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.

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.

Quantitative heterogeneity and subgroup classification based on motility of breast cancer cells

Cancer cell motility and its heterogeneity play an important role in metastasis, which is responsible for death of 90% of cancer patients. Here, in combination with a microfluidic technique, single-cell tracking, and systematic motility analysis,we present a rapid and quantitative approach to judge the motility heterogeneity of breast cancer cells MDA-MB-231 and MCF-7 in a well-defined three-dimensional(3D) microenvironment with controllable conditions. Following this approach,identification of highly mobile active cells in a medium with epithelial growth factor will provide a practical tool for cell invasion and metastasis investigation of multiple cancer cell types, including primary cells. Further, this approach could potentially become a speedy(~ hours) and efficient tool for basic and clinical diagnosis.

Green emitted CdSe@ZnS quantum dots for FLIM and STED imaging applications

Inorganic quantum dots(QDs)have excellent optical properties,such as high°uorescence intensity,excellent photostability and tunable emission wavelength,etc.,facilitating them to be used as labels and probes for bioimaging.In this study,CdSe@ZnS QDs are used as probes for Fluorescence lifetime imaging microscope(FLIM)and stimulated emission depletion(STED)nanoscopy imaging.The emission peak of CdSe@ZnS QDs centered at 526 nm with a narrow width of 19 nm and the photoluminescence quantum yield(PLQY)was 64%.The QDs presented excellent anti-photobleaching property which can be irradiated for 400 min by STED laser with 39.8 mW.The lateral resolution of 42.0 nm is demonstrated for single QDs under STED laser(27.5 mW)irradiation.Furthermore,the CdSe@ZnS QDs were for the first time used to successfully label the lysosomes of living HeLa cells and 81.5 nm lateral resolution is obtained indicating the available super-resolution applications in living cells for inorganic QD probes.Meanwhile,Eca-109 cells labeled with the CdSe@ZnS QDs was observed with FLIM,and their fluorescence lifetime was around 3.1 ns,consistent with the in vitro value,suggesting that the QDs could act as a satisfactory probe in further FLIM-STED experiments.

Laser-Induced Recoverable Fluorescence Quenching of Perovskite Films at a Microscopic Grain Scale

Understanding the fundamental properties of metal-halide perovskite materials is driving the development of novel optoelectronic applications.Here,we report the observation of a recoverable laser-induced fluorescence quenching phenomenon in perovskite films with a microscopic grain-scale restriction,accompanied by spectral variations.This fluorescence quenching depends on the laser intensity and the dwell time under Auger recombination dominated conditions.These features indicate that the perovskite lattice deformation may take the main responsibility for the transient and show a new aspect to understand halide perovskite photostability.We further modulate this phenomenon by adjusting the charge carrier recombination and extraction,revealing that efficient carrier transfer can improve the bleaching resistance of perovskite grains.Our results provide future opportunities to attain high-performance devices by tuning the perovskite lattice disorder and harvesting the energetic carriers.

Encrypted optical fiber tag based on encoded fiber Bragg grating array

Optical fibers are typically used in telecommunications services for data transmission,where the use of fiber tags is essential to distinguish between the different transmission fibers or channels and thus ensure the working functionality of the communication system.Traditional physical entity marking methods for fiber labeling are bulky,easily confused,and,most importantly,the label information can be accessed easily by all potential users.This work proposes an encrypted optical fiber tag based on an encoded fiber Bragg grating(FBG)array that is fabricated using a point-by-point femtosecond laser pulse chain inscription method.Gratings with different resonant wavelengths and reflectivities are realized by adjusting the grating period and the refractive index modulations.It is demonstrated that a binary data sequence carried by a fiber tag can be inscribed into the fiber core in the form of an FBG array,and the tag data can be encrypted through appropriate design of the spatial distributions of the FBGs with various reflection wavelengths and reflectivities.The proposed fiber tag technology can be used for applications in port identification,encrypted data storage,and transmission in fiber networks.

Deep-learning-based methods for super-resolution fluorescence microscopy

The algorithm used for reconstruction or resolution enhancement is one of the factors affectingthe quality of super-resolution images obtained by fluorescence microscopy.Deep-learning-basedalgorithms have achieved stateof-the-art performance in super-resolution fluorescence micros-copy and are becoming increasingly attractive.We firstly introduce commonly-used deep learningmodels,and then review the latest applications in terms of the net work architectures,the trainingdata and the loss functions.Additionally,we discuss the challenges and limits when using deeplearning to analyze the fluorescence microscopic data,and suggest ways to improve the reliability and robustness of deep learning applications.

