHz) laser with sinusoidal (Cosine) modulation or a xenon lamp is used as the light source to excite the sample. Fluorescence is then emitted at the same frequency as the excitation source but with a phase lag and reduced modulation signal. A charge-coupled device or photomultiplier tube (PMT) with the same frequency modulation then is used as a detector to receive and demodulate the°uorescence signal, and the°uorescence lifetime information of the sample is obtained based on the phase di®erence and modulation coe±cient, 19 as indicated in Fig. 1(c).
Using Wide¯eld frequency-domain°uorescence lifetime imaging microscopy (FD-FLIM), Chen et al. 20 tracked the temperature change of living cells via the°uorescence lifetime of Rhodamine B at di®erent frequencies. Wu et al. 21 construct a wideeld frequency domain°uorescence imaging microscopy (FD-FLIM) system to accurately measure oxygen gradient pro¯les in a micro°uidic device.
The time-domain method uses an ultrashort pulse laser to excite the sample. This method measures the°uorescence intensity attenuation after the sample is excited. As the probability of detecting a photon emitted within a certain time period t' and the°uorescence intensity is proportional to time, it is possible to measure the°uorescence lifetime by acquiring the°uorescence intensity attenuation information.
The principle of Time correlated single-photon counting (TCSPC) is presented in Fig. 1(d); a highsensitivity PMT, an avalanche photodiode (APD), or a microchannel plate detector (MCP) has been used as the detector. Each pulse laser excitation is considered a single cycle. Only one°uorescence emission photon can be collected in a single cycle. The time at which the photon appears is recorded. After the accumulation of photons in di®erent cycles, the distribution frequency curve (or the°u orescence lifetime curve) is constructed. Long acquisition time is the most signi¯cant drawback of TCSPC. Becker developed a fast-acquisition°uorescence lifetime microscopy system, as shown in Fig. 1(e). The instrument response function has a width of less than 7 ps and a time channel width as low as 820 fs with nearly no stacking e®ect. TCSPC has a high time resolution, sensitivity, and e±ciency. It can realize multidimensional detection and obtain a better signal-to-noise ratio (SNR) of the°u orescence lifetime. 22,23

Data processing
In FRET-FLIM technology, it is important to get more information from the data. Currently, curvē tting and phasor plot are most commonly used for this purpose. 24 In FRET-FLIM-acquired images measured using TCSPC, each pixel point contains position information and°uorescence decay data. The°uorescence decay curve is typically¯tted by exponential model to estimate its corresponding°uorescence lifetime. This method is performed to optimize the goodness-of-¯t by changing the values of the model parameters. The equation is given as follows: Here, IðtÞ is the°uorescence intensity at time t; n ; n is the°uorescence lifetime and contribution of each component, and C represents other e®ects (e.g., background noise, indoor light, and scattering e®ect). The contribution of each component to the total°uorescence lifetime is estimated over multiple iterations.
When using exponential¯t to process image acquired in using FRET-FLIM technology, it is initially necessary to measure the°uorescence lifetime information of the donor°uorescent molecule. With the occurrence of FRET, various data, such as the°u orescence lifetime of sample and FRET's e±ciency, can be obtained via calculations.
Curve¯tting requires appropriate number of exponential components, then¯tting data in the given sample. More photons and a higher SNR is needed to accurately assume the exponential component; however, this may result in false°uorescence lifetime due to incorporation of nonsample components and system background noise. For FRET e®ect, multi-exponential¯tting has no advantage in demonstrating changes of°uorescence lifetime.
Phasor plot's principle is shown in Fig. 2. In the phasor diagram, the extent and shape of FRET trajectory depends on the contribution of the background°uorescence. Mapping FRET-FLIM data measured to a semicircle, the°uorescence lifetime change of the sample can be more intuitively observed. Semicircle above the X-axis of radius 1 is called the unit circle.
Here, Re and Im are the real and imaginary parts of the Fourier transform, respectively; I is the°u orescence intensity, and m and n are the indices of the rows and columns of the°uorescence image, respectively. 25 Phasor analysis does not rely on input parameters and iterative process, has minimal constraints to provide higher accuracy; this makes the phasor method a reliable analytical method. Furthermore, the phasor method can be used to quickly estimate whether FRET occurs. In addition, it can re°ect the dynamic°uorescence lifetime changes of both the donor and acceptor molecules. However, this method is di±cult to quantify the°uorescence lifetime of each component in the sample. 26-28

Application and Development of FRET-FILM Technology
The intracellular applications of FRET-FLIM technology are illustrated in Figs. 3(a) and 3(b), which describe the principles used to detect protein-protein interactions and changes in protein structure. Figure 3(c) summarizes the common modern applications of FRET-FLIM technology in the biological¯eld.

