Applications,of fluorescence lifetime imaging in clinical medicine

Fluorescence lifetime is not only associated with the molecular structure f fuorophores,but alsostrongly depends on the environment around them,which llows fuorescence lifetime imagingmicroscopy(FLIM)to be used as a tool for precise measurement of the cell or tisue microenvironment,This review introduces the basic principle of fuorescence lifetime imagingtechnology and its application in clinical medicine,including research and diagnosis of diseases inskin,brain,eyes,mouth,bone,blood vessels and cavity organs,and drug evaluation.As anoninvasive,nontoxic and nonionizing radiation technique,FLIM demonstrates excellent per-formance with high sensitivity and specificity,which allows to determine precise position of thelesion and,thus,has good potential for application in biomedical research and clinical diagnosis.

Fluorescence lifetime imaging of fluorescent proteins as an effective quantitative tool for noninvasive study of intracellular processes

Fluorescence litime imaging(FLIM)is an effective noninvasive bioanalytical tol based onmeasuring fuorescent lifetime of fuorophores.A growing number of FLIM studies utilizes ge-netically engineered fluorescent proteins targeted to specific subcellular structures to probe localmolecular environment,which opens new directions in cell science.This paper highlights theunconventional applications of FLIM for studies of molecular processes in diverse organelles oflive cultured cells.

Monitoring microenvironment of Hep G2 cell apoptosis using two-photon fluorescence lifetime imaging microscopy

Apoptosis is very important for the maintenance of cellular homeostasis and is closely related to the occurrence and treatment of many diseases.Mitochondria in cells play a crucial role in programmed cell death and redox processes.Nicotinamide adenine dinucleotide(NAD(P)H)is the primary producer of energy in mitochondria,changing NAD(P)H can directly reflect the physiological state of mitochondria.Therefore,NAD(P)H can be used to evaluate metabolic response.In this paper,we propose a noninvasive detection method that uses two-photon fluorescence lifetime imaging microscopy(TP-FLIM)to characterize apoptosis by observing the binding kinetics of cellular endogenous NAD(P)H.The result shows that the average fluorescence lifetime of NAD(P)H and the fluorescence lifetime of protein-bound NAD(P)H will be affected by the changing pH,serum content,and oxygen concentration in the cell culture environment,and by the treatment with reagents such as H2O2 and paclitaxel.Taxol(PTX).This noninvasive detection method realized the dynamic detection of cellular endogenous substances and the assessment of apoptosis.

Fast fluorescence lifetime imaging techniques:A review on challenge and development

Fluorescence lifetime imaging microscopy(FLIM)is increasingly used in biomedicine,material science,chemistry,and other related research fields,because of its advantages of high specificity and sensitivity in monitoring cellular microenvironments,studying interaction between proteins,metabolic state,screening drugs and analyzing their efficacy,characterizing novel materials,and diagnosing early cancers.Understandably,there is a large interest in obtaining FLIM data within an acquisition time as short as possible.Consequently,there is currently a technology that advances towards faster and faster FLIM recording.However,the maximum speed of a recording technique is only part of the problerm.The acquisition time of a FLIM image is a complex function of many factors.These include the photon rate that can be obtained from the sample,the amount of information a technique extracts from the decay functions,the fficiency at which it determines fluorescence decay parameters from the recorded photons,the demands for the accuracy of these parameters,the number of pixels,and the lateral and axial resolutions that are obtained in biological materials.Starting from a discussion of the parameters which determine the acquisition time,this review will describe existing and emerging FLIM techniques and data analysis algo-rithms,and analyze their performance and recording speed in biological and biomedical applications.

Fluorescence lifetime imaging microscopy and its applications in skin cancer diagnosis

Fluorescence lifetime(FLT)of fluorophores is sensitive to the changes in their surrounding microenvironment,and hence it can quantitatively reveal the physiological characterization of the tissue under investigation.Fluorescence lifetime imaging microscopy(FLIM)provides not only morphological but also functional information of the tisse by producing spatially resolved image of fuorophore lifetime,which can be used as a signature of disorder and/or malignancy in diseased tissues.In this paper,we begin by introducing the basic principle and common detection methods of FLIM.Then the recent advances in the FLIM-based diagnosis of three different skin cancers,including basal cell carcinoma(BCC),squamous cell carcinoma(SCC)and malignant melanoma(MM)are reviewed.Furthermore,the potential advantages of FLIM in skin cancer diagnosis and the challenges that may be faced in the future are prospected.

