Fluorescence lifetime imaging microscopy of flexible and rigid dyes probes the biophysical properties of synthetic and biological membranes.

Sensing of the biophysical properties of membranes using molecular reporters has recently regained widespread attention. This was elicited by the development of new probes of exquisite optical properties and increased performance, combined with developments in fluorescence detection. Here, we report on fluorescence lifetime imaging of various rigid and flexible fluorescent dyes to probe the biophysical properties of synthetic and biological membranes at steady state as well as upon the action of external membrane-modifying agents. We tested the solvatochromic dyes Nile red and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (ammonium salt) (NBD), the viscosity sensor Bodipy C12, the flipper dye FliptR, as well as the dyes 3,3'-dioctadecyloxacarbocyanine perchlorate (DiO), Bodipy C16, lissamine-rhodamine, and Atto647, which are dyes with no previous reported environmental sensitivity. The performance of the fluorescent probes, many of which are commercially available, was benchmarked with well-known environmental reporters, with Nile red and Bodipy C12 being specific reporters of medium hydration and viscosity, respectively. We show that some widely used ordinary dyes with no previous report of sensing capabilities can exhibit competing performance compared to highly sensitive commercially available or custom-based solvatochromic dyes, molecular rotors, or flipper in a wide range of biophysics experiments. Compared to other methods, fluorescence lifetime imaging is a minimally invasive and nondestructive method with optical resolution. It enables biophysical mapping at steady state or assessment of the changes induced by membrane-active molecules at subcellular level in both synthetic and biological membranes when intensity measurements fail to do so. The results have important consequences for the specific choice of the sensor and take into consideration factors such as probe sensitivity, response to environmental changes, ease and speed of data analysis, and the probe's intracellular distribution, as well as potential side effects induced by labeling and imaging.

Time-Resolved Fluorescence Anisotropy and Molecular Dynamics Analysis of a Novel GFP Homo-FRET Dimer.

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 (〈EFRETexp〉 = 0.25 ± 0.05) and those calculated from the molecular dynamics simulations (〈EFRETMD〉 = 0.18 ± 0.14). The relative dipole orientation between the GFPs is best described by the orientation factors 〈κ2〉 = 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.

Time-Resolved Fluorescence Anisotropy of a Molecular Rotor Resolves Microscopic Viscosity Parameters in Complex Environments.

Understanding viscosity in complex environments remains a largely unanswered question despite its importance in determining reaction rates in vivo. Here, time-resolved fluorescence anisotropy imaging (TR-FAIM) is combined with fluorescent molecular rotors (FMRs) to simultaneously determine two non-equivalent viscosity-related parameters in complex heterogeneous environments. The parameters, FMR rotational correlation time and lifetime, are extracted from fluorescence anisotropy decays, which in heterogeneous environments show dip-and-rise behavior due to multiple dye populations. Decays of this kind are found both in artificially constructed adiposomes and in live cell lipid droplet organelles. Molecular dynamics simulations are used to assign each population to nano-environments within the lipid systems. The less viscous population corresponds to the state showing an average 25° tilt to the lipid membrane normal, and the more viscous population to the state showing an average 55° tilt. This combined experimental and simulation approach enables a comprehensive description of the FMR probe behavior within viscous nano-environments in complex, biological systems.

PRODAN differentially influences its local environment.

Environmentally-sensitive membrane dyes have been extensively used to study the different liquid phases, (liquid-ordered (Lo) and liquid-disordered (Ld)) of the heterogenous cellular membrane. However, it is not yet well understood how these dyes affect membrane properties upon and post insertion. Using a combination of molecular dynamics (MD) simulations and fluorescence microscopy, we study the effect of PRODAN insertion upon its local environment. We firstly present the results of the MD simulations of PRODAN interacting with lipid bilayers of various compositions, specifically the resultant hydration and lipid order of the system. Experimentally, the lipid order of Lo and Ld vesicles containing various concentrations of PRODAN are inferred from their Generalised Polarisation (GP) values, calculated using their fluorescence spectra. We then apply the methodology to a more complex biological system, the HeLa cell line. For both systems, the presence of PRODAN influences its local environment differently between the Lo and Ld phases. In the simulated systems, the presence of PRODAN lowers the lipid order in the Ld phase and increases the order in the Lo phase, whilst experimental data demonstrates that even a small increase in PRODAN concentration significantly lowers the order of both phases. We suggest this discrepancy may be ascribed to the differing localisations of the dye molecules within the bilayer, and their effect on the hydration of adjacent lipids.