Internal mechanism of cells
FRET-FLIM technology can analyze the physical and chemical change in intracellular substances, including protein interaction, apoptosis, and gene detection, as shown in Table 1. Trembecka-Lucas 29 used FRET-FLIM technology to verify the interaction between HP1 and PCNA in DNA replication regions in living cells. Using FRET-FLIM technology, Yadav et al. 30 demonstrated that mTOR directly interacts with Rheb or raptor. As shown in Fig. 4, Rheb can accurately locate the cytoplasm of mammalian cells.
Soluble N-ethylmaleimide sensitive fusion protein attachment protein acceptor (SNARE) proteins are key for membrane tra±cking; however, studying the precise functions of SNARE proteins is technically challenge. Verboogen 31 fused the C-terminus of a SNARE protein with a°uorescent protein. By¯tting the°uorescence lifetime histograms by a multicomponent decay model, FRET-FLIM technology allows (semi-)quantitative estimation of the formation rate of SNARE at di®erent vesicles.  FRET-FLIM technology can also be used to study the mechanism of action of speci¯c proteins in plant cells, protein interactions in plant endometrial systems, protein oligomer formation, and biomacromolecular interactions. [35][36][37][38][39] Camborde 40 labeled the target protein with green°u orescent protein (GFP) as a donor and treated the leaves with the nucleic acid dye SYTOX Orange as an acceptor. Detection by FRET-FLIM technology demonstrated a signi¯cant reduction in the GFP lifetime, thereby indicating that the GFP-tagged protein interacted with the SYTOX Orange-stained nucleic acid. Using FRET-FLIM technology, Veerabagu 41 demonstrated that the modi¯ed state of aspartic acid (D70) resulted in homology of the response modi¯er (ARR18). This experiment provides a new perspective on how B-type response regulators bind to their homologous DNA sequences. Dierk 42 labeled heterozygous fusion proteins with GFP/RFP and veri¯ed the formation of parallel dimers in plants using FRET-FLIM technology.
WRKY18 is able to bind directly to di®erent W-boxes in the WRKY53 promoter region and to repress expression of the WRKY53 promoter-driven reporter gene in a transient transformation system using Arabidopsis protoplasts. Potschin 43 used FRET-FLIM technology to detect an interaction between WRKY53 and WRKY18 proteins in transiently transformed tobacco epidermal cells.
Long et al. 44 used FRET-FLIM technology combined with the phasor plot method to reveal the spatial division of protein interactions associated with cell fate speci¯cations. Their results demonstrated that three fully functional°uorescently labeled cell-fate regulators establish cell type-speci¯c interactions at endogenous expression levels and form more advanced complexes. They further tested the interaction between SHORT-ROOT and SCARECROW, indicating that the spatial distribution of the interaction between two transcription factors can be highly regulated in repetitive and structurally similar organs, thereby providing new information about the dynamic redistribution of protein complex con¯gurations at di®erent developmental stages. 45

Disease causes
We can research the role of proteins involved in the disease production process by using FRET-FLIM technology. In the course of disease treatment, drug enters the human body via injection or oral administration. After drug reaches the human body, it uses biomolecules as medium to achieve therapeutic e®ects by inhibiting or promoting a certain protein.
FRET pairs are used to target protein, then the position, conformation, and other information of proteins are obtained by observing the experimental results. Thus, a further understanding of the mechanism and e®ect of the drug therapy can be achieved. The applications of FRET-FLIM technology in pathological research have been presented in Table 2. Cells reduce gap junction coupling during tumor metastasis, Majoul et al. 46 speculated that this might be so to prevent malignant transformation. Using FRET-FLIM technology, they found that Arf6 directs Cx43 to play a role in developing and migrating cells and Cx43 expression inhibits the growth of glioblastoma cells in humans. Waterhouse et al. 47 used primary antibodies from di®erent species together with Alexa488-and Alexa546-conjugated secondary antibodies to validate EGFR in the following three cell lines: EGFR-positive cancer cell line A431, HER2-positive breast cancer cell lines BT474 and SKBR3. HER2 dimerization assay experiments have shown that this method can be applied to tissue microarrays, thereby allowed in situ assessment of the dimerization status of tumor cells and the determination of predicted value of tumor samples measured in the clinical environment.
As shown in Fig. 5, Verboogen 48 used FRET-FLIM technology to describe the transport process of the release of the in°ammatory cytokine interleukin-6 from human blood-derived dendritic cells. This experiment demonstrated the applicability of FRET-FLIM technology to visualize SNARE complexes in living cells at a subcellular spatial resolution.  Beet necrotic yellow vein virus RNA-1 encodes a nonstructural p237 polyprotein processed as two proteins (p150 and p66) by cis-acting protease activity. Pakdel and Mounier 49 experimentally demonstrated the interaction between functional domains of the p150 and p66 proteins and the addressing of benyvirus replicase to the endoplasmic reticulum. They simultaneously used FRET-FLIM technology to indicate the presence of multimeric complexes near the cell membrane network.

