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[Display omitted] • Sonochemical synthesis of methylene blue analogues. • X-Ray Diffraction analysis of 3,7-bis(N -(meta -bromoaryl)amino)phenothiazinium dye. • UV–vis absorption/emission properties of N -(arylamino)phenothiazinium cationic dyes. • Absorption/emission properties by TD-DFT using B3PW91 versus CAM-B3LYP functionals. • Fluorescence Imaging of N -(arylamino)phenothiazinium cationic dyes. Novel (N -arylamino)phenothiazinium dyes containing meta -substituted-arylamine auxochrome units were successfully obtained by applying a sonochemical protocol designed for a more efficient energy usage in the preparation of methylene blue (MB) analogues. Single crystal X-ray diffraction analysis revealed the spatial arrangement in aggregated crystalline state of (N -(meta -bromoaryl)amino)phenothiazinium dye with minor variances induced by the nature of the halogenide counterion (iodide or chloride). The optical UV–vis properties of the novel (N -arylamino)phenothiazinium dyes were comparable to those of the parent MB, with the longest wavelength absorption maxima situated in the visible range (640–680 nm), large molar extinction coefficients (log ε = 4.5–5.1) and weak solvatochromism in polar solvents. Their fluorescence emission in solid state was evidenced by One Photon Excited Fluorescence Lifetime Imaging (OPE-FLIM) and Two Photon Excited Fluorescence Lifetime Imaging (TPE-FLIM) experiments. Theoretical calculations based on Time Dependent-Density Functional Theory (TD-DFT) at B3PW91 and CAM-B3LYP/def2-SV(P) level of theory predicted absorption and fluorescence emission wavelength maxima in reasonable agreement with experimental data. Computational results suggest that the electronic excitations imply a departure from the planar molecular ground state towards geometrically rearranged excited states disfavoring the vibronic couplings due to a high degree of flexibility induced by the conformational motion of the N -arylamino auxochromes. Preliminary studies regarding the dyes' relevance in biological environment indicated lipophilicity (log P octanol/water 0.5–2.3), no aggregation tendency in diluted solutions in the concentration range 10–50 microM and ability for cytoplasmatic staining of D407 human retinal pigment epithelial cells. [ABSTRACT FROM AUTHOR]

Understanding the delivery and diffusion of topically-applied drugs on human skin is of paramount importance in both pharmaceutical and cosmetics research. This information is critical in early stages of drug development and allows the identification of the most promising ingredients delivered at optimal concentrations to their target skin compartments. Different skin imaging methods, invasive and non-invasive, are available to characterize and quantify the spatiotemporal distribution of a drug within ex vivo and in vivo human skin. The first part of this review detailed invasive imaging methods (autoradiography, MALDI and SIMS). This second part reviews non-invasive imaging methods that can be applied in vivo : i) fluorescence (conventional, confocal, and multiphoton) and second harmonic generation microscopies and ii) vibrational spectroscopic imaging methods (infrared, confocal Raman, and coherent Raman scattering microscopies). Finally, a flow chart for the selection of imaging methods is presented to guide human skin ex vivo and in vivo drug delivery studies. Unlabelled Image [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Photophysics of a new fluorescent On/Off compound were studied. • The buffer mediated ESPT reaction has been deeply studied and the kinetic constant recovered. • Competitive studies with other anions present in blood were done. • This dye can be potentially used as acetate probe in blood samples. Abstract Development of biomarkers of analytes with interest in clinic is an important field of study. In this work, we synthesized and analyzed the new fluorescent acetate-biomarker, Iso-PG. The mechanism of detection is the acetate buffer mediated proton transfer reaction. The rate constants involved were obtained, and we measured the change in the fluorescence lifetime produced as a consequence of the presence of acetate in the medium. Finally, we checked its potential use as acetate biomarker in synthetic serum. [ABSTRACT FROM AUTHOR]

Abstract In this paper, a non-iterative large-solution-area fluorescence-lifetime-extraction algorithm applying to the mono-exponential model have been proposed. This algorithm consists of two parts. One is a preprocessing of the decay histogram through first comparing the ratio of the photon counts of the first two original time channels with the corresponding thresholds, and then merging the appropriate number of time channels. The other is a fluorescence-lifetime-extraction formula, which is deduced by expressing the photon counts as an integral based on Boole's rule. Simulation results show its solvable range is 0.003<τ/MW<0.9 with 225 original time channels. With such a large solvable area, the proposed algorithm has applicable value in fluorescence lifetime imaging microscopies. [ABSTRACT FROM AUTHOR]

The rising prevalence of antibiotic-resistant Gram-negative bacteria presents a major challenge for healthcare and requires the development of new and effective detection techniques. In this study, we present a novel and simple method for the selective detection of Gram-negative bacteria using multi-emissive colistin-passivated carbon dots (m-CCD) based on Fluorescence Lifetime (FLT) response and bioimaging. The m-CCD was synthesized using a one-step microwave-assisted hydrothermal process, and its interaction with Gram-negative bacteria was studied using Fluorescence Lifetime Imaging Microscopy (FLIM) and automated Confocal Laser Scanning Microscopy (CLSM). The investigations revealed a notable reduction in the FLT of m-CCD-treated Gram-negative bacteria (average lifetime of m-CCD, (τ avg) m-CCD is 3.91 ns and m-CCD treated Gram-negative bacteria, (τ avg) m-CCD-bac is 1.57 ns), demonstrating the selective interaction between them. Furthermore, the CLSM data revealed high specificity of m-CCD for Gram-negative bacteria, even in polymicrobial samples. Importantly, we also demonstrate that this method can quantify Escherichia coli (E. coli) cells with two linear ranges such as 3.40 × 105 – 9.80 × 105 CFU mL−1 and 6.90 × 107–4.14 × 108 CFU mL−1 in standard culture and real samples such as tap water and human urine with a limit of detection ∼ 3.68–4.89 × 104 CFU mL−1. This straightforward method has great potential for the rapid and selective detection of Gram-negative bacteria, paving the way for early diagnosis and treatment of bacterial infections. [Display omitted] ● FLIM and multicolor imaging based Gram-negative bacteria detection using colistin passivated carbon dots (m-CCD). ● m-CCD showed brilliant photostability, high water dispersibility and quantum yield. ● Average lifetime of m-CCD, (τ avg) m-CCD (3.91 ns) and m-CCD treated E. coli , (τ avg) m-CCD-bac (1.57 ns) was calculated. ● Quantification of E. coli cells (3.40 × 105 to 4.14 × 108 CFU mL-1) in standard and real samples was demonstrated. [ABSTRACT FROM AUTHOR]

