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Mitochondria are dynamic bioenergetic hubs that become compromised with age. In neurons, declining mitochondrial axonal transport has been associated with reduced cellular health. However, it is still unclear to what extent the decline of mitochondrial transport and function observed during ageing are coupled, and if somal and axonal mitochondria display compartment-specific features that make them more susceptible to the ageing process. It is also not known whether the biophysical state of the cytoplasm, thought to affect many cellular functions, changes with age to impact mitochondrial trafficking and homeostasis. Focusing on the mouse peripheral nervous system, we show that age-dependent decline in mitochondrial trafficking is accompanied by reduction of mitochondrial membrane potential and intramitochondrial viscosity, but not calcium buffering, in both somal and axonal mitochondria. Intriguingly, we observe a specific increase in cytoplasmic viscosity in the neuronal cell body, where mitochondria are most polarised, which correlates with decreased cytoplasmic diffusiveness. Increasing cytoplasmic crowding in the somatic compartment of DRG neurons grown in microfluidic chambers reduces mitochondrial axonal trafficking, suggesting a mechanistic link between the regulation of cytoplasmic viscosity and mitochondrial dynamics. Our work provides a reference for studying the relationship between neuronal mitochondrial homeostasis and the viscoelasticity of the cytoplasm in a compartment-dependent manner during ageing., (© 2024 The Author(s). Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.)

A wide-field microscope with epi-fluorescence and selective plane illumination was combined with a single-photon avalanche diode (SPAD) array camera to enable live-cell fluorescence lifetime imaging (FLIM) using time-correlated single-photon counting (TCSPC). The camera sensor comprised of 192 × 128 pixels, each integrating a single SPAD and a time-to-digital converter. Jointly, they produced a stream of single-photon images of photon arrival times with ≈ 38 ps accuracy. The photon arrival times were subject to systematic delays and nonlinearities, which were corrected by a Monte-Carlo algorithm. The SPAD camera was then applied to FLIM where histogramming the resulting photon arrival times in each pixel resulted in decays compatible with common data processing pipelines for fluorescence lifetime analysis. The capabilities of the TCSPC camera-based FLIM microscope were demonstrated by imaging living unicellular photosynthetic algae and artificial lipid vesicles. Epi-fluorescence illumination enabled rapid fluorescence lifetime imaging of living cells and selective-plane illumination enabled 3-dimensional FLIM of stationary samples., (© 2024. The Author(s).)

The removal of varnish from the surface is a key step in painting conservation. Varnish removal is traditionally monitored by examining the painting surface under ultraviolet illumination. We show here that by imaging the fluorescence lifetime instead, much better contrast, sensitivity, and specificity can be achieved. For this purpose, we developed a lightweight (4.8 kg) portable instrument for macroscopic fluorescence lifetime imaging (FLIM). It is based on a time-correlated single-photon avalanche diode (SPAD) camera to acquire the FLIM images and a pulsed 440 nm diode laser to excite the varnish fluorescence. A historical model painting was examined to demonstrate the capabilities of the system. We found that the FLIM images provided information on the distribution of the varnish on the painting surface with greater sensitivity, specificity, and contrast compared to the traditional ultraviolet illumination photography. The distribution of the varnish and other painting materials was assessed using FLIM during and after varnish removal with different solvent application methods. Monitoring of the varnish removal process between successive solvent applications by a swab revealed an evolving image contrast as a function of the cleaning progress. FLIM of dammar and mastic resin varnishes identified characteristic changes to their fluorescence lifetimes depending on their ageing conditions. Thus, FLIM has a potential to become a powerful and versatile tool to visualise varnish removal from paintings., Competing Interests: Competing interestsAuthors declare no competing financial interests., (© The Author(s) 2023.)

