Time-resolved Fluorescence Microscopy Research Articles

Single-Molecule Fluorescence Probes Interactions between Photoactive Protein-Silver Nanowire Conjugate and Monolayer Graphene.

In this work, we apply single-molecule fluorescence microscopy and spectroscopy to probe plasmon-enhanced fluorescence and Förster resonance energy transfer in a nanoscale assemblies. The structure where the interplay between these two processes was present consists of photoactive proteins conjugated with silver nanowires and deposited on a monolayer graphene. By comparing the results of continuous-wave and time-resolved fluorescence microscopy acquired for this structure with those obtained for the reference samples, where proteins were coupled with either a graphene monolayer or silver nanowires, we find clear indications of the interplay between plasmonic enhancement and the energy transfer to graphene. Namely, fluorescence intensities calculated for the structure, where proteins were coupled to graphene only, are less than for the structure playing the central role in this study, containing both silver nanowires and graphene. Conversely, decay times extracted for the latter are shorter compared to a protein-silver nanowire conjugate, pointing towards emergence of the energy transfer. Overall, the results show that monitoring the optical properties of single emitters in a precisely designed hybrid nanostructure provides an elegant way to probe even complex combination of interactions at the nanoscale.

Analysis of individual nanoscale block copolymer vesicles by atomic force microscopy combined with time-resolved fluorescence microscopy

Abstract We report on the analysis of individual dye loaded block copolymer (BCP) vesicles via a combination of confocal laser scanning microscopy (CSLM)/fluorescence lifetime imaging microscopy (FLIM) on the one hand and atomic force microscopy (AFM) on the other hand. AFM measurements on ATTO 647N-loaded poly(styrene-block-poly(acrylic acid)) (PS115-b-PAA15) vesicles were carried out with high spatial resolution and afforded morphology and dimensions of BCP vesicles on solid substrates. By contrast the CSLM and FLIM data are diffraction limited, but from the time resolved fluorescence data information on the local vicinity of the reporter dye can be extracted. In the combined experiment individual dye-loaded vesicles and vesicle aggregates were discerned, analyzed metrologically by AFM and in more detail by CSLM and FLIM. On the basis of FLIM data the partitioning of the reporter dye was analyzed. The dye resides preferentially in the hydrophilic corona inside the vesicles. The dye concentration in the polymersome was about 90 times higher than in the solution used for encapsulation. These results underline that the combination of AFM with sensitive optical techniques, especially FLIM, is a promising approach for obtaining a deeper understanding of molecular interactions and nanoenvironments in supramolecular macromolecular structures and beyond.

Sensing cholesterol-induced rigidity in model membranes with time-resolved fluorescence spectroscopy and microscopy.

Here, we report the characterization of cholesterol levels on membrane fluidity with a twisted intramolecular charge transfer (TICT) membrane dye, namely DI-8-ANEPPS, using fluorescence lifetime techniques such as time-correlated single photon counting (TCSPC) and fluorescence lifetime imaging microscopy (FLIM). The characterized liposomes comprised a 3 : 1 ratio of POPC and POPG, respectively, 1% DI-8-ANEPPS, and increasing cholesterol levels from 0% to 50%. Fluorescence lifetime characterization revealed that increasing the cholesterol levels from 0% to 50% increases the fluorescence lifetime of DI-8-ANEPPS from 2.36 ns to 3.65 ns, a 55% increment. Such lengthening in the fluorescence lifetime is concomitant with reduced Stokes shifts and higher quantum yield, revealing that localized excitation (LE) dominates over TICT states with increased cholesterol levels. Fluorescence anisotropy measurements revealed a less isotropic environment in the membrane upon increasing cholesterol levels, suggesting a shift from liquid-disorder (Lα) to liquid-order (LO) upon adding cholesterol. Local electrostatic and dipole characterization experiments revealed that changes in the zeta-potential (ζ-potential) and transmembrane dipole potential (Ψd) induced by changes in cholesterol levels or the POPC : POPG ratio play a minimal role in the fluorescence lifetime outcome of DI-8-ANEPPS. Instead, these results indicate that the cholesterol's effect in restricting the degree of movement of DI-8-ANEPPS dominates its photophysics over the cholesterol effect on the local dipole strength. We envision that time-resolved spectroscopy and microscopy, coupled with TICT dyes, could be a convenient tool in exploring the complex interplay between membrane lipids, sterols, and proteins and provide novel insights into membrane fluidity, organization, and function.