Objective: To determine whether metabolic imaging with the use of fluorescence lifetime imaging microscopy (FLIM) identifies metabolic differences between normal oocytes and those with metabolic dysfunction.Design: Experimental study.Setting: Academic research laboratories.Patient(s): None.Intervention(s): Oocytes from mice with global knockout of Clpp (caseinolytic peptidase P; n = 52) were compared with wild-type (WT) oocytes (n = 55) as a model of severe oocyte dysfunction. Oocytes from old mice (1 year old; n = 29) were compared with oocytes from young mice (12 weeks old; n = 35) as a model of mild oocyte dysfunction.Main Outcome Measure(s): FLIM was used to measure the naturally occurring nicotinamide adenine dinucleotide dehydrogenase (NADH) and flavin adenine dinucleotide (FAD) autofluorescence in individual oocytes. Eight metabolic parameters were obtained from each measurement (4 per fluorophore): short (τ1) and long (τ2) fluorescence lifetime, fluorescence intensity (I), and fraction of the molecule engaged with enzyme (F). Reactive oxygen species (ROS) levels and blastocyst development rates were measured to assess illumination safety.Result(s): In Clpp-knockout oocytes compared with WT, FAD τ1 and τ2 were longer and I was higher, NADH τ2 was longer, and F was lower. In old oocytes compared with young ones, FAD τ1 was longer and I was lower, NADH τ1 and τ2 were shorter, and I and F were lower. FLIM did not affect ROS levels or blastocyst development rates.Conclusion(s): FLIM parameters exhibit strong differentiation between Clpp-knockout versus WT, and old versus young oocytes. FLIM could potentially be used as a noninvasive tool to assess mitochondrial function in oocytes. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Fluorescence lifetime of lignin was correlated with cellulose conversion and lignin traits. • FRET analysis showed higher interaction of fluorescent PEG probes with chlorite-treated cell-wall. • Fluorescence can provide fast method to estimate biomass susceptibility to enzymatic conversion. Abstract The conversion of cell wall biomass to sugar for fermentation to ethanol requires chemical or physical pretreatments to disrupt the recalcitrant plant cell walls and to make the cellulose accessible to cellulolytic enzymes. Multiscale study of biomass deconstruction gives access to key insights into the cell wall and lignin changes induced by pretreatment. Few studies have compared the effect of pretreatment of different biomass species on the cell wall accessibility. Considering representative softwood (pine) and hardwood (poplar) biomass, we studied the impact of two pretreatments on enzymatic saccharification and cell wall structure and accessibility. Lignin fluorescence properties were investigated by measuring fluorescence lifetime in addition to chemical analysis, and the accessibility of biomass was assessed using fluorescent probes consisting of rhodamine labeled polyethylene glycol (PEG) molecules ranging from 10 to 40 kDa. Hot water treatment and chlorite delignification altered chemical structure and fluorescence lifetime, which was positively correlated with glucose conversion and negatively correlated with lignin and β-O-4′ contents. Imaging distribution of the probes indicated that chlorite pretreatment resulted in a more uniform distribution of probe in the cell wall compared to hot water treatment. The interaction between cell wall and fluorescent PEG probes was evaluated using Förster Resonance Energy Transfer (FRET) and fluorescence microscopy. The FRET efficiency showed a high negative correlation with the probe size and was greatly increased by chlorite delignification, reflecting increased accessibility to the probe and interaction. Thus the accessibility and interactions of small probes in pretreated biomass could be a relevant indicator of potential for saccharification, whereas fluorescence lifetime provides a new criteria for assessing relevant cell wall structural modifications related to enzymatic conversion of lignocellulosic biomass. [ABSTRACT FROM AUTHOR]

Graphical abstract Abstract Quantitative measurement of pH and metabolite gradients by microscopy is one of the challenges in the production of scaffold-grown organoids and multicellular aggregates. Herein, we used the cellulose-binding domain (CBD) of the Cellulomonas fimi CenA protein for designing biosensor scaffolds that allow measurement of pH and Ca2+ gradients by fluorescence intensity and lifetime imaging (FLIM) detection modes. By fusing CBD with pH-sensitive enhanced cyan fluorescent protein (CBD-ECFP), we achieved efficient labeling of cellulose-based scaffolds based on nanofibrillar, bacterial cellulose, and decellularized plant materials. CBD-ECFP bound to the cellulose matrices demonstrated pH sensitivity comparable to untagged ECFP (1.9–2.3 ns for pH 6–8), thus making it compatible with FLIM-based analysis of extracellular pH. By using 3D culture of human colon cancer cells (HCT116) and adult stem cell-derived mouse intestinal organoids, we evaluated the utility of the produced biosensor scaffold. CBD-ECFP was sensitive to increases in extracellular acidification: the results showed a decline in 0.2–0.4 pH units in response to membrane depolarization by the protonophore FCCP. With the intestinal organoid model, we demonstrated multiparametric imaging by combining extracellular acidification (FLIM) with phosphorescent probe-based monitoring of cell oxygenation. The described labeling strategy allows for the design of extracellular pH-sensitive scaffolds for multiparametric FLIM assays and their use in engineered live cancer and stem cell-derived tissues. Collectively, this research can help in achieving the controlled biofabrication of 3D tissue models with known metabolic characteristics. Statement of Significance We designed biosensors consisting of a cellulose-binding domain (CBD) and pH- and Ca2+-sensitive fluorescent proteins. CBD-tagged biosensors efficiently label various types of cellulose matrices including nanofibrillar cellulose and decellularized plant materials. Hybrid biosensing cellulose scaffolds designed in this study were successfully tested by multiparameter FLIM microscopy in 3D cultures of cancer cells and mouse intestinal organoids. [ABSTRACT FROM AUTHOR]

We performed spectroscopic investigations of four novel Sc(III) metal-organic complexes with β -diketonate ligands. It was demonstrated that bonding the ligands with an optically passive Sc(III) ion leads to sensitization of the luminescence of the complexes and affects their optical absorption spectra. Luminescence decays of the complexes exhibit non-exponential behaviour. From spatially-resolved decay measurements we evaluated the number of different sites responsible for the luminescence in the investigated complexes. Small variations of the ligand structure significantly affects the wavelength of emission maximum. This phenomenon may occur due to the difference in crystal packing of the complexes or formation of charge-transfer states. [ABSTRACT FROM AUTHOR]

Lensoside Aβ, representing the flavonol glycosides, is a compound isolated from the aerial parts of edible lentil ( Lens culinaris ) cultivar Tina. This substance arouses interest because so far there is very little data about secondary metabolites isolated from the leaves and stems of this plant. Additionally, bioactive potential of flavonoids is directly coupled with the membranes as a primary target of their physiological and pharmacological activity. The aim of this study was to investigate the effect of lensoside Aβ on lipid membranes. Interaction of examined compound with liposomes formed with dipalmitoylphosphatidylcholine (DPPC) was investigated with application of FTIR spectroscopy and 1 H NMR technique. Molecular localization and orientation of lensoside Aβ in a single lipid bilayer system represented by giant unilamellar vesicles, was also investigated with application of confocal fluorescence lifetime imaging microscopy (FLIM). FTIR analysis revealed that the tested compound incorporates into DPPC membranes via hydrogen bonding to lipid polar head groups in the PO 2 group region and the C O P O C segment. Furthermore 1 H NMR analysis showed ordering effect in both the hydrophobic alkyl chains region and the polar heads of phospholipids. FLIM investigation has revealed roughly parallel orientation of its molecules in the membranes. This suggests that one of the possible physiological functions of this flavonol could be screening a cell against short-wavelength radiation. [ABSTRACT FROM AUTHOR]

[Display omitted] Organoid models have been used to address important questions in developmental and cancer biology, tissue repair, advanced modelling of disease and therapies, among other bioengineering applications. Such 3D microenvironmental models can investigate the regulation of cell metabolism, and provide key insights into the mechanisms at the basis of cell growth, differentiation, communication, interactions with the environment and cell death. Their accessibility and complexity, based on 3D spatial and temporal heterogeneity, make organoids suitable for the application of novel, dynamic imaging microscopy methods, such as fluorescence lifetime imaging microscopy (FLIM) and related decay time-assessing readouts. Several biomarkers and assays have been proposed to study cell metabolism by FLIM in various organoid models. Herein, we present an expert-opinion discussion on the principles of FLIM and PLIM, instrumentation and data collection and analysis protocols, and general and emerging biosensor-based approaches, to highlight the pioneering work being performed in this field. [ABSTRACT FROM AUTHOR]