The cytoplasm is a crowded, visco-elastic environment whose physical properties change according to physiological or developmental states. How the physical properties of the cytoplasm impact cellular functions in vivo remains poorly understood. Here, we probe the effects of cytoplasmic concentration on microtubules by applying osmotic shifts to fission yeast, moss, and mammalian cells. We show that the rates of both microtubule polymerization and depolymerization scale linearly and inversely with cytoplasmic concentration; an increase in cytoplasmic concentration decreases the rates of microtubule polymerization and depolymerization proportionally, whereas a decrease in cytoplasmic concentration leads to the opposite. Numerous lines of evidence indicate that these effects are due to changes in cytoplasmic viscosity rather than cellular stress responses or macromolecular crowding per se. We reconstituted these effects on microtubules in vitro by tuning viscosity. Our findings indicate that, even in normal conditions, the viscosity of the cytoplasm modulates the reactions that underlie microtubule dynamic behaviors., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Purpose: Vitreous humor is a complex biofluid whose composition determines its structure and function. Vitreous viscosity will affect the delivery, distribution, and half-life of intraocular drugs, and key physiological molecules. The central pig vitreous is thought to closely match human vitreous viscosity. Diffusion is inversely related to viscosity, and diffusion is of fundamental importance for all biochemical reactions. Fluorescence Recovery After Photobleaching (FRAP) may provide a novel means of measuring intravitreal diffusion that could be applied to drugs and physiological macromolecules. It would also provide information about vitreous viscosity, which is relevant to drug elimination, and delivery., Methods: Vitreous viscosity and intravitreal macromolecular diffusion of fluorescently labelled macromolecules were investigated in porcine eyes using fluorescence recovery after photobleaching (FRAP). Fluorescein isothiocyanate conjugated (FITC) dextrans and ficolls of varying molecular weights (MWs), and FITC-bovine serum albumin (BSA) were employed using FRAP bleach areas of different diameters., Results: The mean (±standard deviation) viscosity of porcine vitreous using dextran, ficoll and BSA were 3.54 ± 1.40, 2.86 ± 1.13 and 4.54 ± 0.13 cP respectively, with an average of 3.65 ± 0.60 cP., Conclusions: FRAP is a feasible and practical optical method to quantify the diffusion of macromolecules through vitreous., Competing Interests: The authors have declared that no competing interests exist.

Förster resonance energy transfer (FRET) is a powerful tool to investigate the interaction between proteins in living cells. Fluorescence proteins, such as the green fluorescent protein (GFP) and its derivatives, are coexpressed in cells linked to proteins of interest. Time-resolved fluorescence anisotropy is a popular tool to study homo-FRET of fluorescent proteins as an indicator of dimerization, in which its signature consists of a very short component at the beginning of the anisotropy decay. In this work, we present an approach to study GFP homo-FRET via a combination of time-resolved fluorescence anisotropy, the stretched exponential decay model, and molecular dynamics simulations. We characterize a new, to our knowledge, FRET standard formed by two enhanced GFPs (eGFPs) and a flexible linker of 15 aminoacids (eGFP15eGFP) with this protocol, which is validated by using an eGFP monomer as a reference. An excellent agreement is found between the FRET efficiency calculated from the fit of the eGFP15eGFP fluorescence anisotropy decays with a stretched exponential decay model (〈E FRET exp 〉 = 0.25 ± 0.05) and those calculated from the molecular dynamics simulations (〈E FRET MD 〉 = 0.18 ± 0.14). The relative dipole orientation between the GFPs is best described by the orientation factors 〈κ 2 〉 = 0.17 ± 0.16 and 〈|κ|〉 = 0.35 ± 0.20, contextualized within a static framework in which the linker hinders the free rotation of the fluorophores and excludes certain configurations. The combination of time- and polarization-resolved fluorescence spectroscopy with molecular dynamics simulations is shown to be a powerful tool for the study and interpretation of homo-FRET., (Copyright © 2020 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