Flagellar dynamics reveal fluctuations and kinetic limit in the Escherichia coli chemotaxis network

The Escherichia coli chemotaxis network, by which bacteria modulate their random run/tumble swimming pattern to navigate their environment, must cope with unavoidable number fluctuations (“noise”) in its molecular constituents like other signaling networks. The probability of clockwise (CW) flagellar rotation, or CW bias, is a measure of the chemotaxis network’s output, and its temporal fluctuations provide a proxy for network noise. Here we quantify fluctuations in the chemotaxis signaling network from the switching statistics of flagella, observed using time-resolved fluorescence microscopy of individual optically trapped E. coli cells. This approach allows noise to be quantified across the dynamic range of the network. Large CW bias fluctuations are revealed at steady state, which may play a critical role in driving flagellar switching and cell tumbling. When the network is stimulated chemically to higher activity, fluctuations dramatically decrease. A stochastic theoretical model, inspired by work on gene expression noise, points to CheY activation occurring in bursts, driving CW bias fluctuations. This model also shows that an intrinsic kinetic ceiling on network activity places an upper limit on activated CheY and CW bias, which when encountered suppresses network fluctuations. This limit may also prevent cells from tumbling unproductively in steep gradients.

Visualizing the disordered nuclear transport machinery in situ

The approximately 120 MDa mammalian nuclear pore complex (NPC) acts as a gatekeeper for the transport between the nucleus and cytosol1. The central channel of the NPC is filled with hundreds of intrinsically disordered proteins (IDPs) called FG-nucleoporins (FG-NUPs)2,3. Although the structure of the NPC scaffold has been resolved in remarkable detail, the actual transport machinery built up by FG-NUPs—about 50 MDa—is depicted as an approximately 60-nm hole in even highly resolved tomograms and/or structures computed with artificial intelligence4–11. Here we directly probed conformations of the vital FG-NUP98 inside NPCs in live cells and in permeabilized cells with an intact transport machinery by using a synthetic biology-enabled site-specific small-molecule labelling approach paired with highly time-resolved fluorescence microscopy. Single permeabilized cell measurements of the distance distribution of FG-NUP98 segments combined with coarse-grained molecular simulations of the NPC allowed us to map the uncharted molecular environment inside the nanosized transport channel. We determined that the channel provides—in the terminology of the Flory polymer theory12—a ‘good solvent’ environment. This enables the FG domain to adopt expanded conformations and thus control transport between the nucleus and cytoplasm. With more than 30% of the proteome being formed from IDPs, our study opens a window into resolving disorder–function relationships of IDPs in situ, which are important in various processes, such as cellular signalling, phase separation, ageing and viral entry.

Computational based time-resolved multispectral fluorescence microscopy

Multispectral imaging and time-resolved imaging are two common acquisition schemes in fluorescence microscopy, and their combination can be beneficial to increase specificity. The multidimensionality of the dataset (space, time, and spectrum) introduces some challenges, such as the acquisition of big datasets and long measurement times. In this work, we present a time-resolved multispectral fluorescence microscopy system with a short measurement time, achieved by exploiting Compressive Sensing (CS) based on the Single-Pixel Camera (SPC) scheme. Data Fusion (DF) with a high-resolution camera allows us to tackle the problem of low spatial resolution, typical of SPC. The combined use of SPC, CS, and DF, in which hardware and algorithms are integrated, represents a computational imaging framework to reduce the number of measurements while preserving the information content. This approach has been exploited to demonstrate a zoom feature without moving the optical system. We describe and characterize the system in terms of spatial, spectral, and temporal properties, along with validation on a cellular sample.

