Fluorescence Lifetime Distribution Research Articles

Self‐assembled small molecule spherulites under mild conditions: High solid‐state quantum yield and unique interconnected structural and fluorescent colors

AbstractSpherulites are generally fabricated from cooling polymer melts, while their fabrication under mild conditions or from small molecule materials has been barely reported. Besides, organic luminescent molecules typically suffer from low quantum yields in a solid state. Moreover, preparing material with interconnected and simultaneous changes in structural and fluorescent colors is challenging. Here, we present the first solution‐derived spherulites with unique interconnected structural and fluorescent colors, self‐assembled from stearoylated monosaccharides at room temperature. D‐galactose stearoyl ester self‐assembled into banded spherulites, containing twisted nanoplates and interconnected simultaneously changing structural and fluorescent colors. In comparison, D‐mannose stearoyl ester can only form non‐banded spherulites, which contain oriented nanoplates and uniform structural and fluorescent colors. Such materials revealed a novel negative correlation between fluorescence and birefringence, termed alignment‐promoted quenching propensity. Remarkably, the solid‐state fluorescence quantum yields of galactose and mannose‐derived spherulites are as high as 49 ± 2% and 51 ± 2% respectively, approximately ten times higher than those of unmodified monosaccharides. These quantum yield values are among the highest of reported organic nonconventional fluorophores and even comparable to those of conventional aromatic chromophores. Moreover, these spherulites manifested an unexpected excitation‐dependent multicolor photoluminescence with a broad‐spectrum emission (410−620 nm). They show multiple peaks in the photoluminescent emission spectra and broad fluorescence lifetime distributions, which should be attributed to the clustering of a variety of oxygen‐containing functional groups as emissive moieties.

Growth of anthracene microcrystals by the micro-space sublimation method and their photophysical properties

Anthracene and its derivatives are widely utilized in optoelectronic devices due to their unique properties. Generally, single-crystal structures can avoid non-radiative recombination, enhance carrier mobility, and ultimately improve device performance. In this work, anthracene microcrystals were prepared using the micro-space sublimation method. Through real-time in-situ observation, the crystallization dynamics of anthracene molecules were revealed. Unlike traditional vacuum evaporation deposition technique, the close proximity of the substrate to the source facilitates the self-assembly of anthracene molecules into an ordered crystal structure. Six peaks can be observed in the photoluminescence spectrum, corresponding to various lowest excited state decay processes. The fluorescence intensity at the peak of 423 nm decreases significantly with increasing temperature. The reason for this is the relatively high exciton binding energy, which makes excitons more stable and easier to form. The lattice vibrations induced by increased temperature were found to affect the transport and separation of excitons. Time-resolved fluorescence spectroscopy imaging revealed that a relatively uniform distribution of fluorescence lifetimes in the anthracene microcrystals, indicating high crystallization quality. This work provides valuable insights for controlling the morphology and investigating the photophysical properties of organic semiconductors.

Comparison of Retinal Metabolic Activity and Structural Development between rd10 Mice and Normal Mice Using Multiphoton Fluorescence Lifetime Imaging Microscopy.

Fluorescence lifetime imaging microscopy (FLIM) is a technique that analyzes the metabolic state of tissues based on the spatial distribution of fluorescence lifetimes of certain interacting molecules. We used multiphoton FLIM to study the metabolic state of developing C57BL6/J and rd10 retinas based on the fluorescence lifetimes of free versus bound nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate (NAD(P)H), with free NAD(P)H percentages suggesting increased glycolysis and bound NAD(P)H percentages indicating oxidative phosphorylation. The mice were sacrificed and enucleated at various time points throughout their first 3 months of life. The isolated eyecups were fixed, sectioned using a polyacrylamide gel embedding technique, and then analyzed with FLIM. The results suggested that in both C57BL6/J mice and rd10 mice, oxidative phosphorylation initially decreased and then increased, plateauing over time. This trend, however, was accelerated in rd10 mice, with its turning point occurring at p10 versus the p30 turning point in C57BL6/J mice. There was also a noticeable difference in oxidative phosphorylation rates between the outer and inner retinas in both strains, with greater oxidative phosphorylation present in the latter. A greater understanding of rd10 and WT metabolic changes during retinal development may provide deeper insights into retinal degeneration and facilitate the development of future treatments.