A bottom-illuminated orbital shaker designed for the cultivation of microalgae suspensions is described in this open-source hardware report. The instrument agitates and illuminates microalgae suspensions grown inside flasks. It was optimized for low production cost, simplicity, low power consumption, design flexibility, consistent, and controllable growth light intensity. The illuminated orbital shaker is especially well suited for low-resource research laboratories and education. It is an alternative to commercial instruments for microalgae cultivation. It improves on typical do-it-yourself microalgae growth systems by offering consistent and well characterized illumination light intensity. The illuminated growth area is 20 cm × 15 cm, which is suitable for three T75 tissue culture flasks or six 100 ml Erlenmeyer flasks. The photosynthetic photon flux density, is variable in eight steps ( 26 - 800 μ mol · m - 2 · s - 1 ) and programmable in a 24-h light/dark cycle. The agitation speed is variable ( 0 - 210 RPM ). The overall material cost is around £300, including an entry-level orbital shaker. The build takes two days, requiring electronics and mechanical assembly capabilities. The instrument build is documented in a set of open-source protocols, design files, and source code. The design can be readily modified, scaled, and adapted for other orbital shakers and specific experimental requirements. The instrument function was validated by growing fresh-water microalgae Desmodesmus quadricauda and Chlorella vulgaris . The cultivation protocols, microalgae growth curves, and doubling times are included in this report., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2020 The Authors.)

New materials that exhibit tuneable optical properties, notable emission across the visible spectrum, are of immense interest to biologists as they present a broad palette of colours from a single imaging agent that can be utilised in biological detection. Such a flexible system, when combined with the advantages of using conjugated polymer nanoparticles in cell imaging results in a widely useful medical diagnostic system. Here, we describe tuneable emission observed through oxidation of a conjugated polymer followed by the formation of nanoparticles and their subsequent use in cell imaging., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

The only way to visually observe cellular viscosity, which can greatly influence biological reactions and has been linked to several human diseases, is through viscosity imaging. Imaging cellular viscosity has allowed the mapping of viscosity in cells, and the next frontier is targeted viscosity imaging of organelles and their microenvironments. Here we present a fluorescent molecular rotor/FLIM framework to image both organellar viscosity and membrane fluidity, using a combination of chemical targeting and organelle extraction. For demonstration, we image matrix viscosity and membrane fluidity of mitochondria, which have been linked to human diseases, including Alzheimer's Disease and Leigh's syndrome. We find that both are highly dynamic and responsive to small environmental and physiological changes, even under non-pathological conditions. This shows that neither viscosity nor fluidity can be assumed to be fixed and underlines the need for single-cell, and now even single-organelle, imaging., Competing Interests: The authors have declared that no competing interests exist.

Recent evidence suggests that the ion channel TRPA1 is implicated in lung adenocarcinoma (LUAD), where its role and mechanism of action remain unknown. We have previously established that the membrane receptor FGFR2 drives LUAD progression through aberrant protein-protein interactions mediated via its C-terminal proline-rich motif. Here we report that the N-terminal ankyrin repeats of TRPA1 directly bind to the C-terminal proline-rich motif of FGFR2 inducing the constitutive activation of the receptor, thereby prompting LUAD progression and metastasis. Furthermore, we show that upon metastasis to the brain, TRPA1 gets depleted, an effect triggered by the transfer of TRPA1-targeting exosomal microRNA (miRNA-142-3p) from brain astrocytes to cancer cells. This downregulation, in turn, inhibits TRPA1-mediated activation of FGFR2, hindering the metastatic process. Our study reveals a direct binding event and characterizes the role of TRPA1 ankyrin repeats in regulating FGFR2-driven oncogenic process; a mechanism that is hindered by miRNA-142-3p.TRPA1 has been reported to contribute lung cancer adenocarcinoma (LUAD), but the mechanisms are unclear. Here the authors propose that TRPA1/FGFR2 interaction is functional in LUAD and show that astrocytes oppose brain metastasis by mediating the downregulation of TRPA1 through exosome-delivered miRNA-142-3p.

Light-matter interactions can be strongly modified by the surrounding environment. Here, we report on the first experimental observation of molecular spontaneous emission inside a highly non-local metamaterial based on a plasmonic nanorod assembly. We show that the emission process is dominated not only by the topology of its local effective medium dispersion, but also by the non-local response of the composite, so that metamaterials with different geometric parameters but the same local effective medium properties exhibit different Purcell factors. A record-high enhancement of a decay rate is observed, in agreement with the developed quantitative description of the Purcell effect in a non-local medium. An engineered material non-locality introduces an additional degree of freedom into quantum electrodynamics, enabling new applications in quantum information processing, photochemistry, imaging and sensing with macroscopic composites., Competing Interests: The authors declare no conflict of interest.