Investigation of NAD(P)H Fluorescence Decay in Living Cardiomyocytes with Spectrally-resolved Fluorescence Lifetime Spectroscopy

Objective：To study the mitochondrial redox state in experimental animals to sensitively detect early signs of mitochondrial function in pathophysiologieal conditions, such as isehemia. Methods： Fluorescence of nieotinamide adenine dinucleotide （phosphate） , or NAD（P）H, the principal electron donor in mitochondrial respiration responsible for vital ATP supply of cardiomyocytes, is studied for non-invasive fluorescent probing of the mitochondrial function. Examination of NAD （P）H fluorescence in living cardiomyocytes following excitation by UV-pulsed laser diode and detection by spectrally-resolved time-correlated single photon counting （TCSPC） , is based on the simultaneous measurement of the fluorescence spectra and lifetime. Results ： The dynamic characteristics of NAD （P） H fluorescence decay in living rat cardiomyocytes show that at least a 3-exponential decay model, with 0.4 - 0.7 ns, 1.2 - 1.9 ns and 8.0 - 13.0 ns lifetimes, is necessary to describe cardiomyocyte autofluorescenee （AF）. Decay-associated spectra （DSA） revealed the presence of 4 spectrally-distinct populations of NADH molecules in eardiomyocytes with spectral maximum at 470 nm for short-lifetime pool for the first time, and emission peaks at 450 nm, 470 nm and 490 nm for intermediate and long-lifetime pools. Increased mitochondrial NADH content ratio by ketone bodies enhanced the AF intensity, without the significant change in fluorescent lifetimes. Rotenone, the inhibitor of Complex I of the mitochondrial respiratory chain, increased AF and shortened the average fluorescence lifetime. Dinitrophenol （DNP）, an uncoupling agent of the mitochondrial oxidative phosphorylation, lowered AF,broadened the spectral shoulder at 520 nm and increased the average lifetime. These effects, comparable to the changes in the concentration and in the rate of dehydrogenation of NADH in vitro, were also examined under ischemia-mimetic conditions. Conclusion： Our findings anticipate a contribution of both conformational NADH changes and energy transfer from NADH to lipoamide dehydrogenase （LipDH）-bound flavins, to explain observed fluorescence kinetics. Presented spectrally resolved fluorescence lifetime approach provides promising new tool for analysis of mitochondrial NAD （P） H in living cardiomyocytes, and hence for investigation of energy metabolism and mitoehondrial dysfunction at a cellular level.

The LB Films of Dansyl Chloride Labeled Octadecylamine and Its Fluorescence Lifetime

Octadecylamine was derivatized with dansyl chloride (5-dimethylaminonaphthalene-1-sulfonyl chloride) In order to simplify and understand the LB films of fluorescent probe labeling proteins. its monolayer and multilayers in the absence and presence of stearic acid were deposited by LB technique. Fluorescence spectra and lifetimes of the fluorescent products were studied to elucidate the microenvironment of molecules in the LB films.

Iterative multi-photon adaptive compensation technique for deep tissue two-photon fluorescence lifetime imaging

Fluorescence lifetime imaging can reveal the high-resolution structure of various biophysical and chemical parameters in a microenvironment quantitatively.However,the depth of imaging is generally limited to hundreds of micrometers due to aberration and light scattering in biological tissues.This paper introduces an iterative multi-photon adaptive compensation technique(IMPACT)into a two-photon fluorescence lifetime microscopy system to successfully overcome aberrations and multiple scattering problems in deep tissues.It shows that 400 correction modes can be achieved within 5 min,which was mainly limited by the frame rate of a spatial light modulator.This system was used for high-resolution imaging of mice brain tissue and live zebrafish,further verifying its superior performance in imaging quality and photon accumulation speed.

Metabolic state oscillations in cerebral nuclei detected using two-photon fluorescence lifetime imaging microscopy

The fluorescence lifetime of nicotinamide adenine dinucleotide(NADH),a key endogenous coenzyme and metabolic biomarker,can reflect the metabolic state of cells.To implement metabolic imaging of brain tissue at high resolution,we assembled a two-photon fluorescence lifetime imaging microscopy(FLIM)platform and verified the feasibility and stability of NADH-based two-photon FLIM in paraformaldehydefixed mouse cerebral slices.Furthermore,NADH based metabolic state oscillation was observed in cerebral nuclei suprachiasmatic nucleus(SCN).The free NADH fraction displayed a relatively lower level in the daytime than at the onset of night,and an ultradian oscillation at night was observed.Through the combination of high-resolution imaging and immunostaining data,the metabolic tendency of different cell types was detected after the first two hours of the day and at night.Thus,two-photon FLIM analysis of NADH in paraformaldehyde-fixed cerebral slices provides a high-resolution and label-free method to explore the metabolic state of deep brain regions.