Emission Properties of Individual AIE‐Luminogens in Solution at Room Temperature†

Comprehensive SummaryHere, we combined the photon antibunching analysis, fluorescence correlation spectroscopy, and time‐domain fluorescence lifetime imaging microscopy (TD‐FLIM) to study the emission properties of a representative AIE‐luminogen—4,4’‐(benzo[c][1,2,5] thiadiazole‐4,7‐diyl)bis(N,N‐diphenylaniline) (TPA‐BT) at the single emitter level in a tetrahydrofuran (THF)/water solution where water is a non‐solvent for TPA‐BT. Our findings suggest that, at a constant water fraction in the solution, the size of TPA‐BT aggregates increases with the TPA‐BT concentration; TPA‐BT aggregates are not a quantum emitter at room temperature in the solution. Moreover, utilizing TD‐FLIM and a gel trapping technique allowed us to study the fluorescence lifetime of individual TPA‐BT aggregates. Adding a polar solvent like water does not result in an overall decrease in fluorescence lifetime. Rather, it causes the fluorescence lifetime distribution to become wider, and only some molecules experience a decrease in their fluorescence lifetime. These results could represent a step forward in further understanding the photophysics of AIE‐luminogens.

5-Deazaalloxazine as photosensitizer of singlet oxygen and potential redox-sensitive agent

Flavins are a unique class of compounds that combine the features of singlet oxygen generators and redox-dependent fluorophores. From a broad family of flavin derivatives, deazaalloxazines are significantly underdeveloped from the point of view of photophysical properties. Herein, we report photophysics of 5-deazaalloxazine (1a) in water, acetonitrile, and some other solvents. In particular, triplet excited states of 1a in water and in acetonitrile were investigated using ultraviolet–visible (UV–Vis) transient absorption spectroscopy. The measured triplet lifetimes for 1a were all on the microsecond time scale (≈ 60 μs) in deoxygenated solutions. The quantum yield of S1 → T1 intersystem crossing for 1a in water was 0.43 based on T1 energy transfer from 1a to indicaxanthin (5) acting as acceptor and on comparative actinometric measurements using benzophenone (6). 1a was an efficient photosensitizer for singlet oxygen in aerated solutions, with quantum yields of singlet oxygen in methanol of about 0.76, compared to acetonitrile ~ 0.74, dichloromethane ~ 0.64 and 1,2-dichloroethane ~ 0.54. Significantly lower singlet oxygen quantum yields were obtained in water and deuterated water (ФΔ ~ 0.42 and 0.44, respectively). Human red blood cells (RBC) were used as a cell model to study the antioxidant capacity in vitro and cytotoxic activity of 1a. Fluorescence-lifetime imaging microscopy (FLIM) data were analyzed by fluorescence lifetime parameters and distribution for different parts of the emission spectrum. Comparison of multidimensional fluorescent properties of RBC under physiological-like and oxidative-stress conditions in the presence and absence of 1a suggests its dual activity as probe and singlet-oxygen generator and opens up a pathway for using FLIM to analyze complex intracellular behavior of flavin-like compounds. These new data on structure–property relationship contribute to the body of information required for a rational design of flavin-based tools for future biological and biochemical applications.Graphical abstract

Variety of steady and excited state interactions in BODIPY aggregates: Photophysics in antisolvent systems and floating layers

Associative behavior of geometrically anisotropic meso-(4-octadecyloxy-phenyl)-boron-dipyrrin (BODIPY) studied spectroscopically in binary solvent mixtures and upon compression in Langmuir floating layers. Different steady and excited state species were found upon monolayer compression and facilitated aggregation in water/ acetonitrile systems. This discrepancy points to a big concern in possibility of commonly applied generalizations across different aggregating systems. Broad range of decay fitting models were examined to reveal their benefits and pitfalls specific to examination of dye aggregates. Radiative constant gamma-distribution approach and free form fluorescence lifetime distribution with maximum entropy method (MEM) outperformed multiple common techniques for analysis of complex fluorescence decays. MEM could be recommended for analysis of systems where complicated lifetime distributions appear over time or upon external stimuli. Findings and protocols could be utilized as tools in studies of steady and excited-state photophysics of BODIPY aggregates.