Fusion modifies the material properties of membranes.

Membrane fusion is a ubiquitous process in biology and potentially an alternative to endocytic internalization pathways in drug delivery. Due to its high complexity, assays have been developed to disentangle its key aspects, although many of its molecular mechanisms remain elusive. Here, we use a combination of multicolour and time-resolved confocal fluorescence microscopy to study the effects of membrane mechanics on fusion and conversely, how fusion alters membrane mechanics. As a minimal fusion system, we used cationic large unilamellar fusogenic liposomes (LUVs) that rapidly fuse with giant unilamellar vesicles (GUVs) as evidenced by the probes being delivered into the GUVs. Fusion is favoured in fluid membranes and hampered upon an increase in packing. Extensive fusion of a large number of LUVs leads to the creation of area leaflet asymmetry in GUVs, resulting in budding formation driven by spontaneous curvature. In these conditions, the membrane becomes perforated as a result of a decrease in edge tension and the size of the formed pores seems to scale with LUV concentration. Here, the GUVs also display a range of permeability states. In live human embryonic kidney (HEK) cells, lipid mixing and intracellular delivery of LUV encapsulated water-soluble probes are observed, consistent with fusion of the liposomes with the plasma membrane. Furthermore, when exposed to liposomes, cell membrane permeabilization and cell death is observed at liposomal concentrations an order of magnitude higher than pore formation in the GUVs, suggesting an increased resilience of cells compared to GUVs. Fusion also seems to fluidize the membrane and effects are observed within minutes. Overall, the results shed new light onto the effects of liposome-membrane interactions.

Dual-emissive solid-state histidine-stabilized gold nanoclusters for applications in white-light generation.

The majority of present-day white-light emitting devices (WLEDs) are built upon the use of rare-earth elements, which have a short supply, are expensive and can become extremely toxic. Thus, in this work, we synthesized an eco-friendly, efficient and cheap white-light emitting material (WLEM) based on solid-state histidine-stabilized gold nanoclusters (His-AuNCs), obtained through the lyophilization of microwave-synthesized photoluminescent His-AuNCs. Their morphological and structural characterization was followed by thorough evaluation of their intrinsic solid-state photoluminescence properties via steady-state and time-resolved fluorescence spectroscopy and microscopy, at multiple excitation wavelengths. A white-light emission was observed under UV light excitation due to the two-band broad emission, with maxima at 475 and 520 nm, covering a large area of the visible spectrum. In order to evaluate the purity of the white-light emission we calculated the chromaticity coordinates, at different wavelengths, and displayed them on a CIE (Commision Internationale d'Eclairage) diagram. An excellent value of (0.36, 0.33) was found at 420 nm excitation, which falls within the range of pure white-light emission. Moreover, the His-AuNCs show great photo- and thermo-stability, thus proving their ability to perform as a reliable WLEM with potential use in the development of eco-friendly WLEDs.

Focus on lasers, imaging, microscopy, and nanoscience

Focus on lasers, imaging, microscopy, and nanoscience

Self-Enhancement of Efficiency and Self-Attenuation of Hysteretic Behavior of Perovskite Solar Cells with Aging

Spontaneous enhancement of the photovoltaic performance of perovskite solar cells (PSCs) after aging has been reported, but the underlying reasons for such behavior are still under debate. Herein, we demonstrate that this spontaneous improvement effect accompanied by self-attenuation of hysteresis in the current-voltage characteristics is time- and temperature-dependent. Moreover, it is universal to PSCs based on a range of mixed-ion perovskites and coupled to different hole- and electron-transporting materials. Time-resolved confocal fluorescence microscopy and other characterization techniques suggest that the "self-healing" phenomenon is accompanied by the homogenization and enhancement of the charge extraction efficiency as well as suppressed recombination throughout cm2-scale perovskite layers. These dynamic effects need to be accounted for when assessing the initial and stabilized performance of PSCs.

