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.

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.

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.

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.

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.

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.

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.

Sidelobe suppression in light-sheet fluorescence microscopy with Bessel beam plane illumination using subtractive imaging

The fluorescence from the out-of-focus region excited by the sidelobes of a Bessel beam is the major concern for light-sheet fluorescence microscopy （LSFM） with Bessel beam plane illumination. Here, we propose a method of applying the subtractive imaging to overcome the limitation of the conventional LSFM with Bessel beam plane illumination. In the proposed method, the sample is imaged twice by line scanning using the extended solid Bessel beam and the ring-like Bessel beam. By subtracting between the two images with similar out-of-focus blur, the improved image quality with the suppression of the Bessel beam sidelobes and enhanced sectioning ability with improved contrast are demonstrated.

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.

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.

Recent progress on single-molecule nanocatalysis based on single-molecule fluorescence microscopy

Understanding the heterogeneous catalytic properties of nanoparticles is of great significance for the development of high efficient nanocatalysts, but the intrinsic heterogeneities of nanocatalysts were always covered in traditional ensemble studies. This issue can be overcome if one can follow the catalysis of individual nanoparticles in real time. This paper mainly summarizes recent developments in single- molecule nanocatalysis at single particle level in Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. These developments include the revealing of catalytic kinetics of different types （plane ＆ edge） of surface atoms on individual Pd nanocubes, the observing of in situ deactivation of indi- vidual carbon-supported Pt nanoparticles during the electrocatalytic hydrogen-oxidation reaction, and the measurement of catalytic activation energies on single nanocatalysts for both product formation process and dissociation process, etc. These studies further indicate the advantages or unique abilities of single-molecule methods in the studies of nanocatalvsis or even chemical reactions.

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.

Highly selective two-photon fluorescent probe for imaging of nitric oxide in living cells

A new two-photon fluorescent probe, ADNO, for nitric oxide （NO） based on intramolecular photoinduced electron transfer （PET） mechanism d/splays a rapid response to NO with a remarkable fluorescent enhancement in PBS buffer. The excellent chemoselectivity of ADNO for NO over other ROS/RNS （reactive oxygen species or nitrogen species） and common metal ions was observed. Moreover, ADNO has been successfully applied in fluorescence imaging of NO of living cells using both one-photon microscopy （OPM） and two-~hoton microscopy （TPM）,

Recent advances in optical-based and force-based single nucleic acid imaging

The capability to image, as well as control and manipulate single molecules such as nucleic acids(DNA or RNA) can greatly enrich our knowledge of the roles of individual biomolecules in cellular processes and their behavior in native environments. Here we summarize the recent advances of single nucleic acid imaging based on optical observation and force manipulation. We start by discussing the superiority of single molecule image, the central roles nucleic acids play in biosystems, and the significance of single molecule image towards nucleic acids. We then list a series of representative examples in brief to illustrate how nucleic acid of various morphologies has been imaged from different aspects, and what can be learned from such characterizations. Finally,concluding remarks on parts of which should be improved and outlook are outlined.

Superresolution imaging of telomeres with continuous wave stimulated emission depletion (STED) microscope

The significant role of telomeres in cells has attracted much attention since they were discovered.Fluorescence imaging is an effective method to study subcellular structures like telomeres.However,the diffraction limit of traditional optical microscope hampers further investigation on them.Recent progress on superresolution fluorescence microscopy has broken this limit.In this work,we used stimulated emission depletion(STED) microscope to observe fluorescence-labeled telomeres in interphase cell nuclei.The results showed that the size of fluorescent puncta representing telomeres under the STED microscope was much smaller than that under the confocal microscope.Two adjacent telomeres were clearly separated via STED imaging,which could hardly be discriminated by confocal microscopy due to the diffraction limit.We conclude that STED microscope is a more powerful tool that enable us to obtain detailed information about telomeres.

DeepNoise: Signal and Noise Disentanglement Based on Classifying Fluorescent Microscopy Images via Deep Learning

The high-content image-based assay is commonly leveraged for identifying the phenotypic impact of genetic perturbations in biology field.However,a persistent issue remains unsolved during experiments:the interferential technical noises caused by systematic errors(e.g.,temperature,reagent concentration,and well location)are always mixed up with the real biological signals,leading to misinterpretation of any conclusion drawn.Here,we reported a mean teacher-based deep learning model(Deep Noise)that can disentangle biological signals from the experimental noises.Specifically,we aimed to classify the phenotypic impact of 1108 different genetic perturbations screened from 125,510 fluorescent microscopy images,which were totally unrecognizable by the human eye.We validated our model by participating in the Recursion Cellular Image Classification Challenge,and Deep Noise achieved an extremely high classification score(accuracy:99.596%),ranking the 2nd place among 866 participating groups.This promising result indicates the successful separation of biological and technical factors,which might help decrease the cost of treatment development and expedite the drug discovery process.The source code of Deep Noise is available at https://github.com/Scu-sen/Recursion-Cellular-Image-Classification-Challenge.

Luminescent probes for luminescence lifetime sensing and imaging in live cells:a narrative review

Luminescence(mostly fluorescence and phosphorescence)probes are a powerful tool widely used in the life sciences research.They can be used,for example,in the quantitative analysis of physiological parameters,visualization of different cell organelles,and measurement of drug transportation.The luminescence intensity and lifetime of these probes are among the main signals recorded and evaluated in these applications.Other reviews have discussed optical probes from the perspective of their luminescence intensity.However,the luminescence lifetime,which depends on the molecular microenvironment but not the probe concentration,is another promising metric for biological sensing and imaging applications.In this review,we aim to introduce the basic strategies of FLIM probe design.We also present applications of these probes,including sensing of intracellular pH,cation/anion concentrations,oxygen levels,biomolecule contents,and physiological parameters,as well as live-cell imaging based on luminescence lifetime.Studies based on FLIM imaging of cells or tissues with endogenous organic molecules are not included in this mini review.With the rapid development of microscopy technology for fluorescence lifetime imaging,fluorescence lifetime-based probes have shown great potential in a variety of biological applications.

Emission Properties of Individual AIE-Luminogens in Solution at Room Temperature

Here,we combined the photon antibunching analysis,fluorescence correlation spectroscopy,and time-domain fluorescence lifetime imaging microscopy(TD-FLIM)to study the emission properties of a representative AIE-luminogen—4,4’-(benzo[c][1,2,5]thiadiazole-4,7-diyl)bis(N,N-diphenylaniline)(TPA-BT)at the single emitter level in a tetrahydrofuran(THF)/water solution where water is a non-solvent for TPA-BT.Our findings suggest that,at a constant water fraction in the solution,the size of TPA-BT aggregates increases with the TPA-BT concentration;TPA-BT aggregates are not a quantum emitter at room temperature in the solution.Moreover,utilizing TD-FLIM and a gel trapping technique allowed us to study the fluorescence lifetime of individual TPA-BT aggregates.Adding a polar solvent like water does not result in an overall decrease in fluorescence lifetime.Rather,it causes the fluorescence lifetime distribution to become wider,and only some molecules experience a decrease in their fluorescence lifetime.These results could represent a step forward in further understanding the photophysics of AIE-luminogens.