Genetically encoded sensors of protein hydrodynamics and molecular proximity.

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.

Quantitative Live Cell FLIM Imaging in Three Dimensions.

In this chapter, the concept of fluorescence lifetime and its utility in quantitative live cell imaging will be introduced, along with methods to record and analyze FLIM data. Relevant applications in 3D tissue and live cell imaging, including multiplexed FLIM detection, will also be detailed.

Hydrodynamic Radii of Ranibizumab, Aflibercept and Bevacizumab Measured by Time-Resolved Phosphorescence Anisotropy.

PURPOSE: To measure the hydrodynamic radii of intravitreal anti-VEGF drugs ranibizumab, aflibercept and bevacizumab with µs time-resolved phosphorescence anisotropy. METHODS: Ruthenium-based dye Ru(bpy)2(mcbpy - O - Su - ester)(PF6)2, whose lifetime of several hundred nanoseconds is comparable to the rotational correlation time of these drugs in buffer, was used as a label. The hydrodynamic radii were calculated from the rotational correlation times of the Ru(bpy)2(mcbpy - O - Su - ester)(PF6)2-labelled drugs obtained with time-resolved phosphorescence anisotropy measurements in buffer/glycerol solutions of varying viscosity. RESULTS: The measured radii of 2.76±0.04 nm for ranibizumab, 3.70±0.03 nm for aflibercept and 4.58±0.01 nm for bevacizumab agree with calculations based on molecular weight and other experimental measurements. CONCLUSIONS: Time-resolved phosphorescence anisotropy is a relatively simple and straightforward method that allows experimental measurement of the hydrodynamic radius of individual proteins, and is superior to theoretical calculations which cannot give the required accuracy for a particular protein.

Photon Counting Imaging with an Electron-Bombarded Pixel Image Sensor.

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.

Single-molecule localization software applied to photon counting imaging.

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.

Fluorescence lifetime imaging and FRET-induced intracellular redistribution of Tat-conjugated quantum dot nanoparticles through interaction with a phthalocyanine photosensitiser.

The interaction of Tat-conjugated PEGylated CdSe/ZnS quantum dots (QD) with the amphiphilic disulfonated aluminium phthalocyanine photosensitiser is investigated in aqueous solution and in a human breast cancer cell line. In aqueous solution, the QDs and phthalocyanine form stable nanocomposites. Using steady-state and time-resolved fluorescence measurements combined with singlet oxygen detection, efficient Förster resonance energy transfer (FRET) is observed with the QDs acting as donors, and the phthalocyanine photosensitiser, which mediates production of singlet oxygen, as acceptors. In cells, the Tat-conjugated QDs localise in lysosomes and the QD fluorescence lifetimes are close to values observed in aqueous solution. Strong FRET-induced quenching of the QD lifetime is observed in cells incubated with the nanocomposites using fluorescence lifetime imaging microscopy (FLIM). Using excitation of the QDs at wavelengths where phthalocyanine absorption is negligible, FRET-induced release of QDs from endo/lysosomes is confirmed using confocal imaging and FLIM, which is attributed to photooxidative damage to the endo/lysosomal membranes mediated by the phthalocyanine acceptor.

Wide-field time-correlated single-photon counting (TCSPC) lifetime microscopy with microsecond time resolution.

A 1 MHz frame rate complementary metal-oxide semiconductor (CMOS) camera was used in combination with an image intensifier for wide-field time-correlated single-photon counting (TCSPC) imaging. The system combines an ultrafast frame rate with single-photon sensitivity and was employed on a fluorescence microscope to image decays of ruthenium compound Ru(dpp) with lifetimes from around 1 to 5 µs. A submicrowatt excitation power over the whole field of view is sufficient for this approach, and compatibility with live-cell imaging was demonstrated by imaging europium-containing beads with a lifetime of 570 µs in living HeLa cells. A standard two-photon excitation scanning fluorescence lifetime imaging (FLIM) system was used to independently verify the lifetime for the europium beads. This approach brings together advantageous features for time-resolved live-cell imaging such as low excitation intensity, single-photon sensitivity, ultrafast camera frame rates, and short acquisition times.

Time-resolved multifocal multiphoton microscope for high speed FRET imaging in vivo.