Femtosecond imaging of spatial deformation of surface plasmon polariton wave packet during resonant interaction with nanocavity

Abstract The spatiotemporal dynamics of a surface plasmon polariton (SPP) wave packet (WP) that interacts with a plasmonic nanocavity on a metal surface are investigated via femtosecond time-resolved two-photon fluorescence microscopy and numerical calculations. The nanocavity, which consists of a metal–insulator–metal (MIM) laminar structure (longitudinal length: ∼100 nm), behaves as a subwavelength meta-atom possessing discretized eigenenergies. When a chirp-induced femto-second SPP WP is incident on the nanocavity, only the spectral component matching a particular eigenenergy is transmitted to continue propagation on the metal surface. This spectral clipping induces a spatial peak shift in the WP. The shift can be controlled by tuning the eigenenergy or chirp.

Self-aggregation of the proteolytic forms of Verteporfin: An in silico and in vitro study

Verteporfin is a benzoporphyrin derivative monoacid ring A that has been historically and successfully used as a photosensitizer in clinical applications and in clinical trials including for cancer phototherapy. However, the photo-efficiency of Verteporfin is limited by self-aggregation in water, in which the various proteolytic forms present distinct self-aggregation tendencies. In this work, spectroscopic and computational studies were used to elucidate the self-aggregation behavior of the three proteolytic forms of Verteporfin. Additionally, the intracellular self-aggregation of Verteporfin was evaluated in U87MG cells line, a type of brain cancer considered incurable. The results indicated that the self-aggregation of Verteporfin in water is most likely modulated by formal charge effects following the order: neutral > monoanionic > dicationic. UV–vis electronic spectroscopy and computational dynamics analyses suggested that J-type aggregates were primarily formed. Time-resolved fluorescence and fluorescence lifetime imaging microscopy (FLIM) confirmed that although such aggregates exist in solution and inside cells, there is no evidence of preferential self-aggregation in acidic organelles, such as lysosomes. The present approach allows a better understanding of the self-aggregation of other photosensitizers, particularly the influence of different proteolytic forms and organelles on the self-aggregation of compounds that can be protonated, and the heterogeneity of photosensitizers within intracellular compartments.

Time-Resolved Fluorescence Microscopy Screens on Host Protein Subversion During Bacterial Cell Invasion.

Intracellular bacterial pathogens have evolved a plethora of strategies to invade eukaryotic cells. By manipulating host signaling pathways, in particular vesicular trafficking, these microbes subvert host functions to promote their internalization and to establish an intracellular niche. During these events, host endomembrane compartments are dynamically reorganized. Shigella flexneri, the causative agent of bacillary dysentery, recruits components of the host recycling pathway and the exocyst of non-phagocytic enterocytes in the vicinity of its entry site to facilitate its access to the host cytosol. These factors are either dynamically tethered to in situ formed macropinosomes or to the bacteria-containing vacuole itself. The underlying interactions cannot readily be monitored as individual bacterial infection events take place without synchronicity using cellular infection models. Therefore, time-resolved screens by fluorescence microscopy represent a powerful tool for the study of host subversion. Such screens can be performed with libraries of fluorescently tagged host factors. Using the cytosolic pathogenic agent Shigella flexneri as a model, we provide detailed protocols for such medium-to-high throughput multidimensional imaging screening of the dynamic host-pathogen cross talk. Our workflow is designed to be easily adapted for the study of different host factor libraries and different pathogen models.