SOFFLFM:Super-resolution optical fluctuation Fourierlight-field microscopy

Fourier light-field microscopy(FLFM)uses a microlens aray(MLA)to segment the Fourierplane of the microscopic objective lens to generate multiple two-dimensional perspective views,thereby reconstructing the threedimensional(3D)structure of the sample using 3D deconvo-lution calculation without scanning.However,the resolution of FLFM is stil limited by dif-fraction,and furthermore,it is dependent on the aperture division.In order to improve itsresolution,a super-resolution opticai fuctuation Fourier light-field microscopy(SOFFLFM)wasproposed here,in which the super-resolution optical fluctuation imaging(SOFI)with the abilityof super-resolution was introduced into FLFM.SOFFLFM uses higher-order cumulants statis-tical analysis on an image sequence collected by FLFM,and then carries out 3D deconvolutioncalculation to reconstruct the 3D structure of the sample.The theoretical basis of SOFFLFM onimproving resolution was explained and then verified with the simulations.Simulation resultsdemonstrated that SOFFLFM improved the lateral and axial resolution by more than V2 and 2times in the second-and fourth-order accumulations,compared with that of FLFM.

Comparison of point detection and area detection for point-scanning structured illumination microscopy

Structured illumination microscopy(SIM)is suitable for biological samples because of its relatively low-peak illumination intensity requirement and high imaging speed.The system resolution is affected by two typical detection modes:Point detection and area detection.However,a systematic analysis of the imaging performance of the different detection modes of the system has rarely been conducted.In this study,we compared laser point scanning point detection(PS-PD)and point scanning area detection(PS-AD)imaging in nonconfocal microscopy through theoretical analysis and simulated imaging.The results revealed that the imaging resolutions of PSPD and PS-AD depend on excitation and emission point spread functions(PSFs),respectively.Especially,we combined the second harmonic generation(SHG)of point detection(P-SHG)and area detection(A-SHG)with SIM to realize a nonlinear SIM-imaging technique that improves the imaging resolution.Moreover,we analytically and experimentally compared the nonlinear SIM performance of P-SHG with that of A-SHG.

Intermolecular hydrogen-bonding as a robust tool toward significantly improving the photothermal conversion efficiency of a NIR-II squaraine dye

The photothermal therapy(PTT) has come across as a promising noninvasive therapeutic strategy for tumor treatment. However, low photothermal conversion efficiency(PCE) and hydrophobicity may impede the therapeutic efficacy of organic photothermal agents and an efficient PTT-agent must overcome these two major challenges. In this work, we developed a new strategy to promote higher PCE wherein the intermolecular hydrogen-bonding interaction between the single dye molecule and water facilitated the transformation of the absorbed energy into the heat. A hydrophilic squaraine dye(SCy1) with the second near-infrared region(NIR-II) absorption and extremely low emission were designed to exhibit much higher PCE than that of the analogues of pentamethine-dyes(PCy1, PCy2). The presence of the ‘–O-' at middle of squaric cycle enabled the intermolecular H-bonding formation between the SCy1 and water to promote the energy dissipation channel. Moreover, the introduction of long-chain phenylsulfonate groups helped in to improve the water solubility apart from serving as an additional means of further enhancing PCE through fluorescence quenching. Therefore, SCy1 with a squaraine backbone and long-chain sulfonate moieties revealed outstanding photothermal stability and anti-aggregation activity apart from showing exceptionally high PCE(74%) in water. SCy1 demonstrated excellent therapeutic efficacy when applied in the PTT treatment of tumor-bearing mice under a laser irradiation of 915 nm.

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.

In vivo mice brain microcirculation monitoring based on contrast-enhanced SD-OCT

In this paper,we proposed a contrast-enhanced homemade spectral domain optical coherence tomography(SD-OCT)method for monitoring of brain microcirculation.We used the polyethylene glycol(PEG)-ylated gold nanorods(GNRs)as a contrast-enhanced agent,obtained clearly 2D and 3D OCT images of blood vessels and dynamic changes of probes in mouse blood vessels.Owing to high scattering of the PEG-GNRs,more tiny blood vessels can be imaged and the OCT signal can be enhanced by 5.87 dB after injection of PEG-GNRs for 20 min,the enhancement then declined gradually for 60 min.Our results demonstrate an effective technique for the enhanced imaging of blood vessels in vivo,especially for studies of the brain microcirculation,which could be serviced for disease mechanism research and therapeutic drug monitoring.

Recent advances in nonlinear optics for bio‐imaging applications

Nonlinear optics,which is a subject for studying the interaction between intense light and materials,has great impact on various research fields.Since many structures in biological tissues exhibit strong nonlinear optical effects,nonlinear optics has been widely applied in biomedical studies.Especially in the aspect of bio-imaging,nonlinear optical techniques can provide rapid,label-free and chemically specific imaging of biological samples,which enable the investigation of biological processes and analysis of samples beyond other microscopy techniques.In this review,we focus on the introduction of nonlinear optical processes and their applications in bio-imaging as well as the recent advances in this filed.Our perspective of this field is also presented.