Conventional antimicrobial strategies have become increasingly ineffective due to the rapid emergence of antibiotic resistance among pathogenic bacteria. In order to overcome this problem, antimicrobial PhotoDynamic Therapy (PDT) is considered a promising alternative therapy. PDT has a broad spectrum of action and low mutagenic potential. It is particularly effective when microorganisms present endogenous photosensitizing pigments. Helicobacter pylori (Hp), a pathogen notoriously responsible of severe gastric infections (chronic gastritis, peptic ulcer, MALT lymphoma and gastric adenocarcinoma), produces and accumulates the photosensitizers protoporphyrin IX and coproporphyrin, thus it might be a suitable target of antimicrobial PDT. With the aim to design and develop an ingestible LED-based robotic pill for intragastric phototherapy, so that irradiation can be performed in situ without the use of invasive endoscopic light, photophysical studies on the Hp endogenous photosensitizers were carried out. These studies represent an important prerequisite in order to select the most effective irradiation conditions for Hp eradication. The photophysical characterization of Hp porphyrins, including their spectroscopic features in terms of absorption, steady-state and time-resolved fluorescence, was performed on bacterial extracts as well as within planktonic and biofilm growing Hp cells. [ABSTRACT FROM AUTHOR]

The emerging field of nanomedicine provides new approaches for the diagnosis and treatment of diseases, for symptom relief, and for monitoring of disease progression. Topical application of drug-loaded nanoparticles for the treatment of skin disorders is a promising strategy to overcome the stratum corneum, the upper layer of the skin, which represents an effective physical and biochemical barrier. The understanding of drug penetration into skin and enhanced penetration into skin facilitated by nanocarriers requires analytical tools that ideally allow to visualize the skin, its morphology, the drug carriers, drugs, their transport across the skin and possible interactions, as well as effects of the nanocarriers within the different skin layers. Here, we review some recent developments in the field of fluorescence microscopy, namely Fluorescence Lifetime Imaging Microscopy (FLIM)), for improved characterization of nanocarriers, their interactions and penetration into skin. In particular, FLIM allows for the discrimination of target molecules, e.g. fluorescently tagged nanocarriers, against the autofluorescent tissue background and, due to the environmental sensitivity of the fluorescence lifetime, also offers insights into the local environment of the nanoparticle and its interactions with other biomolecules. Thus, FLIM shows the potential to overcome several limits of intensity based microscopy. [ABSTRACT FROM AUTHOR]

Actinic Keratosis’ (AKs) are small skin lesions that are related to a prolonged sun-damage, which can develop into invasive squamous cell carcinoma (SCC) when left untreated. Effective, specific and well tolerable therapies to cure AKs are still of great interest. Diclofenac (DCF) is the current gold standard for the local treatment of AKs in terms of costs, effectiveness, side effects and tolerability. In this work, an electrospun polylactic acid (PLA) scaffold loaded with a synthetic DCF prodrug was developed and characterized. Specifically, the prodrug was successfully synthetized by binding DCF to a glycine residue via solid phase peptide synthesis (SPPS) and then incorporated in an electrospun PLA scaffold. The drug encapsulation was verified using multiphoton microscopy (MPM) and its scaffold release was spectrophotometrically monitored and confirmed with MPM. The scaffold was further characterized with scanning electron microscopy (SEM), tensile testing and contact angle measurements. Its biocompatibility was verified by performing a cell proliferation assay and compared to PLA scaffolds containing the same amount of DCF sodium salt (DCFONa). Finally, the effect of the electrospun scaffolds on human dermal fibroblasts (HDFs) morphology and metabolism was investigated by combining MPM with fluorescence lifetime imaging microscopy (FLIM). The obtained results suggest that the obtained scaffold could be suitable for the controlled and targeted delivery of the synthesized prodrug for the treatment of AKs. Statement of Significance Electrospun scaffolds are of growing interest as materials for a controlled drug delivery. In this work, an electrospun polylactic acid scaffold containing a synthetically obtained Diclofenac prodrug is proposed as a novel substrate for the topical treatment of actinic keratosis. A controlled drug delivery targeted to the area of interest could enhance the efficacy of the therapy and favor the healing process. The prodrug was synthesized via solid phase, employing a clean and versatile approach to obtain Diclofenac derivatives. Here, we used multiphoton microscopy to image drug encapsulation within the fibrous scaffold and fluorescence lifetime imaging microscopy to investigate Diclofenac effects and potential mechanisms of action. [ABSTRACT FROM AUTHOR]

We report the use of the ‘grafting from’ approach to produce inherently rod-shaped polymer nanoparticles with triggered drug release. Cylindrical polymer brushes (CPBs) can be directly used to yield functional polymer nanorods for pH-sensitive drug release of doxorubicin (DOX). Water-soluble CPBs have been produced via a straightforward one-step grafting of vinyl benzaldehyde (VBA) and poly(ethylene glycol) methyl ether methacrylate (PEGMA) comonomers, in which the VBA distributed throughout the CPBs provides a cost-effective and simple functionality for the subsequent conjugation of DOX using imine chemistry. Atomic force microscopy (AFM) underlined the rod-like conformation of the CPBs prior and after drug conjugation. Fluorescence spectroscopy studies revealed faster drug release in acidic environments (pH 5.0) compared to physiological pH conditions (pH 7.4). Fluorescence lifetime imaging microscopy (FLIM) and in vitro cell studies further highlighted the intracellular DOX release from the CPB drug carriers within MCF-7 breast cancer cells. [ABSTRACT FROM AUTHOR]

Cyclometallated iridium(III) complexes have unique photophysical properties that turn them into promising photosensitizers and potential fluorescence lifetime imaging probes. Herein, we developed a novel cyclometallated iridium(III) complex (Ir–OH) with aggregation-induced emission (AIE) properties for FLIM imaging-guided photodynamic therapy. Studies on photophysical properties show that Ir–OH exhibits excellent fluorescence response to in situ mitochondrial viscosity with varied lifetime. In addition, Ir–OH shows satisfactory multiphoton absorption properties in the near-infrared region (Two-photon: 700–800 nm; Three-photon: 1150–1450 nm). More importantly, changes in mitochondrial viscosity during photodynamic processes can be quantified in real-time by FLIM. Thus, our work illustrates that cyclometallated iridium(III) complexes can simultaneously induce and precisely detect microenvironmental changes at the subcellular level using FLIM, leading to self-reporting PDT outcomes. Procedure for photodynamic therapy with Ir–OH. [Display omitted] • Ir–OH demonstrated the difference in fluorescence lifetime at different intracellular viscosities. • Ir–OH has outstanding nonlinear optical properties due to its octupolar structures, long excited wavelength allows for deep tissue penetration. • Ir–OH exhibits good photodynamic effects both in vivo and in vitro. [ABSTRACT FROM AUTHOR]