Phototherapy of Brain Tumours Using a Fibre Optic Neurosystem

In this work, a new approach was tested to assess the cellular composition of tissues by time-resolved methods of fluorescence analysis of exogenous and endogenous fluorophores. First of all, the differences in fluorescence kinetics of endogenous fluorophores (coenzymes NADH and FAD) in tumour and immunocompetent cells were determined. After that, differences in fluorescence kinetics of photosensitizer 5 ALA-induced protoporphyrin IX were established due to its different metabolism in cells of different phenotypes. Kinetics of photoluminescence of NADH and FAD coenzymes as well as photosensitizer were studied by means of two different methods: time-resolved spectroscopy based on a streak-camera and fibre optic neuroscopy, which served to perform process monitoring and regular fluorescence diagnosis of the probed region. Time-resolved fluorescence microscopy (FLIM) was used as a control technique. Time-resolved spectroscopic fluorescence lifetime analysis was performed on sexually mature female rats induced with glioma C6 brain tumour under in vivo conditions; thus, under conditions where the immune system actively intervenes in the process of oncogenesis. In this regard, the aim of the study was to recognize the cellular composition of the brain tumour tissue, namely the ratio of cancer and immunocompetent cells and their mutual localization. Understanding the role of the immune system thus provides new ways and approaches for further diagnosis and therapy, making tumour-associated immune cells a prime target for modern therapies.

Time-resolved fluorescence imaging technique for rat brain tumors analysis

The paper presents a new approach to assessing the state of tissues that differ in phenotype and in the degree of immunocompetent cells activity using photosensitizers (PS) and time-resolved fluorescence analysis methods. The main attention is paid to the detection of differences between tumor cells and tumor-associated macrophages (TAM) using spectroscopic and microscopic methods by the fluorescent kinetics signal and the difference in the accumulation of PS (the accumulation is several times greater in macrophages). The results of the PS photoluminescence study were obtained using two different techniques: time-resolved spectroscopy and time-resolved fluorescence microscopy (FLIM). Time-resolved spectroscopic analysis of the PS fluorescence lifetime was performed on adult female rats with induced C6 glioma in vivo. 5-ALA-induced Pp IX, which is widely used in clinical practice for carrying out effective conduction photodiagnostics and PDT, was used as the PS.

Applying TADF Emitters in Bioimaging and Sensing-A Novel Approach Using Liposomes for Encapsulation and Cellular Uptake.

A new method for facilitating the delivery, uptake and intracellular localisation of thermally activated delayed fluorescence (TADF) complexes was developed. First, confinement of TADF complexes in liposomes was demonstrated, which were subsequently used as the delivery vehicle for cellular uptake. Confocal fluorescence microscopy showed TADF complexes subsequently localise in the cytoplasm of HepG2 cells. The procedures developed in this work included the removal of molecular oxygen in the liposome preparation without disrupting the liposome structures. Time-resolved fluorescence microscopy (point scanning) showed initial prompt fluorescence followed by a weak, but detectable, delayed fluorescence component for liposomal TADF internalised in HepG2 cells. By demonstrating that it is possible to deliver un-functionalised and/or unshielded TADF complexes, a sensing function for TADFs, such as molecular oxygen, can be envisaged.

Non-glandular trichomes of sunflower are important in the absorption and translocation of foliar-applied Zn.

Trichomes are potentially important for absorption of foliar fertilizers. A study has shown that the non-glandular trichromes (NGTs) of sunflower (Helianthus annuus) accumulated high concentrations of foliar-applied zinc (Zn); however, the mechanisms of Zn accumulation in the NGTs and the fate of this Zn are unclear. Here we investigated how foliar-applied Zn accumulates in the NGTs and the subsequent translocation of this Zn. Time-resolved synchrotron-based X-ray fluorescence microscopy and transcriptional analyses were used to probe the movement of Zn in the NGTs, with the cuticle composition of the NGTs examined using confocal Raman microscopy. The accumulation of Zn in the NGTs is both an initial preferential absorption process and a subsequent translocation process. This preferred absorption is likely because the NGT base has a higher hydrophilicity, whilst the subsequent translocation is due to the presence of plasmodesmata, Zn-chelating ligands, and Zn transporters in the NGTs. Furthermore, the Zn sequestered in the NGTs was eventually translocated out of the trichome once the leaf Zn concentration had decreased, suggesting that the NGTs are also important in maintaining leaf Zn homeostasis. This study demonstrates for the first time that trichomes have a key structural and functional role in the absorption and translocation of foliar-applied Zn.