Nonlinear scanning structured illumination microscopy based on nonsinusoidal modulation

Structured illumination microscopy(SIM)is an essential super-resolution microscopy technique that enhances resolution.Several images are required to reconstruct a super-resolution image.However,linear SIM resolution enhancement can only increase the spatial resolution of micros-copy by a factor of two at most because the frequency of the structured illumination pattern is limited by the cutoff frequency of the excitation point spread function.The frequency of the pattern generated by the nonlinear response in samples is not limited;therefore,nonlinear SIM(NL-SIM),in theory,has no inherent limit to the resolution.In the present study,we describe a two-photon nonlinear SIM(2P-SIM)technique using a multiple harmonics scanning pattern that employs a composite structured illumination pattern,which can produce a higher order harmonic pattern based on the fluorescence nonlinear response in a 2P process.The theoretical models of super-resolution imaging were established through our simulation,which describes the working mechanism of the multi-frequency structure of the nonsinusoidal function to improve the reso-lution.The simulation results predict that a 5-fold improvement in resolution in the 2P-SIM is possible.

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.

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.

Improvement in Resolution of Multiphoton Scanning Structured Illumination Microscopy via Harmonics

We describe a multiphoton(mP)-structured illumination microscopy(SIM)technique,which demonstrates substantial improvement in image resolution compared with linear SIM due to the nonlinear response of fluorescence.This nonlinear response is caused by the effect of nonsinusoidal structured illumination created by scanning a sinusoidally modulated illumination to excite an mP fluorescence signal.The harmonics of the structured fluorescence illumination are utilised to improve resolution.We present an mP-SIM theory for reconstructing the super-resolution image of the system.Theoretically,the resolution of our m P-SIM is unlimited if all the high-order harmonics of the nonlinear response of fluorescence are considered.Experimentally,we demonstrate an 86 nm lateral resolution for two-photon(2P)-SIM and a 72 nm lateral resolution for second-harmonic-generation(SHG)-SIM.We further demonstrate their application by imaging cells stained with F-actin and collagen fibres in mouse-tail tendon.Our method can be directly used in commercial mP microscopes and requires no specific fluorophores or high-intensity laser.

Development of a hydrogen peroxide-responsive and oxygen-carrying nanoemulsion for photodynamic therapy against hypoxic tumors using phase inversion composition method

Photodynamic therapy(PDT)has become an attractive tumor treatment modality because of its noninvasive feature and low side effects.However,extreme hypoxia inside solid tumors severely impedes PDT therapeutic outcome.To overcome this obstacle,various strategies have been developed recently.Among them,in situ oxygen generation,which relies on the decom-position of tumor endogenous H_(2)O_(2),and oxygen delivery tactic using high oxygen loading capacity of hemoglobin or perfluorocarbons,have been widely studied.The in situ oxygen generation strategy has high specificity to tumors,but its oxygen-generating efficiency is lim-ited by the intrinsically low tumor H_(2)O_(2)level.In contrast,the oxygen delivery approach holds advantage of high oxygen loading efficiency,nevertheless lacks tumor specificity.In this work,we prepared a nanoemulsion system containing H_(2)O_(2)-responsive catalase,highly efficient ox-ygen carrier perfiuoropolyether(PFPE),and a near-infrared(NIR)light activatable photo-sensitizer IR780,to combine the high tumor specificity of the in situ oxygen generation strategy and the high efficiency of the oxygen delivery strategy.This concisely prepared nanoplatform exhibited enhanced and H_(2)O_(2)-controllable production of singlet oxygen under light excitation,satisfactory cytocompatibility,and ability to kill cancer cells under NIR light excitation.This highlights the potential of this novel nanoplatform for highly efficient and selective NIR light mediated PDT against hypoxic tumors.This research provides new insight into the design of intelligent nanoplatform for relieving tumor hypoxia and enhancing the oxygen-dependent PDT effects in hypoxic tumors.

RECENT PROGRESS IN MULTIFOCAL MULTIPHOTON MICROSCOPY

Multifocal multiphoton microscopy(MMM)has recently become an important tool in biomedicine for performing three-dimensional fastfluorescence imaging.Using various beamsplitting techniques,MMM splits the near-infrared laser beam into multiple beamlets and produces a multifocal array on the sample for parallel multiphoton excitation and then recordsfluorescence signal from all foci simultaneously with an area array detector,which significantly improves the imaging speed of multiphoton microscopy and allows for high efficiency in use of the excitation light.In this paper,we discuss the features of several MMM setups using different beamsplitting devices,including a Nipkow spinning disk,a microlens array,a set of beamsplitting mirrors,or a diffractive optical element(DOE).In particular,we present our recent work on the development of an MMM using a spatial light modulator(SLM).

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.