Neural activity relies on molecular diffusion within nanoscopic spaces outside and inside nerve cells, such as synaptic clefts or dendritic spines. Measuring diffusion on this small scale in situ has not hitherto been possible, yet this knowledge is critical for understanding the dynamics of molecular events and electric currents that shape physiological signals throughout the brain. Here we advance time-resolved fluorescence anisotropy imaging combined with two-photon excitation microscopy to map nanoscale diffusivity in ex vivo brain slices. We find that in the brain interstitial gaps small molecules move on average ~30% slower than in a free medium whereas inside neuronal dendrites this retardation is ~70%. In the synaptic cleft free nanodiffusion is decelerated by ~46%. These quantities provide previously unattainable basic constrains for the receptor actions of released neurotransmitters, the electrical conductance of the brain interstitial space and the limiting rate of molecular interactions or conformational changes in the synaptic microenvironment., Competing Interests: The authors declare no competing financial interests.

Quantum dots (QDs) are attractive photoluminescence probes for biomedical imaging due to their unique photophysical properties. However, the potential toxicity of QDs has remained a major obstacle to their clinical use because they commonly incorporate the toxic heavy metal cadmium within the core of the QDs. In this work, we have evaluated a novel type of heavy metal-free/cadmium-free and biocompatible QD nanoparticles (bio CFQD(®) nanoparticles) with a good photoluminescence quantum yield. Sentinel lymph node mapping is an increasingly important treatment option in the management of breast cancer. We have demonstrated their potential for lymph node mapping by ex vivo imaging of regional lymph nodes after subcutaneous injection in the paw of rats. Using photoluminescence imaging and chemical extraction measurements based on elemental analysis by inductively coupled plasma mass spectroscopy, the quantum dots are shown to accumulate quickly and selectively in the axillary and thoracic regional lymph nodes. In addition, lifetime imaging microscopy of the QD photoluminescence indicates minimal perturbation to their photoluminescence properties in biological systems., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Electron-bombarded pixel image sensors, where a single photoelectron is accelerated directly into a CCD or CMOS sensor, allow wide-field imaging at extremely low light levels as they are sensitive enough to detect single photons. This technology allows the detection of up to hundreds or thousands of photon events per frame, depending on the sensor size, and photon event centroiding can be employed to recover resolution lost in the detection process. Unlike photon events from electron-multiplying sensors, the photon events from electron-bombarded sensors have a narrow, acceleration-voltage-dependent pulse height distribution. Thus a gain voltage sweep during exposure in an electron-bombarded sensor could allow photon arrival time determination from the pulse height with sub-frame exposure time resolution. We give a brief overview of our work with electron-bombarded pixel image sensor technology and recent developments in this field for single photon counting imaging, and examples of some applications.

Guanine-rich oligonucleotides can fold into quadruple-stranded helical structures known as G-quadruplexes. Mounting experimental evidence has gathered suggesting that these non-canonical nucleic acid structures form in vivo and play essential biological roles. However, to date, there are no small-molecule optical probes to image G-quadruplexes in live cells. Herein, we report the design and development of a small fluorescent molecule, which can be used as an optical probe for G-quadruplexes. We demonstrate that the fluorescence lifetime of this new probe changes considerably upon interaction with different nucleic acid topologies. Specifically, longer fluorescence lifetimes are observed in vitro for G-quadruplexes than for double- and single-stranded nucleic acids. Cellular studies confirm that this molecule is cell permeable, has low cytotoxicity and localizes primarily in the cell nucleus. Furthermore, using fluorescence lifetime imaging microscopy, live-cell imaging suggests that the probe can be used to study the interaction of small molecules with G-quadruplexes in vivo.