Structure\u2013function analysis of oncogenic EGFR Kinase Domain Duplication reveals insights into activation and a potential approach for therapeutic targeting

Mechanistic understanding of oncogenic variants facilitates the development and optimization of treatment strategies. We recently identified in-frame, tandem duplication of EGFR exons 18 - 25, which causes EGFR Kinase Domain Duplication (EGFR-KDD). Here, we characterize the prevalence of ERBB family KDDs across multiple human cancers and evaluate the functional biochemistry of EGFR-KDD as it relates to pathogenesis and potential therapeutic intervention. We provide computational and experimental evidence that EGFR-KDD functions by forming asymmetric EGF-independent intra-molecular and EGF-dependent inter-molecular dimers. Time-resolved fluorescence microscopy and co-immunoprecipitation reveals EGFR-KDD can form ligand-dependent inter-molecular homo- and hetero-dimers/multimers. Furthermore, we show that inhibition of EGFR-KDD activity is maximally achieved by blocking both intra- and inter-molecular dimerization. Collectively, our findings define a previously unrecognized model of EGFR dimerization, providing important insights for the understanding of EGFR activation mechanisms and informing personalized treatment of patients with tumors harboring EGFR-KDD. Finally, we establish ERBB KDDs as recurrent oncogenic events in multiple cancers.

The Dynamism of Intrinsically Disordered Proteins: Binding-Induced Folding, Amyloid Formation, and Phase Separation.

Intrinsically disordered proteins (IDPs) or natively unfolded proteins do not undergo autonomous folding into a well-defined 3-D structure and challenge the conventional structure-function paradigm. They are involved in a multitude of critical physiological functions by adopting various structural states via order-to-disorder transitions or by maintaining their disordered characteristics in functional complexes. In recent times, there has been a burgeoning interest in the investigation of intriguing behavior of IDPs using highly multidisciplinary and complementary approaches due to the pivotal role of this unique class of protein chameleons in physiology and disease. Over the past decade or so, our laboratory has been actively investigating the unique physicochemical properties of this class of highly dynamic, flexible, rapidly interconverting proteins. We have utilized a diverse array of existing and emerging tools involving steady-state and time-resolved fluorescence, Raman spectroscopy, circular dichroism, light scattering, fluorescence microscopy, and atomic force microscopy coupled with site-directed mutagenesis and other biochemical and biophysical tools to study a variety of interesting and important aspects of IDPs. In this Feature Article, I describe our work on the conformational characteristics, solvation dynamics, binding-induced folding, amyloid formation, and liquid-liquid phase separation of a number of amyloidogenic IDPs. A series of these studies described here captures the role of conformational plasticity and dynamics in directing binding, folding, assembly, aggregation, and phase transitions implicated in physiology and pathology.

Time-Resolved Fluorescence Microscopy Combined with Scanning Electrochemical Microscopy: A New Way to Visualize Photo-Induced Electron Transfer Quenching with an Electrofluorochromic Probe

The combination of Scanning Electrochemical Microscopy (SECM) and Time-Resolved Fluorescence Microscopy was successfully implemented to investigate the control of the fluorescence of a tetrazine derivative in solution according to its redox state, a phenomenon called electrofluorochromism. The normalized fluorescence intensity difference between the 'on' and 'off' states can be tuned by the tip potential and the distance between the polarized tip and the substrate. It is demonstrated for the first time that luminescence lifetime can be modulated as well, an evidence that a quenching process contributes to the fluorescence intensity drop. The experimental results demonstrate that this quenching is due to the electron transfer reaction between the excited fluorophore and the electrogenerated anion radical. A mixed dynamic and static quenching mechanism is considered to fit the variations of both luminescence intensity and lifetime ratios vs. the coulombic charge. The pertinence of a static quenching is validated by DFT calculations. Insulating (glass) and conductive (ITO) substrates have been tested and optical approach curves based on fluorescence lifetime modulation have been obtained in each case that are more sensitive than the electrochemical one based on the tip current. Finally, numerical simulations have been performed to fit these optical approach curves and validate the role of the electron transfer quenching in the variation of fluorescence intensity and lifetime.