Evaluation of drug treatment e®ects
Cancerous cells are generally produced due to errors in gene expression, and metastasis and infection are achieved by biological macromolecules. Highly targeted drugs have become a hot topic relative to the treatment of cancer. Detecting the therapeutic effect of drugs at the molecular level is a signi¯cant challenge. FRET-FLIM technology can image deep tissues of cancer, thereby permitting the use of rapid target-modi¯cation and high-level drug screening, FRET-FLIM technology is expected to become an important means of detecting the therapeutic e®ects of drugs on targeted detect proteins in tumor cells or tissue specimens. 50 Coban 51 used FRET-FLIM technology two-color single-molecule tracking to study the e®ects of tyrosine kinase inhibitors and phosphatase-based manipulation of EGFR phosphorylation on live cells. The results indicated that a modest druginduced increase in the fraction/stability of EGFR homodimer may have signi¯cant biological impact on the proliferation potential of tumor cells.

Application of FRET-FLIM in biosensors
Monitoring di®erent signaling enzymes in a single assay using multiplex biosensor provides a multidimensional workspace to elucidate biological process, signal pathway crosstalk, and determine precise sequence of events at the single living-cell level. Demeautis52 proposed a validated method for multi-parametric kinase biosensor in living cells using FRET-FLIM technology. Using single excitation wavelength dual-color FLIM technology and FRET biosensor, they could detect donor molecules as to measure PKA and ERK1&2 kinase activities in the same cellular location.

Summary and Outlook of FRET-FLIM Technology
Nowadays, FRET-FLIM technology is mainly used to solve various biological problems that need to meet the requirement of high precision and multiple dimensions. Although biochemical tests such as coimmunoprecipitation can also be used to study protein interactions, this method lacks spatial information. The resolution of the Confocal laser scanning microscope can reach 250 nm. FRET-FLIM technology breaks through this limit, achieving a high resolution of intracellular interactions of the molecular scale in cells. It has little dependence on the°uorescence intensity, on comma and has a high sensitivity to detect the interaction between low expression proteins under natural conditions. This technology is noninvasive thus dose not in°uence cell morphology. FRET-FLIM technology is mainly divided into three parts:°uorescent labels, detection means and data analysis. Fluorescent molecules as a marker should have good optical properties such as higher quantum yield, light stability and biological a±nity. Nowadays, the choice of labels is no longer limited to°uorescent dyes and probes. Researchers use new materials to accomplish the aim, such as graphene quantum dots and gold nanorods. To achieve biomicroscopic imaging and detection in vivo, FRET-FLIM technology should have the advantages of short measurement time, high precision, be simple to operate and low in cost. Among all the methods, TCSPC has the highest time resolution, but its acquisition speed is slow. The frequency-domain method has low requirements on equipment, low cost, and high data processing speed, but its performance index is not as good as the time-domain method. Using algorithms can get lots of information from the acquired data.
The potential value of FRET-FLIM technology in the biomedical¯eld has not been fully explored, and the practical application of this technology still faces challenges. First, the FRET-FLIM system is relatively complex and has high requirement for the light source, expensive detectors, its large hardware system limits the°exibility of clinical diagnostic techniques. FRET pairs that have high light stability and biological a±nity are also a key for this method of transforming to the clinical. Second, it usually takes a few seconds to acquire a high-quality image, and several tens of minutes are needed to obtain a certain depth of three-dimensional image. Increasing the quantum yield of°uorescent particles can improve this problem to some extent, and the breakthrough in imaging speed depends on the further improvement of detection method. New algorithms are needed to solve multi-component problems in biomedical samples. Third, high biological tissue penetration and super-resolution are also the development orientation of FRET-FLIM technology. It can combine with other technologies which can provide deep tissue information such as photoacoustic imaging. The development of new multimodal imaging systems is also expected to expand diagnosis capabilities of FRET-FLIM technology.