Fluorescence lifetime distribution in phakic and pseudophakic healthy eyes.

To investigate the influence of the lens status and to describe fundus autofluorescence lifetimes (FLT) in a large cohort of healthy eyes across a wide age range. FLT data were acquired from healthy phakic and pseudophakic eyes using fluorescence lifetime imaging ophthalmoscopy (FLIO). Retinal autofluorescence was excited with a 473 nm laser and emitted autofluorescence was detected in a short and a long spectral channel (SSC: 498-560 nm; LSC: 560-720 nm). 141 healthy eyes from 141 participants (56 ± 18 years) were included. The shortest mean FLTs were measured within the macular center, followed by the temporal inner and outer ETDRS (Early Treatment Diabetic Retinopathy Study) grid segments, and the remaining areas of the inner and the outer ETDRS ring. In phakic participants (81%), mean, short and long FLTs correlated with the age (SSC: r2 = 0.54; LSC: r2 = 0.7; both p<0.0001) with an increase of about 33 ps in the SSC resp. 28 ps in the LSC per decade. In pseudophakic subjects (19%), mean FLTs only correlated with age in the long spectral channel (r2 = 0.44; p = 0.0002) but not in the short spectral channel (r2 = 0.066; p = 0.2). Fundus autofluorescence lifetimes are age dependent. FLTs in the SSC are more susceptible to lens opacities but less dependent on age changes, whereas FLTs in the LSC are largely independent of the lens status but display a higher degree of age dependency. ClinicalTrials.gov NCT01981148.

Reconstruction of fluorophore absorption and fluorescence lifetime using early photon mesoscopic fluorescence molecular tomography: a phantom study.

Fluorescence molecular lifetime tomography (FMLT) plays an increasingly important role in experimental oncology. The article presents and experimentally verifies an original method of mesoscopic time domain FMLT, based on an asymptotic approximation to the fluorescence source function, which is valid for early arriving photons. The aim was to justify the efficiency of the method by experimental scanning and reconstruction of a phantom with a fluorophore. The experimental facility included the TCSPC system, the pulsed supercontinuum Fianium laser, and a three-channel fiber probe. Phantom scanning was done in mesoscopic regime for three-dimensional (3D) reflectance geometry. The sensitivity functions were simulated with a Monte Carlo method. A compressed-sensing-like reconstruction algorithm was used to solve the inverse problem for the fluorescence parameter distribution function, which included the fluorophore absorption coefficient and fluorescence lifetime distributions. The distributions were separated directly in the time domain with the QR-factorization least square method. 3D tomograms of fluorescence parameters were obtained and analyzed using two strategies for the formation of measurement data arrays and sensitivity matrices. An algorithm is developed for the flexible choice of optimal strategy in view of attaining better reconstruction quality. Variants on how to improve the method are proposed, specifically, through stepped extraction and further use of a posteriori information about the object. Even if measurement data are limited, the proposed method is capable of giving adequate reconstructions but their quality depends on available a priori (or a posteriori) information. Further research aims to improve the method by implementing the variants proposed.