Self-confocal NIR-II fluorescence microscopy for multifunctional in vivo imaging

Fluorescence imaging in the second near-infrared window(NIR-II,900–1880 nm)with less scattering background in biological tissues has been combined with the confocal microscopic system for achieving deep in vivo imaging with high spatial resolution.However,the traditional NIR-IIfluorescence confocal microscope with separate excitation focus and detection pinhole makes it possess low confocal e±ciency,as well as di±cultly to adjust.Two types of upgraded NIR-IIfluorescence confocal microscopes,sharing the same pinhole by excitation and emission focus,leading to higher confocal e±ciency,are built in this work.One type is-ber-pinhole-based confocal microscope applicable to CW laser excitation.It is constructed forfluorescence intensity imaging with large depth,high stabilization and low cost,which could replace multiphotonfluorescence microscopy in some applications(e.g.,cerebrovascular and hepatocellular imaging).The other type is air-pinhole-based confocal microscope applicable to femtosecond(fs)laser excitation.It can be employed not only for NIR-IIfluorescence intensity imaging,but also for multi-channelfluorescence lifetime imaging to recognize different structures with similarfluorescence spectrum.Moreover,it can be facilely combined with multiphotonfluorescence microscopy.A single fs pulsed laser is utilized to achieve up-conversion(visible multiphotonfluorescence)and down-conversion(NIR-II one-photonfluorescence)excitation simultaneously,extending imaging spectral channels,and thus facilitates multi-structure and multi-functional observation.

Layer-dependent signatures for exciton dynamics in monolayer and multilayer WSe2 revealed by fluorescence lifetime imaging measurement

Two-dimensional(2D)transition-metal dichalcogenide(TMD)materials have aroused noticeable interest due to their distinguished electronic and optical properties.However,little is known about their complex exciton properties together with the exciton dynamics process which have been expected to influence the performance of optoelectronic devices.The process of fluorescence can well reveal the process of exciton transition after excitation.In this work,the room-temperature layer-dependent exciton dynamics properties in layered WSe2 are investigated by the fluorescence lifetime imaging microscopy(FLIM)for the first time.This paper focuses on two mainly kinds of excitons including the direct transition neutral excitons and trions.Compared with the lifetime of neutral excitons(<0.3 ns within four-layer),trions possess a longer lifetime(~6.6 ns within four-layer)which increases with the number of layers.We attribute the longer-lived lifetime to the increasing number of trions as well as the varieties of trion configurations in thicker WSe2.Besides,the whole average lifetime increases over 10%when WSe2 flakes added up from monolayer to four-layer.This paper provides a novel tuneable layer-dependent method to control the exciton dynamics process and finds a relatively longer transition lifetime of trions at room temperature,enabling to investigate in the charge transport in TMD-based optoelectronics devices in the future.

NIR-Ⅱ Excitation and NIR-I Emission Based Two-photon Fluorescence Lifetime Microscopic Imaging Using Aggregation-induced Emission Dots

Near-infrared(NIR)lights are powerful tools to conduct deep-tissue imaging since NIR-Ⅰ wavelengths hold less photon absorption and NIR-Ⅱ wavelengths serve low photon scattering in the biological tissues compared with visible lights.Two-photon fluorescence lifetime microscopy(2PFLM)can utilize NIR-Ⅱ excitation and NIR-Ⅰ emission at the same time with the assistance of a well-designed fluorescent agent.Aggregation induced emission(AIE)dyes are famous for unique optical properties and could serve a large two-photon absorption(2PA)cross-section as aggregated dots.Herein,we report two-photon fluorescence lifetime microscopic imaging with NIR-Ⅱ excitation and NIR-Ⅰ emission using a novel deep-red AIE dye.The AIE-gens held a 2PA cross-section as large as 1.61×10^(4)GM at 1040 nm.Prepared AIE dots had a two-photon fluorescence peak at 790 nm and a stable lifetime of 2.2 ns under the excitation of 1040 nm femtosecond laser.The brain vessels of a living mouse were vividly reconstructed with the two-photon fluorescence lifetime information obtained by our home-made 2PFLM system.Abundant vessels as small as 3.17µm were still observed with a nice signal-background ratio at the depth of 750µm.Our work will inspire more insight into the improvement of the working wavelength of fluorescent agents and traditional 2PFLM.