We present a novel integrated multimodal fluorescence microscopy technique for simultaneous fluorescence recovery after photobleaching (FRAP), fluorescence lifetime imaging (FLIM) and fluorescence anisotropy imaging (FAIM). This approach captures a series of polarization-resolved fluorescence lifetime images during a FRAP recovery, maximizing the information available from a limited photon budget. We have applied this method to analyse the behaviour of GFP-labelled coxsackievirus and adenovirus receptor (CAR) in living human epithelial cells. Our data reveal that CAR exists in oligomeric states throughout the cell, and that these complexes occur in conjunction with high immobile fractions of the receptor at cell-cell junctions. These findings shed light on previously unknown molecular associations between CAR receptors in intact cells and demonstrate the power of combined FRAP, FLIM and FAIM microscopy as a robust method to analyse complex multi-component dynamics in living cells.

Spectrally resolved confocal microscopy and fluorescence lifetime imaging have been used to measure the polarity of lipid-rich regions in living HeLa cells stained with Nile red. The emission peak from the solvatochromic dye in lipid droplets is at a shorter wavelength than other, more polar, stained internal membranes, and this is indicative of a low polarity environment. We estimate that the dielectric constant, ϵ , is around 5 in lipid droplets and 25<ϵ<40 in other lipid-rich regions. Our spectrally resolved fluorescence lifetime imaging microscopy (FLIM) data show that intracellular Nile red exhibits complex, multiexponential fluorescence decays due to emission from a short lifetime locally excited state and a longer lifetime intramolecular charge transfer state. We measure an increase in the average fluorescence lifetime of the dye with increasing emission wavelength, as shown using phasor plots of the FLIM data. We also show using these phasor plots that the shortest lifetime decay components arise from lipid droplets. Thus, fluorescence lifetime is a viable contrast parameter for distinguishing lipid droplets from other stained lipid-rich regions. Finally, we discuss the FLIM of Nile red as a method for simultaneously mapping both polarity and relative viscosity based on fluorescence lifetime measurements.

Centroiding in photon counting imaging has traditionally been accomplished by a single-step, noniterative algorithm, often implemented in hardware. Single-molecule localization techniques in superresolution fluorescence microscopy are conceptually similar, but use more sophisticated iterative software-based fitting algorithms to localize the fluorophore. Here, we discuss common features and differences between single-molecule localization and photon counting imaging and investigate the suitability of single-molecule localization software for photon event localization. We find that single-molecule localization software packages designed for superresolution microscopy-QuickPALM, rapidSTORM, and ThunderSTORM-can work well when applied to photon counting imaging with a microchannel-plate-based intensified camera system: photon event recognition can be excellent, fixed pattern noise can be low, and the microchannel plate pores can easily be resolved.

The specialized light organ of the ponyfish supports the growth of the bioluminescent symbiont Photobacterium leiognathi. The bioluminescence of P. leiognathi is generated within a heteromeric protein complex composed of the bacterial luciferase and a 20-kDa lumazine binding protein (LUMP), which serves as a Förster resonance energy transfer (FRET) acceptor protein, emitting a cyan-colored fluorescence with an unusually long excited state lifetime of 13.6 ns. The long fluorescence lifetime and small mass of LUMP are exploited for the design of highly optimized encoded sensors for quantitative fluorescence anisotropy (FA) measurements of protein hydrodynamics. In particular, large differences in the FA values of the free and target-bound states of LUMP fusions appended with capture sequences of up to 20 kDa are used in quantitative FA imaging and analysis of target proteins. For example, a fusion protein composed of LUMP and a 5-kDa G protein binding domain is used as an FA sensor to quantify the binding of the GTP-bound cell division control protein 42 homolog (Cdc42) (21 kDa) in solution and within Escherichia coli. Additionally, the long fluorescence lifetime and the surface-bound fluorescent cofactor 6,7-dimethyl-8- (1'-dimethyl-ribityl) lumazine in LUMP are utilized in the design of highly optimized FRET probes that use Venus as an acceptor probe. The efficiency of FRET in a zero-length LUMP-Venus fusion is 62% compared to ∼ 31% in a related CFP-Venus fusion. The improved FRET efficiency obtained by using LUMP as a donor probe is used in the design of a FRET-optimized genetically encoded LUMP-Venus substrate for thrombin.

