Fluorescence life-time imaging microscopy(FLIM)monitors tumor cell death triggered by photothermal therapy with MoS_(2) nanosheets

Recently,photothermal therapy(PTT)has been proved to have great potential in tumor therapy.In the last several years,MoS_(2),as one novel member of nanomaterials,has been applied into PTT due to its excellent photothermal conversion efficacy.In this work,we applied fuorescence lifetime imaging microscopy(FLIM)techniques into monitoring the PPT-triggered cell death under MoS_(2) nanosheet treatment.Two types of MoS_(2) nanosheets(single layer nanosheets and few layer nanosheets)were obtained,both of which exhibited presentable photothermal conversion fficacy,leading to high cell death rates of 4T1 cells(mouse breast cancer cells)under PTT.Next,live cell images of 4T1 cells were obtained via directly labeling the mitochondria with Rodamine123,which were then continuously observed with FLIM technique.FLIM data showed that the fuorescence lifetimes of mitochondria targeting dye in cells treated with each type of MoS_(2) nanosheets significantly increased during PTT treatment.By contrast,the fuorescence lifetime of the same dye in control cells(without nanomaterials)remained constant after laser irradiation.These findings suggest that FLIM can be of great value in monitoring cell death process during PTT of cancer cells,which could provide dynamic data of the cellular microenvironment at single cell level in multiple biomedical applications.

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.

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.

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.

Aggregation-induced emission luminogen for in vivo three-photon fuorescence lifetime microscopic imaging

Compared with visible light,near infrared(NIR)light has deeper penetration in biological tisues.Three-photon fuorescence microscopy(3PFM)can effectively utilize the NIR excitation to obtain high-contrast images in the deep tisue.However,the weak three photon fluorescence signals may be not well presented in the traditional fuorescence intensity imaging mode.Fluorescence lifetime of certain probes is insensitive to the intensity of the excitation laser.Moreover,fluorescence lifetimne imaging microscopy(FLIM)can detect weak signals by utilizing time correlated single photon counting(TCSPC)technique.Thus,it would be an improved strategy to combine the 3PFM imaging with the FLIM together.Herein,DCDPP-2TPA,a novel agegation-induced emission luminogen(AIEgen),was adopted as the fluorescent probes.The three-photon absorption cros-section of the AlEgen,which has a deep-red fluorescence emission,was proved to be large.DCDPP-2TPA nanoparticles were synthesized,and the three photon fluorescence lifetime of which was measured in water.Moreover,in vrivo thre-photon fuorescence lifetime microscopic imaging of a craniotomy mouse was conducted via a home made optical system.High contrast cerebrovascular images of different vertical depths were obtained and the maximun depth was about 600 pumn.Even reaching the depth of 600 pum,tiny capillary vessels as small as 1.9 pum could still be distinguished.The three photon fuorescence lifetimes of the capillaries in some representative images were in accord with that of DCDPP-2TPA nanoparticles in water.A vivid 3D reconstruction was further organized to present a wealth of lifetime information.In the future,the combination strategy of 3PFM and FLIM could be further applied in the brain functional imaging.

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.

荧光共振能量转移-荧光寿命显微成像(FRET-FLIM)技术在生命科学研究中的应用进展

细胞是动植物结构和生命活动的基本单位。细胞过程的一个重要特点就是其生化组分在时空调控上的相互作用关系。然而,利用传统的生化方法(如酵母双杂交系统、pull-down系统等)很难在空间上评估活细胞内分子间的相互作用。光学技术的快速发展,为研究活细胞中生物分子的时空动态提供了新的遗传研究工具,其中荧光共振能量转移-荧光寿命显微成像(FRET-FLIM)技术在实时探测分析活细胞中生物大分子构象变化和分子间动态相互作用过程具有独特的优势,如:实现对活细胞的实时“可视化”研究,同时具有高时空分辨率;检测更加灵敏、结果可信度高;且基于简易的数学运算完成简单快捷的分析程序。介绍FRET-FLIM技术的理论背景知识,对比了该技术与传统蛋白相互作用技术研究的利弊,同时归纳了其在蛋白相互作用、细胞生物学和疾病诊断等方面的最新应用研究进展,最后总结和讨论了FRET-FLIM技术的未来发展趋势,以期能够为揭示活细胞的结构和细胞过程相关研究提供新的见解。

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.

FLIM as a Promising Tool for Cancer Diagnosis and Treatment Monitoring

Fluorescence lifetime imaging microscopy(FLIM)has been rapidly developed over the past 30 years and widely applied in biomedical engineering.Recent progress in fluorophore-dyed probe design has widened the application prospects of fluorescence.Because fluorescence lifetime is sensitive to microenvironments and molecule alterations,FLIM is promising for the detection of pathological conditions.Current cancer-related FLIM applications can be divided into three main categories:(i)FLIM with autofluorescence molecules in or out of a cell,especially with reduced form of nicotinamide adenine dinucleotide,and flavin adenine dinucleotide for cellular metabolism research;(ii)FLIM with Förster resonance energy transfer for monitoring protein interactions;and(iii)FLIM with fluorophore-dyed probes for specific aberration detection.Advancements in nanomaterial production and efficient calculation systems,as well as novel cancer biomarker discoveries,have promoted FLIM optimization,offering more opportunities for medical research and applications to cancer diagnosis and treatment monitoring.This review summarizes cutting-edge researches from 2015 to 2020 on cancer-related FLIM applications and the potential of FLIM for future cancer diagnosis methods and anti-cancer therapy development.We also highlight current challenges and provide perspectives for further investigation.