Study of genetic evolution of oil inclusion and density of surface oil by measurement of fluorescence lifetime of crude oil and oil inclusion

By using fluorescence lifetime image microscope （FLIM） and time-correlated single photon counting （TCSPC） technique, we measured fluorescence lifetime of crude oils with density of 0.9521-0.7606 g/cm3 and multiple petroleum inclusions from Tazhong uplift of Tarim Basin. As indicated by the test results, crude oil density is closely correlated with average fluorescence lifetime following the regression equation Y=-0.0319X＋0.9411, which can thus be used to calculate density of oil inclusions in relation to fluorescence lifetime and density of corresponding surface crude. For type A oil inclusions showing brown-yellow fluorescence from Tazhong 1 well in Tarim Basin, their average fluorescence lifetime was found to be 2.144-2.765 ns, so the density of surface crude corresponding to crude trapping these oil inclusions is 0.852-0.873 g/cm3, indicating that they are matured oil inclusions trapped at earlier stage ofoil formation. For type B oil inclusions with light yellow-white fluorescence, their average fluorescence lifetime was found to be 4.0294.919 ns, so the density of surface crude corresponding to crude trapping these oil inclusions is 0.784-0.812 g/cm3, indicating that they are higher matured oil inclusions trapped at the second stage of oil formation. For type C oil inclusions showing light blue-green fluorescence, their average fluorescence lifetime was found to be 5.0634.168 ns, so the density of surface crude corresponding to crude trapping these oil inclusions is 0.743-0.779 g/cm3, indicating that they are highly-matured light oil inclusions trapped at the third stage of oil formation.

A near-infrared plasma membrane-specific AIE probe for fluorescence lifetime imaging of phagocytosis

Phagocytosis is a biological process that plays a key role in host defense and tissue homeostasis.Efficient approaches for real-time imaging of phagocytosis are highly desired but limited.Herein,an AIE-active near-infrared fluorescent probe,named TBTCP,was developed for fluorescence lifetime imaging of phagocytosis.TBTCP could selectively label the cell plasma membrane with fast staining,wash-free process,high signal-to-background ratio,and excellent photostability.Cellular membrane statuses under different osmolarities as well as macrophage phagocytosis of bacteria or large silica particles in early stages could be reported by the fluorescence lifetime changes of TBTCP.Compared with current fluorescence imaging methods,which target the bioenvironmental changes in the late phagocytosis stage,this approach detects the changes in the cell membrane,thus giving a faster response to phagocytosis.This article provides a functional tool to report the phagocytic dynamics of macrophages which may greatly contribute to the studies of phagocytic function-related diseases.

Fluorescence lifetime measurement from a designated single-bunch in the BEPCⅡ colliding mode

Fluorescence lifetime measurement in the time domain requires excitation from a well separated single bunch using synchrotron light sources. In the colliding mode of the Beijing Electron Positron Collider Ⅱ （BEPCⅡ）, a hybrid filling pattern was realized such that a single bunch was placed in the middle of a large gap between two multi-bunch groups. Detection of fluorescence lifetime, based on the excitation of the light pulse from this designated single-bunch, was established at Beamline 4B8 of the Beijing Synchrotron Radiation Facility （BSRF）. The timing signal of the BEPCII was utilized as a trigger to gate this fluorescence event. L-Tryptophan amino acid, a known lifetime standard, was selected to assess the lifetime measurement performance. The measured lifetime was consistent in both colliding and single-bunch mode with the time resolution down to 450 ps. Moreover, both the bunch purity and the fine structure of the hybrid filling pattern were characterized.

Conformational change of E.coli sulfurtransferase YgaP upon SCN- in intact native membrane revealed by fluorescence lifetime and anisotropy

Fluorescence lifetime and anisotropy has become a prevalent tool to detect the structure change and motility property of proteins. YgaP is the only membrane-integrated rhodanese in E. coli. The sulfur transfer process has been characterized by various studies. However, the mechanism of the outward transportation of SCN-remains unclear. In this work, we examined the fluorescence lifetime and anisotropy of site-specific incorporated unnatural amino acid 7-HC to study the conformational change of YgaP upon SCN-binding. We also compared the fluorescence changes between detergent-wrapped environment in DPC and intact native membrane environment in SMA. Our results suggested the presence of at least two different conformations in YgaP protein. Both the residues in the middle of TMH2 and the residues near extracellular side play important roles in the binding and/or output of SCN-. SMA is a good material to reflect the in situ conformation changes of protein than micelles.