We demonstrate diffraction limited multiphoton imaging in a massively parallel, fully addressable time-resolved multi-beam multiphoton microscope capable of producing fluorescence lifetime images with sub-50ps temporal resolution. This imaging platform offers a significant improvement in acquisition speed over single-beam laser scanning FLIM by a factor of 64 without compromising in either the temporal or spatial resolutions of the system. We demonstrate FLIM acquisition at 500 ms with live cells expressing green fluorescent protein. The applicability of the technique to imaging protein-protein interactions in live cells is exemplified by observation of time-dependent FRET between the epidermal growth factor receptor (EGFR) and the adapter protein Grb2 following stimulation with the receptor ligand. Furthermore, ligand-dependent association of HER2-HER3 receptor tyrosine kinases was observed on a similar timescale and involved the internalisation and accumulation or receptor heterodimers within endosomes. These data demonstrate the broad applicability of this novel FLIM technique to the spatio-temporal dynamics of protein-protein interaction.

Constitutive receptor tyrosine kinase phosphorylation requires regulation of kinase and phosphatase activity to prevent aberrant signal transduction. A dynamic mechanism is described here in which the adaptor protein, growth factor receptor-bound protein 2 (Grb2), controls fibroblast growth factor receptor 2 (FGFR2) signaling by regulating receptor kinase and SH2 domain-containing protein tyrosine phosphatase 2 (Shp2) phosphatase activity in the absence of extracellular stimulation. FGFR2 cycles between its kinase-active, partially phosphorylated, nonsignaling state and its Shp2-dephosphorylated state. Concurrently, Shp2 cycles between its FGFR2-phosphorylated and dephosphorylated forms. Both reciprocal activities of FGFR2 and Shp2 were inhibited by binding of Grb2 to the receptor. Phosphorylation of Grb2 by FGFR2 abrogated its binding to the receptor, resulting in up-regulation of both FGFR2's kinase and Shp2's phosphatase activity. Dephosphorylation of Grb2 by Shp2 rescued the FGFR2-Grb2 complex. This cycling of enzymatic activity results in a homeostatic, signaling-incompetent state. Growth factor binding perturbs this background cycling, promoting increased FGFR2 phosphorylation and kinase activity, Grb2 dissociation, and downstream signaling. Grb2 therefore exerts constitutive control over the mutually dependent activities of FGFR2 and Shp2.

IgE binding to its high affinity receptor FcεRI on mast cells and basophils is a key step in the mechanism of allergic disease and a target for therapeutic intervention. Early indications that IgE adopts a bent structure in solution have been confirmed by recent x-ray crystallographic studies of IgEFc, which further showed that the bend, contrary to expectation, is enhanced in the crystal structure of the complex with receptor. To investigate the structure of IgEFc and its conformational changes that accompany receptor binding in solution, we created a Förster resonance energy transfer (FRET) biosensor using biologically encoded fluorescent proteins fused to the N- and C-terminal IgEFc domains (Cε2 and Cε4, respectively) together with the theoretical basis for quantitating its behavior. This revealed not only that the IgEFc exists in a bent conformation in solution but also that the bend is indeed enhanced upon FcεRI binding. No change in the degree of bending was seen upon binding to the B cell receptor for IgE, CD23 (FcεRII), but in contrast, binding of the anti-IgE therapeutic antibody omalizumab decreases the extent of the bend, implying a conformational change that opposes FcεRI engagement. HomoFRET measurements further revealed that the (Cε2)(2) and (Cε4)(2) domain pairs behave as rigid units flanking the conformational change in the Cε3 domains. Finally, modeling of the accessible conformations of the two Fab arms in FcεRI-bound IgE revealed a mutual exclusion not seen in IgG and Fab orientations relative to the membrane that may predispose receptor-bound IgE to cross-linking by allergens.