[Display omitted] • Radachlorin fluorescence lifetime and quantum yield vary with microenvironment pH. • Radachlorin fluorescence lifetimes differ in different cell compartments. • Radachlorin localizes predominantly in lysosomes. • Changes of Radachlorin fluorescence lifetimes with pH are reversible. Intracellular localization of photosensitizer molecules is influential on cell death pathway at photodynamic treatment and is thus an important aspect in achieving enhanced efficacy of photodynamic therapy. In this paper we performed thorough studies of the distribution of Radachlorin photosensitizer in three established cell lines: HeLa, A549, and 3T3 with fluorescence lifetime imaging microscopy through the analysis of lifetime distributions. Experiments carried out in Radachlorin solutions in phosphate buffered saline revealed the pronounced dependence of the fluorescence quantum yield and lifetime on solution pH. This finding was used for analysis of lifetime images of living cells and their phasor plot representations and allowed us to suggest that Radachlorin localized predominantly in lysosomes, known to have acidic pH values. Experiments on co-localization of Radachlorin fluorescence lifetimes and LysoTracker fluorescence intensity supported this suggestion. The results obtained show that the inhomogeneity of fluorescence quantum yield within a cell can be significant due to lower pH values in lysosomes than in other intracellular compartments. This finding suggests that the actual amount of accumulated Radachlorin can be underestimated if being evaluated solely by comparison of fluorescence intensities. [ABSTRACT FROM AUTHOR]

The non-diffracting Airy beams have been reported to increase acquisition speed by projecting the axially significantly elongated information to a two-dimensional (2D) image. Due to the elongated focal length, an Airy volumetric image can cover information as much as stack images with multiple Gaussian images at different imaging depths, which significantly increases the volumetric imaging rate. However, the projection of three-dimensional (3D) structures on a 2D plane is usually indistinguishable, especially those that overlap along the axis. Therefore, we proposed an axially resolved volume imaging technique based on a two-photon fluorescence lifetime microscopy imaging (FLIM) by using Airy beams. [ABSTRACT FROM AUTHOR]

The decay time of the fluorescence of excited molecules, called fluorescence lifetime, can provide information about the cuticle composition additionally to widely used spectral characteristics. We compared autofluorescence lifetimes of different cuticle regions in the copulatory organ of females of the bedbug, Cimex lectularius . After two-photon excitation at 720 nm, regions recently characterised as being rich in resilin showed a longer bimodal distribution of the mean autofluorescence lifetime τ m (tau-m) at 0.4 ns and 1.0–1.5 ns, while resilin-poor sites exhibited a unimodal pattern with a peak around 0.8 ns. The mean lifetime, and particularly its second component, can be useful to distinguish resilin-rich from resilin-poor parts of the cuticle. The few existing literature data suggest that chitin is unlikely responsible for the main autofluorescent component observed in the resilin-poor areas in our study and that melanin requires further scrutiny. Autofluorescence lifetime measurements can help to characterise properties of the arthropod cuticle, especially when coupled with multiphoton excitation to allow for deeper tissue penetration. [ABSTRACT FROM AUTHOR]

Inflammatory disorders of the skin pose particular therapeutic challenges due to complex structural and functional alterations of the skin barrier. Penetration of several anti-inflammatory drugs is particularly problematic in psoriasis, a common dermatitis condition with epidermal hyperplasia and hyperkeratosis. Here, we tested in vivo dermal penetration and biological effects of dendritic core-multishell-nanocarriers (CMS) in a murine skin model of psoriasis and compared it to healthy skin. In both groups, CMS exclusively localized to the stratum corneum of the epidermis with only very sporadic uptake by Langerhans cells. Furthermore, penetration into the viable epidermis of nile red as a model for lipophilic compounds was enhanced by CMS. CMS proved fully biocompatible in several in vitro assays and on normal and psoriatic mouse skin. The observations support the concept of CMS as promising candidates for drug delivery in inflammatory hyperkeratotic skin disorders in vivo . [ABSTRACT FROM AUTHOR]

Intracellular fluorescence lifetime images of the endogenous fluorophores of nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD), which are well known as autofluorescence chromophores, were obtained from rat normal fibroblast cells (WFB) and H- ras oncogene-transfected cancer cells among WFB (W31). The average lifetime of the NADH and FAD autofluorescence was shorter in cancer cells than in normal cells, indicating that the difference in metabolism between healthy and cancer cells alters the conditions for coenzymes such as NADH and FAD and that the autofluorescence lifetime measurement of NADH and FAD is applicable for the noninvasive diagnosis of cancer cells. The pico- and nano-second time-resolved fluorescence spectra of NADH obtained with different time windows were similar in normal and cancer cells, indicating that every fluorescence decay component gives the same spectrum in both cell types. These results as well as the fluorescence lifetime images of exogenous fluorophores stained with sodium pheophorbide a in normal and cancer cells suggest that the difference in the fluorescence lifetime between normal and cancer cells cannot be attributed to a difference in the intracellular pH or refractive index but to the difference in the bound condition between proteins and NADH or FAD under the different intracellular environments of normal and cancer cells. [ABSTRACT FROM AUTHOR]

The redox states of the NAD and NADP pyridine nucleotide pools play critical roles in defining the activity of energy producing pathways, in driving oxidative stress and in maintaining antioxidant defences. Broadly speaking, NAD is primarily engaged in regulating energy-producing catabolic processes, whilst NADP may be involved in both antioxidant defence and free radical generation. Defects in the balance of these pathways are associated with numerous diseases, from diabetes and neurodegenerative disease to heart disease and cancer. As such, a method to assess the abundance and redox state of these separate pools in living tissues would provide invaluable insight into the underlying pathophysiology. Experimentally, the intrinsic fluorescence of the reduced forms of both redox cofactors, NADH and NADPH, has been used for this purpose since the mid-twentieth century. In this review, we outline the modern implementation of these techniques for studying mitochondrial redox state in complex tissue preparations. As the fluorescence spectra of NADH and NADPH are indistinguishable, interpreting the signals resulting from their combined fluorescence, often labelled NAD(P)H, can be complex. We therefore discuss recent studies using fluorescence lifetime imaging microscopy (FLIM) which offer the potential to discriminate between the two separate pools. This technique provides increased metabolic information from cellular autofluorescence in biomedical investigations, offering biochemical insights into the changes in time-resolved NAD(P)H fluorescence signals observed in diseased tissues. [ABSTRACT FROM AUTHOR]

Comparable intracellular concentrations (≈ 30 pmol/10 6 cells) of bovine serum albumin-ZnPc (BSA) adduct outperformed dipalmitoyl-phosphatidyl-choline (DPPC) liposomes containing ZnPc at photodynamic-killing of human cervical cancer cells (HeLa) after only 15 min of irradiation using red light (λ > 620 nm, 30 W/cm 2 ). This result could not be simply explained in terms of dye aggregation within the carrier, since in the liposomes the dye was considerably less aggregated than in bovine serum albumin, formulation that was capable to induce cell apoptosis upon red light exposure. Thus, using specific organelle staining, our cumulative data points towards intrinsic differences in intra-cellular localization depending on the cargo vehicle used, being ZnPc:BSA preferentially located in the near vicinity of the nucleus and in the Golgi structures, while the liposomal formulation ZnPc:DPPC was preferentially located in cellular membrane and cytoplasm. In addition to those differences, using real-time advanced fluorescence lifetime imaging of HeLa cells loaded with the photosensitizer contained in the different vehicles, we have found that only for the ZnPc:BSA formulation, there was no significant changes in the fluorescence lifetime of the photosensitizer inside the cells. This contrasts with the in situ ≈ two-fold reduction of the fluorescence lifetime measured for the liposomal ZnPc formulation. Those observations point towards the superiority of the protein to preserve dye aggregation, and its photochemical activity, post-cell uptake, demonstrating the pivotal role of the delivery vehicle at determining the ultimate fate of a photosensitizer. [ABSTRACT FROM AUTHOR]