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.

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.

蟾酥灵诱导细胞内caspase-3活化特性荧光分析

采用多光子激发荧光光谱和寿命成像显微技术,在单个活细胞内实时检测蟾酥灵(bufalin,Bu)诱导人类肺腺癌(ASTC-a-1)细胞死亡过程中caspase-3的活化特性.利用CCK-8(Cell Counting Kit-8)检测蟾酥灵对人类肺腺癌细胞活性的抑制效应.蟾酥灵处理稳定表达FRET质粒SCAT3的人类肺腺癌后,在不同时间点检测单个活细胞中SCAT3的多光子激发荧光光谱及其供体ECFP的荧光寿命,从而检测bufa-lin诱导细胞凋亡过程中caspase-3活化特性.实验结果表明:蟾酥灵以浓度依赖性的方式显著地抑制人类肺腺癌细胞的生长;蟾酥灵处理细胞24h后,细胞内SCAT3未被切割,而蟾酥灵作用细胞48h后SCAT3被切割.说明caspase-3参与调控蟾酥灵诱导人类肺腺癌细胞凋亡的过程.

荧光寿命成象显微技术及其应用

本文综述了荧光寿命成象显微技术的概念、原理及实现方法，介绍了荧光寿命成象显微技术在生物物理、生物化学及临床医学诊断等领域的最新研究成果和发展现状，并就其未来的发展及应用研究进行了讨论．

单组分荧光寿命成象显微的研究

激发光的调制频率和CCD所记录到的光强度是影响荧光寿命成象显微频域法测量和数据处理精度的主要因素．本文采用MonteCarlo方法，以单组分问题为例，研究了在用频域外差法进行荧光寿命成象显微测量中激发光的调制频率以及CCD所记录到的光强度对数据处理精度的影响，给出了当调制频率不变时，在不同荧光强度下，对于不同的荧光寿命范围、荧光寿命计算的统计结果，给出了改进测量精度的方法．

基于多光子激光扫描技术的人体皮肤黑色素含量和分布的在体探究

采用多光子激光扫描技术(MPT)和荧光寿命成像技术(FLIM),通过荧光图像和FLIM图像可视化,以及荧光信号强度和短寿命组分a1的计算,对不同光暴露区域皮肤表皮层黑色素的含量和分布进行定性和定量在体探究。试验结果表明:皮肤表皮浅层中黑色素含量的分布呈现光暴露区域高于非光暴露区域的趋势;皮肤基底层中黑色素含量在不同光暴露区域无明显差异。该探索研究为化妆品美白功效评价提供了新的测试方法。

荧光共振能量转移显微术及其新进展

本文叙述荧光共振能量转移显微术及荧光寿命成像显微术的原理、方法及特点。同时介绍利用荧光共振能量转移显微术研究信号分子Rac蛋白在3T3成纤维细胞内的定位及活化过程,以及利用荧光共振能量转移—荧光寿命成像显微术研究转录因子CAATT/增强子结合蛋白α在小鼠垂体细胞内的二聚化现象。

金属酞菁光敏剂的三阶非线性光学性能研究

从光动力治疗癌症的疗效着眼研究酞菁配合物的三阶非线性光学性能。用时间分辨简并四波混频方法测量苯硫基钛菁锌(C56H32S4Zn),苯硫基铝酞菁(C56N32AlClN8S4)以及烷氧基铝酞菁(C56H32AlN8O4)的三阶非线性光极化率;测量四波混频响应的衰减过程;研究时间响应的超快过程和慢过程及其动力学机制,它们分别对应于单态和三线态的寿命。在荧光显微成像系统中观察三种酞菁光敏剂对人肝癌细胞杀伤的形态变化,并用MTT方法检测细胞存活率。对三种酞菁配合物的三线态量子产率和寿命进行测定,结果与它们对人肝癌细胞的光动力杀伤作用相关联。

荧光寿命显微成像技术及应用的最新研究进展

由于荧光寿命不受探针浓度、激发光强度和光漂白效应等因素影响,荧光寿命显微成像技术(fluorescence lifetime imaging microscopy, FLIM)在监测微环境变化、反映分子间相互作用方面具有高特异性、高灵敏度、可定量测量等优点,近年来已被广泛应用于生物医学等领域.然而,尽管FLIM的发明和发展已历经数十年时间,其在实际应用中仍然面临着许多挑战.例如,其成像分辨率受衍射极限限制,而其成像速度与成像质量和寿命测量精度则存在相互制约的关系.近几年来,相关硬件和软件的快速发展及其与其他光学技术的结合,极大地推动了FLIM技术及其应用的新发展.本文简要介绍了基于时域和频域的不同寿命探测方法的FLIM技术的基本原理及特点,在此基础上概述了该技术的最新研究进展,包括其成像性能的提升和在生物医学应用中的研究现状,详细阐述了近几年来研究者们通过硬件和软件算法的改进以及与自适应光学、超分辨成像技术等新型光学技术的结合来提升FLIM的成像速度、寿命测量精度、成像质量和空间分辨率等方面所做的努力,以及FLIM在生物医学基础研究、疾病诊断与治疗、纳米材料的生物医学研究等方面的应用,最后对其未来发展趋势进行了展望.