Classical FRET (Förster Resonance Energy Transfer) using two fluorescent labels (one for the donor and another one for the acceptor) is not efficient for studying the homodimerization of a protein as only half of the homodimers formed can be identified by this technique. We thus resorted to homoFRET detected by time-resolved Fluorescence Anisotropy IMaging (tr-FAIM). To specifically image the plasma membrane of living cells, an original combination of tr-FAIM and Total Internal Reflection Fluorescence Lifetime Imaging Microscope (TIRFLIM) was implemented. The correcting factor accounting for the depolarization due to the high numerical aperture (NA) objective, mandatory for TIRF microscopy, was quantified on fluorescein solutions and on HEK293 cells expressing enhanced Green Fluorescence Protein (eGFP). Homodimerization of Amyloid Precursor Protein (APP), a key mechanism in the etiology of Alzheimer's disease, was measured on this original set-up. We showed, both in epifluorescence and under TIRF excitation, different energy transfer rates associated with the homodimerization of wild type APP-eGFP or of a mutated APP-eGFP, which forms constitutive dimers. This original set-up thus offers promising prospects for future studies of protein homodimerization in living cells in control and pathological conditions.

We demonstrate that an ultra-fast CMOS camera combined with a photon counting image intensifier can be used to determine photon arrival times well below the exposure time of the camera. We can obtain a time resolution down to around 1% of the exposure time, i.e. of the order of 40 ns with microsecond exposure times. This is achieved by exploiting the invariant phosphor decay of the image intensifier's phosphor screen: Developing a suitable mathematical framework, we show that the relative intensities of the phosphor decay in successive frames following the photon detection uniquely determine the photon arrival time. This approach opens a way to measuring fast luminescence decays in parallel in many pixels. Possible applications include oxygen and ion concentration imaging using probes with luminescence lifetimes in the range of 100 ns to microseconds.

We report a novel wide-field imaging method capable of time-correlated single photon counting. It is based on a photon counting image intensifier coupled to an ultra-fast CMOS camera running at 40 kHz frame rate. Using a pulsed excitation source and decaying luminescent sample, the arrival times of hundreds of photons can be determined simultaneously in many pixels with microsecond resolution and reduced photon pile-up. The detection system is mounted on an inverted microscope and applied to time-resolved imaging of Europium-containing polyoxometalate nanoparticles.

The sol-to-gel transition was monitored via the use of time-resolved recording of the fluorescence emission of viscosity-sensitive probes. Two dyes were chosen for the study, water-soluble DASPMI and a hydrophobic BODIPY, and steady-state, time-resolved and time-tagged fluorescence measurements were performed. These techniques, coupled with the probes different solubility, allowed complementary fluorescence lifetime and intensity data to be obtained from the dyes introduced into the matrix-forming mixture to produce sol-gel derived monoliths. Two different precursors were used as examples. A hydrogel was formed from a commercially available gellan gum (Gelrite), and a glass-like monolith was formed using tetraethyl orthosilicate. Changes in fluorescence lifetime could be related to those in the local viscosity sensed by the probe. The combination of this type of probe with time-resolved measurements is extremely useful in monitoring the microscopic changes that occur during the sol-to-gel transition within this important class of materials.

Fluorescence lifetime imaging (FLIM) and fluorescence polarization imaging are complementary techniques that can be used to extract information about macromolecules from biological samples. Owing to the sensitivity of fluorescence to the physicochemical environment, and nanometer-scale interactions via Förster resonance energy transfer (FRET), FLIM has been implemented in many laboratories for numerous applications in the life sciences and beyond. This review seeks to provide a brief overview of some of the recent advances in the techniques and more pertinently their applications in cell and tissue imaging. The particular merits of polarization-resolved fluorescence measurements are highlighted, including the unique ability to elucidate the occurrence of homo-FRET.