Imaging the spatiotemporal interaction of proteins in vivo is essential to understanding the complexities of biological systems. The highest accuracy monitoring of protein-protein interactions is achieved using Förster resonance energy transfer (FRET) measured by fluorescence lifetime imaging, with measurements taking minutes to acquire a single frame, limiting their use in dynamic live cell systems. We present a diffraction limited, massively parallel, time-resolved multifocal multiphoton microscope capable of producing fluorescence lifetime images with 55 ps time-resolution, giving improvements in acquisition speed of a factor of 64. We present demonstrations with FRET imaging in a model cell system and demonstrate in vivo FLIM using a GTPase biosensor in the zebrafish embryo.

Fixed pattern noise in localization microscopy.

Localization microscopy vastly improves the resolution achieved by fluorescence microscopy by fitting the positions of individual fluorophores. We examine the reconstructions produced by different fitting algorithms for instances of fixed pattern noise--systematic tendencies to alter estimated emitter positions according to their subpixel location in a way that does not reflect the ground truth structure. We show that while not readily visible at standard empirical signal strengths, fixed pattern noise can occur when performing sub-pixel fitting, and that its degree varies according to the algorithm used and the relative size of the pixels compared to the point spread function. For pixel sizes in the range 80-170 nm, this results in variations in accuracy of the order of 2-4 nm-comparatively small for many applications, but non-negligible in scenarios where very high accuracy is sought.

A time-correlated single photon counting SPAD array camera with a bespoke data-processing algorithm for lightsheet fluorescence lifetime imaging (FLIM) and FLIM videos.

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.

Age-specific and compartment-dependent changes in mitochondrial homeostasis and cytoplasmic viscosity in mouse peripheral neurons.

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.

A fluorescent biosensor reveals conformational changes in human immunoglobulin E Fc: implications for mechanisms of receptor binding, inhibition, and allergen recognition.

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.

Visualising varnish removal for conservation of paintings by fluorescence lifetime imaging (FLIM).

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.

Influence of molecular shape, conformability, net surface charge, and tissue interaction on transscleral macromolecular diffusion.

The purpose of this study was to investigate the influence of molecular shape, conformability, net surface charge and tissue interaction on transscleral diffusion. Unfixed, porcine sclera was clamped in an Ussing chamber. Fluorophore-labelled neutral albumin, neutral dextran, or neutral ficoll were placed in one hemi-chamber and the rate of transscleral diffusion was measured over 24 h using a spectrophotometer. Experiments were repeated using dextrans and ficoll with positive or negative net surface charges. Fluorescence recovery after photobleaching (FRAP) was undertaken to compare transscleral diffusion with diffusion through a solution. All molecules were 70 kDa. With FRAP, the diffusion coefficient (D) of neutral molecules was highest for albumin, followed by ficoll, then dextran (p < 0.0001). Positive dextrans diffused fastest, followed by negative, then neutral dextrans (p = 0.0004). Neutral ficoll diffused the fastest, followed by positive then negative ficoll (p = 0.5865). For the neutral molecules, transscleral D was highest for albumin, followed by dextran, then ficoll (p < 0.0001). D was highest for negative ficoll, followed by neutral, then positive ficoll (p < 0.0001). By contrast, D was highest for positive dextran, followed by neutral, then negative dextran (p = 0.0021). In conclusion, diffusion in free solution does not predict transscleral diffusion and the molecular-tissue interaction is important. Molecular size, shape, and charge may all markedly influence transscleral diffusion, as may conformability to a lesser degree, but their effects may be diametrically opposed in different molecules, and their influence on diffusion is more complex than previously thought. Each variable cannot be considered in isolation, and the interplay of all these variables needs to be tested, when selecting or designing drugs for transscleral delivery.

Unlocking the potential of forensic traces: Analytical approaches to generate investigative leads.

Forensic investigation involves gathering the information necessary to understand the criminal events as well as linking objects or individuals to an item, location or other individual(s) for investigative purposes. For years techniques such as presumptive chemical tests, DNA profiling or fingermark analysis have been of great value to this process. However, these techniques have their limitations, whether it is a lack of confidence in the results obtained due to cross-reactivity, subjectivity and low sensitivity; or because they are dependent on holding reference samples in a pre-existing database. There is currently a need to devise new ways to gather as much information as possible from a single trace, particularly from biological traces commonly encountered in forensic casework. This review outlines the most recent advancements in the forensic analysis of biological fluids, fingermarks and hair. Special emphasis is placed on analytical methods that can expand the information obtained from the trace beyond what is achieved in the usual practices. Special attention is paid to those methods that accurately determine the nature of the sample, as well as how long it has been at the crime scene, along with individualising information regarding the donor source of the trace.