A novel 1,8-naphthalimide-based chemosensor was designed and synthesized for rapid recognition of Cr 3+ . The desired sensor showed off-on fluorescent and lifetime-based response upon Cr 3+ and S 2− in solution, on test paper and in cells. With the intensity-based method, the limit of quantification (LOQ) value was 0–5.5 × 10 −5 M and the detection limit could be as low as 0.60 ppm. The ' in situ ' prepared Cr 3+ complex can recognize S 2− among a series of common anions with high selectivity and sensitivity, the LOD can be as low as 307 nM. The lifetime of the sensor changes from 4.95 to 4.89 and further to 5.88 ns upon addition of Cr 3+ and S 2− in turn. [ABSTRACT FROM AUTHOR]

According to the recent developments in imaging strategies and in tailoring fluorescent molecule as probe for monitoring biological systems, we coupled a Bodipy-based molecular rotor ( BoMe ) with FLIM phasor approach to evaluate the viscosity in different intracellular domains. BoMe rapidly permeates cells, stains cytoplasmic as well as nuclear domains, and its optical properties make it perfectly suited for widely diffused confocal microscopy imaging setups. The capability of BoMe to report on intracellular viscosity was put to the test by using a cellular model of a morbid genetic pathology (Hutchinson–Gilford progeria syndrome, HGPS). Our results show that the nucleoplasm of HGPS cells display reduced viscosity as compared to normal cells. Since BoMe displays significant affinity towards DNA, as demonstrated by an in vitro essay, we hypothesize that genetic features of HGPS, namely the misassembly of lamin A protein within the nuclear lamina, modulates chromatin compaction. This hypothesis nicely agrees with literature data. [ABSTRACT FROM AUTHOR]

Luminescence decay functions describe the time dependence of the intensity of radiation emitted by electronically excited species. Decay phasor plots (plots of the Fourier sine transform vs. the Fourier cosine transform, for one or several angular frequencies) are being increasingly used in fluorescence, namely in lifetime imaging microscopy (FLIM). In this work, a detailed study of the sum of two exponentials decay function is carried out revealing that sub-exponential, super-exponential and unimodal decays have different phasor signatures. A generalization of the lever rule is obtained, and the existence of an outermost phasor curve corresponding to intermediate-like decays is demonstrated. A study of the behavior of more complex decay functions (sum of three exponentials, stretched and compressed exponentials, phosphorescence with reabsorption and triplet–triplet annihilation, fluorescence with quantum beats) allows concluding that a rich diversity of phasor plot patterns exists. In particular, super-exponential decays can present complex shapes, spiraling at high frequencies. The concept of virtual phasor is also introduced. [ABSTRACT FROM AUTHOR]

Intracellular chloride ion concentration ([Cl–] i) homeostasis is critical for excitatory/inhibitory balance and volume regulation in neurons. We quantitatively map spatiotemporal dendritic [Cl–] i dynamics during N -methyl- d -aspartate (NMDA) excitotoxicity to determine how Cl– changes contribute to localized dendritic swelling (blebbing) in stroke-like conditions. Whole-cell patch clamp electrophysiology combined with simultaneous fluorescence lifetime imaging (FLIM) of the Cl– dye N -(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE; MQAE-FLIM) reliably report resting and dynamic [Cl–] i shifts in dendrites. NMDA application generates spatially restricted and persistent high [Cl–] i subdomains at dendritic blebs in a process that requires Ca2+ influx and the subsequent opening of small-conductance Ca2+-activated K+ (SK) channels. We propose sustained and localized K+ efflux increased extracellular K+ concentrations ([K+] o) sufficiently at discrete regions to reverse K+-Cl– cotransporter (KCC2) transport and trigger synaptic swelling. Together, our data establish a mechanism for KCC2 to generate pathological [Cl–] i microdomains in blebbing with relevance for multiple neurological disorders. [Display omitted] • Spatiotemporal [Cl–] i dynamics during dendrite swelling were imaged using FLIM • Pathological high [Cl–] i microdomains triggered dendritic swelling and blebbing • Cl– entry and blebbing required NMDAR Ca2+ influx and subsequent SK channel opening • Sustained K+ efflux via SK channels leads to KCC2 reversal and pathological Cl– uptake Neuropathological dendritic swelling and blebbing are shown by Weilinger et al. to be triggered by pathological, non-uniform [Cl–] i accumulation driven by NMDAR overstimulation and Ca2+ entry that leads to Ca2+-dependent opening of SK channels, K+ efflux, and Cl– dysregulation via KCC2 reversed uptake. [ABSTRACT FROM AUTHOR]

Time-resolved fluorescence spectroscopy and microscopy is increasingly used to probe molecular interactions and dynamics events in vitro and in vivo. We point out some pitfalls to avoid in the data acquisition procedure using time correlated single photon counting. A good accuracy in fluorescence decay measurements is not only linked to the counts in the peak channel but also to the statistics at the end of the curve. A too narrow time interval between successive excitation leads to an overlap of decays, and that should be taken into account in data analysis. The counting rate in the peak channel of the excitation profile should be close to the one of the fluorescence decay. Many distributions of lifetimes can fit an incomplete or noisy data set to satisfying precisions corresponding to close values of χ 2 . The maximum entropy principle is appropriate to distinguish among these in a consistent way. It is also shown that encoding a prior knowledge about the system study dramatically improves the quality of the recovered distribution, particularly in case of a set of discrete components. Based on simulated noisy quantify maximum entropy method (MEM) data analysis, we propose a simple strategy for estimating the quality of information and inferences we can draw from experimental results. [ABSTRACT FROM AUTHOR]

The transcription factor NF-E2 p45-related factor 2 (Nrf2) and its negative regulator Kelch-like ECH associated protein 1 (Keap1) control the expression of nearly 500 genes with diverse cytoprotective functions. Keap1, a substrate adaptor protein for Cullin3/Rbx1 ubiquitin ligase, normally continuously targets Nrf2 for degradation, but loses this ability in response to electrophiles and oxidants (termed inducers). Consequently, Nrf2 accumulates and activates transcription of its downstream target genes. Many inducers are phytochemicals, and cruciferous vegetables represent one of the richest sources of inducer activity among the most commonly used edible plants. Here we summarize the discovery of the isothiocyanate sulforaphane as a potent inducer which reacts with cysteine sensors of Keap1, leading to activation of Nrf2. We then describe the development of a quantitative Förster resonance energy transfer (FRET)-based methodology combined with multiphoton fluorescence lifetime imaging microscopy (FLIM) to investigate the interactions between Keap1 and Nrf2 in single live cells, and the effect of sulforaphane, and other cysteine-reactive inducers, on the dynamics of the Keap1–Nrf2 protein complex. We present the experimental evidence for the “cyclic sequential attachment and regeneration” or “conformation cycling” model of Keap1-mediated Nrf2 degradation. Finally, we discuss the implications of this mode of regulation of Nrf2 for achieving a fine balance under normal physiological conditions, and the consequences and mechanisms of disrupting this balance for tumor biology. [ABSTRACT FROM AUTHOR]