The average fluorescence lifetime of the green fluorescent protein (GFP) in solution is a function of the refractive index of its environment. We report that this is also the case for GFP-tagged proteins in cells. Using time-correlated single-photon counting (TCSPC)-based fluorescence lifetime imaging (FLIM) with a confocal scanning microscope, images of GFP-tagged proteins in cells suspended in different refractive index media are obtained. It is found that the average fluorescence lifetime of GFP decreases on addition of glycerol or sucrose to the media in which the fixed cells are suspended. The inverse GFP lifetime is proportional to the refractive index squared. This is the case for GFP-tagged major histocompatibility complex (MHC) proteins with the GFP located inside the cytoplasm, and also for GPI-anchored GFP that is located outside the cell membrane. The implications of these findings are discussed with regard to total internal reflection fluorescence (TIRF) techniques where the change in refractive index is crucial in producing an evanescent wave to excite fluorophores near a glass interface. Our findings show that the GFP fluorescence lifetime is shortened in TIRF microscopy in comparison to confocal microscopy.

The study of protein adsorption and any associated conformational changes on interaction with biomaterials is of great importance in the area of implants and tissue constructs. This study aimed to evaluate some fluorescent techniques to probe protein conformation on a selection of biodegradable polymers currently under investigation for biomedical applications. Because of the fluorescence emanating from the polymers, the use of monitoring intrinsic protein fluorescence was precluded. A highly solvatochromic fluorescent dye, Nile red, and a well-known protein label, fluorescein isothiocyanate, were employed to study the adsorption of serum albumin to polycaprolactone and to some extent also to two starch-containing polymer blends (SPCL and SEVA-C). A variety of fluorescence techniques, steady state, time resolved, and imaging were employed. Nile red was found to leach from the protein, while fluorescein isothiocyanate proved useful in elucidating a conformational change in the protein and the observation of protein aggregates adsorbed to the polymer surface. These effects were seen by making use of the phenomenon of energy migration between the fluorescent tags to monitor interprobe distance and the use of fluorescence lifetime imaging to ascertain the surface packing of the protein on polymer.

Time-resolved fluorescence anisotropy and fluorescence recovery after photobleaching were applied to study the diffusion of dyes and a fluorescence-labeled enzyme in a sol-gel-derived medium. This type of medium exhibits attractive properties such as robustness, low processing temperature, high porosity, large internal surface area, and can act as protective immobilization media for biologically active molecules. This makes it a suitable candidate for biosensor applications. The glasslike nature and good optical quality allows for light addressable entities to be incorporated and accessed using spectroscopy. This type of matrix, once formed, can be anything from an ordered gel to a robust glassy block depending on the aging process. In this work we apply confocal microscopy and time-resolved fluorescence techniques to study both rotational and lateral diffusion with aging time within a silica sol-gel derived monolith. An enzyme, horseradish peroxidase, was labeled with Alexa Fluor 488 and rotation related to both the enzyme and the probe monitored during the matrix aging process. Diffusion coefficients of between ca. 0.5 x 10(-7) and 4 x 10(-7) cm2 s(-1) were obtained from preliminary FRAP measurements of fluorescein and correlated to differences in the catalytic activity of HRP incorporated in the monolith.

The novel five- and seven-membered ring appended aminoporphyrazines 3 and 12 have been prepared via mixed Linstead macrocyclization. The structures of both have been unequivocally established by X-ray crystallographic studies. Reductive deselenation of selenadiazole 3 in the presence of 9,10-phenanthrenequinone or 2,3-butanedione results in the formation of pyrazines 6a,b, whereas oxidation of porphyrazine 12 gave the corresponding seco derivative 14. seco-Porphyrazine 14 mediates the generation of singlet oxygen with a quantum yield of 0.74.

An emerging theme in cell biology is that cell surface receptors need to be considered as part of supramolecular complexes of proteins and lipids facilitating specific receptor conformations and distinct distributions, e.g., at the immunological synapse. Thus, a new goal is to develop bioimaging that not only locates proteins in live cells but can also probe their environment. Such a technique is demonstrated here using fluorescence lifetime imaging of green fluorescent protein (GFP). We first show, by time-correlated single-photon counting, that the fluorescence decay of GFP depends on the local refractive index. This is in agreement with the Strickler Berg formula, relating the Einstein A and B coefficients for absorption and spontaneous emission in molecules. We then quantitatively image, by wide-field time-gated fluorescence lifetime imaging, the refractive index of the environment of GFP. This novel approach paves the way for imaging the biophysical environment of specific GFP-tagged proteins in live cells.