In cell measurement of fluorescence lifetime imaging microscopy revealed C-terminal conformation changes of Ferroportin upon addition of Mn^2＋

Fluorescence microscopy, as a sensitive method to detect microenvironment of molecules, is widely used in protein conformation and dynamic studies in live cells. Fluorescence lifetime imaging microscopy（FLIM）, which is independent of fluorophore concentrations, scattering and bleaching, is a suitable tool to analyze membrane proteins in a single cell. Ferroportin（FPN）, a multi-ion exporter in vertebrates, was modulated by metal ions with unknown mechanism. Herein, we fused green fluorescence protein on Cterminal of FPN（FPN-eGFP） and applied fluorescence lifetime to monitor conformation changes of FPN in a live cell. The fluorescence lifetime distribution showed a shift to shorter lifetime upon Mn^（2＋） treatment,suggesting a preference conformation of FPN in Mn^（2＋） exposure. It is also observed that the lifetime（rather than intensity） measurement was not strongly influenced by laser power. The observed fluorescence lifetime changes of FPN-eGFP upon Mn^（2＋） treatments indicated that extracellular metal ions can modulate FPN through conformation exchanges between several different states.

Quantitative deconvolution of autocorrelations and cross correlations from two-dimensional lifetime decay maps in fluorescence lifetime correlation spectroscopy

Fluorescence correlation spectroscopy （FCS） is a widely used method for measuring molecular diffusion and chemical kinetics. However, when a mixture of fluorescent species is taken into account, the conven- tional FCS method has limitations in extracting autocorrelations for different species and cross correla- tions between different species. Recently developed fluorescence lifetime correlation spectroscopy （FLCS） based on time-tagged time-resolved （TITR） photon recording, which can record the global and micro arrival time for each individual photon, has been used to discriminate different species according to fluorescence lifetime. Here, based on two-dimensional lifetime decay maps constructed from TITR photon stream, we have developed a quantitative lifetime-deconvolution FCS model （LDFCS） to extract precise chemical rates for chemical conversions in multi-species systems. The key point of LDFCS model is separation of different species according to the global distribution of fluorescence lifetime and then deconvolution of autocorrelations and cross-correlations from the two-dimensional lifetime decay maps constructed bv the micro arrival times of photon pairs at each delay time.

AgInS_(2)/ZnS quantum dots for noninvasive cervical cancer screening with intracellular pH sensing using fluorescence lifetime imaging microscopy

Intracellular pH plays a critical role in biological functions,and abnormal pH values are related to various diseases.Here,we report on an intracellular pH sensor AgInS_(2)(AIS)/ZnS quantum dots(QDs)that show long fluorescence lifetimes of hundreds of nanoseconds and low toxicity.Fluorescence lifetime imaging microscopy(FLIM)combined with AIS/ZnS QDs is used for the imaging of live cells in different pH buffers and different cell lines.The FLIM images of AIS/ZnS QDs in live cells demonstrate different intracellular pH values in different regions,such as in lysosomes or cytoplasm.This method can also distinguish cancer cells from normal cells,and the fluorescence lifetime difference of the AIS/ZnS QDs between the two types of cells is 100±7 ns.Most importantly,the exfoliated cervical cells from 20 patients are investigated using FLIM combined with AIS/ZnS QDs.The lifetime difference value between the normal and cervical cancer(CC)groups is 115±9 ns,and the difference between the normal and the precancerous lesion group is 64±9 ns.For the first time,the noninvasive method has been used for cervical cancer screening,and it has shown great improvement in sensitivity compared with a clinical conventional cytology examination.

Deep-UV fluorescence lifetime imaging microscopy

A novel fluorescence lifetime imaging microscopy(FLIM) working with deep UV 240–280 nm wavelength excitations has been developed. UV-FLIM is used for measurement of defect-related fluorescence and its changes upon annealing from femtosecond laser-induced modifications in fused silica. This FLIM technique can be used with microfluidic and biosamples to characterize temporal characteristics of fluorescence upon UV excitation, a capability easily added to a standard microscope-based FLIM. UV-FLIM was tested to show annealing of the defects induced by silica structuring with ultrashort laser pulses. Frequency-domain fluorescence measurements were converted into the time domain to extract long fluorescence lifetimes from defects in silica.