The skin is a potential site of entry for nanoparticles (NP) but the role of disease-associated barrier disturbances on the path and extent of skin penetration of NP remains to be characterized. Silica nanoparticles (SiO 2 -NP) possess promising potential for various medical applications. Here, effects of different skin barrier disruptions on the penetration of N -(6-aminohexyl)-aminopropyltrimethoxysilane (AHAPS) functionalized SiO 2 -NP were studied. AHAPS-SiO 2 -NP (55 ± 6 nm diameter) were topically applied on intact, tape stripped or on inflamed skin of SKH1 mice with induced allergic contact dermatitis for one or five consecutive days, respectively. Penetration of AHAPS-SiO 2 -NP through the skin was not observed regardless of the kind of barrier disruption. However, only after subcutaneous injection, AHAPS-SiO 2 -NP were incorporated by macrophages and transported to the regional lymph node only. Adverse effects on cells or tissues were not observed. In conclusion, AHAPS-SiO 2 -NP seem to not cross the normal or perturbed mouse skin. From the Clinical Editor Skin is a potential site of entry for nanoparticles; however, it is poorly understood how skin diseases may alter this process. In tape-stripped skin and allergic contact dermatitis models the delivery properties of AHAPS-SiO2 nanoparticles remained unchanged, and in neither case were these NP-s able to penetrate the skin. No adverse effects were noted on the skin in these models and control mice. [ABSTRACT FROM AUTHOR]

The determination of pH in the cell cytoplasm or in intracellular organelles is of high relevance in cell biology. Also in plant cells, organelle-specific pH monitoring with high spatial precision is an important issue, since e.g. ΔpH across thylakoid membranes is the driving force for ATP synthesis critically regulating photoprotective mechanisms like non-photochemical quenching (NPQ) of chlorophyll (Chl) fluorescence or the xanthophyll cycle. In animal cells, pH determination can serve to monitor proton permeation across membranes and, therefore, to assay the efficiency of drugs against proton-selective transporters or ion channels. In this work, we demonstrate the applicability of the pH-sensitive GFP derivative (eGFP-pHsens, originally termed deGFP4 by Hanson et al. [1]) for pH measurements using fluorescence lifetime imaging microscopy (FLIM) with excellent precision. eGFP-pHsens was either expressed in the cytoplasm or targeted to the mitochondria of Chinese hamster ovary (CHO-K1) cells and applied here for monitoring activity of the M2 proton channel from influenza A virus. It is shown that the M2 protein confers high proton permeability of the plasma membrane upon expression in CHO-K1 cells resulting in rapid and strong changes of the intracellular pH upon pH changes of the extracellular medium. These pH changes are abolished in the presence of amantadine, a specific blocker of the M2 proton channel. These results were obtained using a novel multi-parameter FLIM setup that permits the simultaneous imaging of the fluorescence amplitude ratios and lifetimes of eGFP-pHsens enabling the quick and accurate pH determination with spatial resolution of 500 nm in two color channels with time resolution of below 100 ps. With FLIM, we also demonstrate the simultaneous determination of pH in the cytoplasm and mitochondria showing that the pH in the mitochondrial matrix is slightly higher (around 7.8) than that in the cytoplasm (about 7.0). The results obtained for CHO-K1 cells without M2 channels in comparison to M2-expressing cells show that the pH dynamics is determined by the specific H+ permeability of the membrane, the buffering of protons in the internal cell lumen and/or an outwardly directed proton pump activity that stabilizes the interior pH at a higher level than the external acidic pH. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy. [ABSTRACT FROM AUTHOR]

In engineered cells, endothelin ETA and ETB receptors can heterodimerize. We tested whether this can also be observed in native tissue. Main methods Rat mesenteric resistance arteries (rMRA) were maintained in organ culture for 24h to upregulate ETB-mediated contractions in addition to their normal ETA-mediated responses. They were then exposed to 100nM linear ET-1 (ETB-agonist) labeled with Oregon Green 488 (OG488/L.-ET-1) and/or to 16nM intact ET-1 (ETA/ETB-agonist) labeled with the rhodamine dye TAMRA (TAMRA/ET-1). Two photon laser scanning microscopy (TPLSM) was used for the visualization of their binding in the tissue. Fluorescence Lifetime Imaging Microscopy (FLIM) was employed for measurements of the OG488/L.-ET-1 lifetime in the absence and presence of TAMRA/ET-1. Key findings After incubation with the labeled ligands, medial smooth muscle cells (SMCs) were efficiently stained and became visible under TPLSM. TAMRA/ET-1 bound to all SMCs whereas OG488/L.-ET-1 stained only groups of SMCs. Interaction of the two receptor subtypes in SMC was investigated in double staining experiments. Fluorescence lifetime of OG488/L.-ET-1 was reduced in the presence of TAMRA/ET-1, which indicates the occurrence of Fluorescence Resonant Energy Transfer (FRET) and suggests close proximity of the two receptor subtypes within the arterial wall. Significance The methodology that is introduced by these new observations may be useful to assess ET-receptor heterodimerization in biopsies from relevant experimental animal models and human patients. [ABSTRACT FROM AUTHOR]

Prion diseases are associated to the conversion of the prion protein into a misfolded pathological isoform. The mechanism of propagation of protein misfolding by protein templating remains largely unknown. Neuroblastoma cells were transfected with constructs of the prion protein fused to both CFP-GPI-anchored and to YFP-GPI-anchored and directed to its cell membrane location. Live-cell FRET imaging between the prion protein fused to CFP or YFP was measured giving consistent values of 10±2%. This result was confirmed by fluorescence lifetime imaging microscopy and indicates intermolecular interactions between neighbor prion proteins. In particular, considering that a maximum FRET efficiency of 17±2% was determined from a positive control consisting of a fusion CFP-YFP-GPI-anchored. A stable cell clone expressing the two fusions containing the prion protein was also selected to minimize cell-to-cell variability. In both, stable and transiently transfected cells, the FRET efficiency consistently increased in the presence of infectious prions — from 4±1% to 7±1% in the stable clone and from 10±2% to 16±1% in transiently transfected cells. These results clearly reflect an increased clustering of the prion protein on the membrane in the presence of infectious prions, which was not observed in negative control using constructs without the prion protein and upon addition of non-infected brain. Our data corroborates the recent view that the primary site for prion conversion is the cell membrane. Since our fluorescent cell clone is not susceptible to propagate infectivity, we hypothesize that the initial event of prion infectivity might be the clustering of the GPI-anchored prion protein. [ABSTRACT FROM AUTHOR]

Abstract: Photosystem II (PSII) is the pigment–protein complex which converts sunlight energy into chemical energy by catalysing the process of light-driven oxidation of water into reducing equivalents in the form of protons and electrons. Three-dimensional structures from x-ray crystallography have been used extensively to model these processes. However, the crystal structures are not necessarily identical to those of the solubilised complexes. Here we compared picosecond fluorescence of solubilised and crystallised PSII core particles isolated from the thermophilic cyanobacterium Thermosynechococcus elongatus. The fluorescence of the crystals is sensitive to the presence of artificial electron acceptors (K3Fe(CN)3) and electron transport inhibitors (DCMU). In PSII with reaction centres in the open state, the picosecond fluorescence of PSII crystals and solubilised PSII is indistinguishable. Additionally we compared picosecond fluorescence of native PSII with PSII in which Ca2 in the oxygen evolving complex (OEC) is biosynthetically replaced by Sr2+. With the Sr2+ replaced OEC the average fluorescence decay slows down slightly (81ps to 85ps), and reaction centres are less readily closed, indicating that both energy transfer/trapping and electron transfer are affected by the replacement. [Copyright &y& Elsevier]

In Alzheimer's disease (AD), the initial stages of the aggregation process of the β-amyloid peptide (Aβ) present a particular interest to understand the origin of the development of this disease. In this work, we present a sensor with the property of solvatofluorochromism that allows a fluorophore to undergo a shift in its emission wavelengths when placed in environments of different polarity. We used the natural adsorption binding of the dye 2-Me-4-OMe-TM, a silicon-modified xanthene derivative, to macrostructures and its solvatofluorochromic behavior to detect, in real time, modifications in the environment polarity around the dye during the initial stages of Aβ aggregation process, using steady-state, time resolved fluorescence spectroscopy and advanced fluorescence imaging techniques as fluorescence lifetime imaging microscopy (FLIM). During this progression, formation of pre-amyloid aggregates produces strong local changes in hydrophobicity that can be monitored by the fluorescent dye 2-Me-4-OMe-TM. In particular, this dye has two different emission bands and by establishing a polarity scale based on the ratiometric value of the average fluorescence lifetimes between both bands, we were able to quantify the hydrophobicity of the detected aggregates and established a method for distinguishing between aggregates of different types. • A silicon-modified xanthene derivative capable of marking amyloid aggregates. • The solvatochromism of the dye allows establishing a microenvironment polarity scale. • FLIM microscopy of Aβ aggregates shows different stages of the aggregation process. [ABSTRACT FROM AUTHOR]

Abstract: We report on development and characterization of Whispering Gallery Modes spherical microcavities integrated with organic dye molecules in a J-aggregate state. The microcavities are studied using micro-photoluminescence spectroscopy, and fluorescence lifetime imaging confocal microscopy. Directional emission of light from the microcavity is also experimentally demonstrated and attributed to the photonic jets generated in the microsphere. [Copyright &y& Elsevier]

Abstract: In bacterial chemotaxis, the levels of phosphorylated CheY in association with FliM determine the sense of the flagella rotation, which in turn controls the bacterial swimming behavior. We report the 1.7Å resolution crystallographic structure of the Thermotoga maritima BeF3 −-activated CheY in complex with the CheY-binding N-terminal region of FliM. Analysis of the structure in comparison to the previously reported Escherichia coli counterpart reveals that similar regions of H4–β5–H5 in CheY and the helix in FliM are used for the complex interfaces. Our structure also indicates that the correlated movement of Phe101 and Ser82 (F–S coupling) in T. maritima CheY upon phosphorylation and FliM binding, parallels that of Tyr106 and Thr87 (Y–T coupling) demonstrated in E. coli CheY. Furthermore, significant displacements of the β4–H4 loop in both CheYs impose a crucial role of this loop, which can be related to flagellar switch component binding or to propagating changes that is necessary during the CheY-mediated reversal of the motor. [Copyright &y& Elsevier]

Abstract: Citrus psorosis (CPsV) and Mirafiori lettuce big-vein virus (MiLBVV) belong to the family Ophioviridae, plant viruses with filamentous nucleocapsids and segmented genomes of negative polarity, causing the worldwide distributed citrus psorosis and lettuce big-vein diseases, respectively. To gain insight into the replication cycle of these viruses, the subcellular localization of the viral coat proteins (CP) was studied. Immunoblot analysis of fractionated extracts derived from natural and experimental infected hosts indicated that the CP of CPsV occurs in the soluble cytoplasmic fraction. The cytoplasmic localization of this protein was confirmed by confocal microscopy of fluorescent protein (FP)-tagged CP following its expression in either CPsV-infected and healthy Citrus sinensis plants or in Nicotiana benthamiana plants. The same localization was observed for FP-tagged CP of MiLBVV. The CPs of CPsV and MiLBBV can undergo homologous and heterologous interactions as revealed by fluorescent lifetime imaging microscopy and co-immunoprecipitation analysis. A putative leucine zipper motif that is conserved among ophiovirus CP sequences may account for these interactions. [Copyright &y& Elsevier]

Abstract: Fluorescent nanoparticles (NPs), including semiconductor NPs (Quantum Dots), metal NPs, silica NPs, polymer NPs, etc., have been a major focus of research and development during the past decade. The fluorescent nanoparticles show unique chemical and optical properties, such as brighter fluorescence, higher photostability and higher biocompatibility, compared to classical fluorescent organic dyes. Moreover, the nanoparticles can also act as multivalent scaffolds for the realization of supramolecular assemblies, since their high surface to volume ratio allow distinct spatial domains to be functionalized, which can provide a versatile synthetic platform for the implementation of different sensing schemes. Their excellent properties make them one of the most useful tools that chemistry has supplied to biomedical research, enabling the intracellular monitoring of many different species for medical and biological purposes. In this review, we focus on the developments and analytical applications of fluorescent nanoparticles in chemical and biological sensing within the intracellular environment. The review also points out the great potential of fluorescent NPs for fluorescence lifetime imaging microscopy (FLIM). Finally, we also give an overview of the current methods for delivering of fluorescent NPs into cells, where critically examine the benefits and liabilities of each strategy. [Copyright &y& Elsevier]

Abstract: Small fluorescent organic molecules based on [1,3]dioxolo[4,5-f][1,3]benzodioxole (DBD) could be used as probes for lipophillic microenvironments in aqueous solutions by indicating the critical micelles concentration of detergents and staining cell organelles. Their fluorescence lifetime decreases drastically by the amount of water in their direct environment. Therefore they are potential probes for fluorescence lifetime imaging microscopy (FLIM). [Copyright &y& Elsevier]

Abstract: Addition of Vpr C-terminus to various cell types provokes cell apoptosis. This property was recently shown useful to develop inhibitors of cell proliferation. In that context, we investigated the cellular uptake of rhodamine- and fluorescein-labeled Vpr(52–96) peptides to understand the mechanism of Vpr C-terminus entry into cells. Dynamic light scattering data indicated that this peptide spontaneously formed polydispersed aggregates in cell culture medium. The fluorescently labeled Vpr(52–96) peptide was efficiently internalized, appearing either as large fluorescent patches in the cytoplasm or in a more diffuse form throughout the cell. Using isothermal titration calorimetry, we demonstrated that Vpr(52–96) can tightly associate with heparin, a glycosaminoglycan analog of heparan sulphate, suggesting a central role of the ubiquitous cell surface-associated heparan sulphate proteoglycans for the internalization of Vpr C-terminus. Fluorescently-labeled transferrin and methyl-β-cyclodextrin showed that the Vpr C-terminus was mediated through clathrin- and caveolae/raft-dependent endocytosis. We found that Vpr C-terminus uptake was partly blocked at 4°C suggesting the importance of membrane fluidity for Vpr C-terminus entry. In fact, atomic force microscopy and liposome leakage further indicated that the Vpr peptide can destabilize and disrupt model membrane bilayers, suggesting that this mechanism may contribute to the passive entry of the peptide. Finally, using fluorescence lifetime imaging, we found that the Vpr(52–96) peptide was stable in cells for at least 48h, probably as a consequence of the poor accessibility of the peptide to proteolytic enzymes in aggregates. [Copyright &y& Elsevier]

Abstract: Excitation of the major photosynthetic antenna complex of plants, LHCII, with blue light (470nm) provides an advantage to plants, as it gives rise to chlorophyll a fluorescence lifetimes shorter than with excitation with red light (635nm). This difference is particularly pronounced in fluorescence emission wavelengths longer than 715nm. Illumination of LHCII preparation with blue light additionally induces fluorescence quenching, which develops on a minute timescale. This effect is much less efficient when induced by red light, despite the equalized energy absorbed in both the spectral regions. Simultaneous analysis of the fluorescence and photoacoustic signals in LHCII demonstrated that the light-driven fluorescence quenching is not associated with an increase in heat emission. Instead, a reversible light-induced conformational transformation of the protein takes place, as demonstrated by the FTIR technique. These findings are discussed in terms of the blue-light-specific excitation energy quenching in LHCII, which may have photoprotective applications. [Copyright &y& Elsevier]

Abstract: There is a wide range of techniques utilizing fluorescence of doxorubicin (Dox) commonly used for analysis of intracellular accumulation and destiny of various drug delivery systems containing this anthracycline antibiotic. Unfortunately, results of these studies can be significantly influenced by doxorubicin degradation product, 7,8-dehydro-9,10-desacetyldoxorubicinone (D*) forming spontaneously in aqueous environment, whose fluorescence strongly interfere with that of doxorubicin. Here, we define two microscopy techniques enabling to distinguish and separate Dox and D* emission based either on its spectral properties or on fluorescence lifetime analysis. To analyze influx and nuclear accumulation of Dox (free or polymer-bound) by flow cytometry, we propose using an indirect method based on its DNA intercalation competition with Hoechst 33342 rather than a direct measurement of doxorubicin fluorescence inside the cells. [ABSTRACT FROM AUTHOR]

Abstract: Luminescent sensors with improved performance for monitoring dissolved oxygen in microfluidic devices were developed. Our brightness enhanced sensing films with reduced film thickness showed higher signals than standard sensors and responded in real-time. Sensor layers were integrated into microfluidic chips and readout off-chip on a fluorescence microscope with lifetime imaging or ratiometric imaging using the color channels of a CCD camera. Cell respirometry of model culture systems was monitored with this sensor setup. [ABSTRACT FROM AUTHOR]

Abstract: In tauopathies including Alzheimer''s disease (AD) tau molecules have lost their normal spatial distance to each other and appear in oligomeric or aggregated forms. Conventional immunostaining methods allow detection of abnormally phosphorylated or conformationally altered aggregated tau proteins, but fail to visualize oligomeric forms of tau. Here we show that tau molecules that lost their normal spatial localization can be detected on a subcellular level in postmortem central nervous system (CNS) tissue sections of AD patients by fluorescence lifetime-based Förster resonance energy transfer (FRET). Paraffin sections were co-immunostained with two tau-specific monoclonal antibodies recognizing the same epitope, but labeled with distinct fluorescence dyes suitable for spatial resolution at a nanometer scale by lifetime-based FRET. A FRET signal was detected in neuritic plaques and neurofibrillary tangles of CNS tissue sections of AD patients, showing associated tau proteins typically reflecting either fibrillary, oligomeric or aggregated tau. The ‘pretangle-like’ structures within the neuronal perikarya did not contain spatially pathogenic forms of tau accordingly to this method. Data demonstrate that fluorescence lifetime-based FRET can be applied to human brain tissue sections to detect pathogenic forms of tau molecules that lost their normal spatial distance. [Copyright &y& Elsevier]

Abstract: The discovery that the lipids constituting the plasma membrane are not randomly distributed, but instead are able to form laterally segregated lipid domains with different properties has given hints how the formation of such lipid domains influences and regulates many processes occurring at the plasma membrane. While in model systems these lipid domains can be easily accessed and their properties studied, it is still challenging to determine the properties of cholesterol rich lipid domains, the so called “Rafts”, in the plasma membrane of living cells due to their small size and transient nature. One promising technique to address such issues is fluorescence lifetime imaging (FLIM) microscopy, as spatially resolved images make the visualization of the lateral lipid distribution possible, while at the same time the fluorescence lifetime of a membrane probe yields information about the bilayer structure and organization of the lipids in lipid domains and various properties like preferential protein–protein interactions or the enrichment of membrane probes. This review aims to give an overview of the techniques underlying FLIM probes which can be applied to investigate the formation of lipid domains and their respective properties in model membrane and biological systems. Also a short technical introduction into the techniques of a FLIM microscope is given. [Copyright &y& Elsevier]

Abstract: The adaptor protein Grb2 is recruited to intracellular early signalling complexes of many receptor tyrosine kinases and plays an important role transducing signals leading to MAP kinase activation. To date the SH2 domain of Grb2 has been shown to mediate receptor interactions with phosphorylated tyrosine residues sited directly on the receptor or on auxiliary docking proteins. Here we report that FGFR2 recruits Grb2 through its C-terminal SH3 domain. The binding site of this domain was mapped to the proline-rich C-terminus of the receptor. Deletion of the last 10 amino acids of FGFR2 abrogates interaction with Grb2. Synthetic peptides based on the C-terminus of FGFR2 bind to full length Grb2 with low micromolar affinity. The function of this novel mode of Grb2 binding provides resistance to site-specific Shp2-mediated receptor dephosphorylation. [Copyright &y& Elsevier]

Abstract: Plants have developed several adaptive regulatory mechanisms, operating at all the organization levels, to optimize utilization of light energy and to protect themselves against over-excitation-related damage. We report activity of a previously unknown possible regulatory mechanism that operates at the molecular level of the major photosynthetic pigment–protein complexes of plants, LHCII. This mechanism is driven exclusively by blue light, operates in the trimeric but not in the monomeric complex, and results in singlet excitation quenching leading to thermal energy dissipation. The conclusions are based on single molecule fluorescence lifetime analysis, direct measurements of thermal energy dissipation by photo-thermal spectroscopy, and on fluorescence spectroscopy. Possible molecular mechanisms involved in the blue-light-induced photoprotective effect are discussed, including xanthophyll photo-isomerization and the thermo-optic effect. [Copyright &y& Elsevier]

Abstract: Confocal fluorescence imaging and fluorescence resonance energy transfer (FRET) technology have been widely used to study protein–protein interactions in living cells. However, it is very difficult to quantitatively analyze FRET efficiency due to the excitation spectral crosstalk and emission spectral crosstalk between donor and acceptor. In this study, we developed a novel method to quantitatively obtain the FRET efficiency by fitting the emission spectra (FES) of donor–acceptor pair, and this method is free from both excitation and emission spectral crosstalk. We used the FES method to quantitatively monitor the FRET efficiency of SCAT3, a caspase-3 indicator based on FRET, inside living cells stably expressing SCAT3 during STS-induced apoptosis. At 0, 6 and 12h after STS treatment, the FRET efficiency of SCAT3 obtained by FES are consistent with that by two-photon excitation (TPE) fluorescence lifetime imaging microscopy (FLIM) in living cells stably expressing SCAT3. In this study, the FES was also used to analyze the caspase-3 activation in living cells during anti-cancer drug such as taxol, Artesunate (ART) or Dihydroartemisinin (DHA) treatment. Our results showed that ART or DHA induced apoptosis by a caspase-3-dependent manner, while caspase-3 was not involved in taxol-induced cell death. [Copyright &y& Elsevier]