Contribution of autofluorescence from intracellular proteins in multiphoton fluorescence lifetime imaging

Multiphoton fluorescence lifetime imaging microscopy (MPM-FLIM) is extensively proposed as a non-invasive optical method to study tissue metabolism. The approach is based on recording changes in the fluorescence lifetime attributed to metabolic co-enzymes, of which nicotinamide adenine dinucleotide (NADH) is of major importance. However, intrinsic tissue fluorescence is complex. Particularly when utilizing two-photon excitation, as conventionally employed in MPM. This increases the possibility for spectral crosstalk and incorrect assignment of the origin of the FLIM signal. Here we demonstrate that in keratinocytes, proteins such as keratin may interfere with the signal usually assigned to NADH in MPM-FLIM by contributing to the lifetime component at 1.5 ns. This is supported by a change in fluorescence lifetime distribution in KRT5- and KRT14-silenced cells. Altogether, our results suggest that the MPM-FLIM data originating from cellular autofluorescence is far more complex than previously suggested and that the contribution from other tissue constituents should not be neglected—changing the paradigm for data interpretation in this context.

Deciphering the conformational dynamics of gephyrin-mediated collybistin activation

Efficient neuronal signaling depends on the proper assembly of the postsynaptic neurotransmitter machinery. The majority of inhibitory synapses feature γ-aminobutyric acid type A (GABAA) receptors. The function of these GABAergic synapses is controlled by the scaffolding protein gephyrin and collybistin, a Dbl family guanine nucleotide exchange factor and neuronal adaptor protein. Specifically, collybistin interacts with small GTPases, cell adhesion proteins, and phosphoinositides to recruit gephyrin and GABAA receptors to postsynaptic membrane specializations. Collybistin usually contains an N-terminal SH3 domain and exists in closed/inactive or open/active states. Here, we elucidate the molecular basis of the gephyrin-collybistin interaction with newly designed collybistin Förster resonance energy transfer (FRET) sensors. Using fluorescence lifetime-based FRET measurements, we deduce the affinity of the gephyrin-collybistin complex, thereby confirming that the C-terminal dimer-forming E domain binds collybistin, an interaction that does not require E domain dimerization. Simulations based on fluorescence lifetime and sensor distance distributions reveal at least a two-state equilibrium of the SH3 domain already in the free/unbound collybistin, thereby illustrating the accessible volume of the SH3 domain. Finally, our data provide strong evidence for a tightly regulated collybistin-gephyrin interplay, where, unexpectedly, switching of collybistin from closed/inactive to open/active states is efficiently triggered by gephyrin.

Encoder-decoder deep learning network for simultaneous reconstruction of fluorescence yield and lifetime distributions.

A time-domain fluorescence molecular tomography in reflective geometry (TD-rFMT) has been proposed to circumvent the penetration limit and reconstruct fluorescence distribution within a 2.5-cm depth regardless of the object size. In this paper, an end-to-end encoder-decoder network is proposed to further enhance the reconstruction performance of TD-rFMT. The network reconstructs both the fluorescence yield and lifetime distributions directly from the time-resolved fluorescent signals. According to the properties of TD-rFMT, proper noise was added to the simulation training data and a customized loss function was adopted for self-supervised and supervised joint training. Simulations and phantom experiments demonstrate that the proposed network can significantly improve the spatial resolution, positioning accuracy, and accuracy of lifetime values.

Comparative Study Between a Customized Bimodal Endoscope and a Benchtop Microscope for Quantitative Tissue Diagnosis

Nowadays, surgical removal remains the standard method to treat brain tumors. During surgery, the neurosurgeon may encounter difficulties to delimitate tumor boundaries and the infiltrating areas as they have a similar visual appearance to adjacent healthy zones. These infiltrating residuals increase the tumor recurrence risk, which decreases the patient’s post-operation survival time. To help neurosurgeons improve the surgical act by accurately delimitating healthy from cancerous areas, our team is developing an intraoperative multimodal imaging tool. It consists of a two-photon fluorescence fibered endomicroscope that is intended to provide a fast, real-time, and reliable diagnosis information. In parallel to the instrumental development, a large optical database is currently under construction in order to characterize healthy and tumor brain tissues with their specific optical signature using multimodal analysis of the endogenous fluorescence. Our previous works show that this multimodal analysis could provide a reliable discrimination response between different tissue types based on several optical indicators. Here, our goal is to show that the two-photon fibered endomicroscope is able to provide, based on the same approved indicators in the tissue database, the same reliable response that could be used intraoperatively. We compared the spectrally resolved and time-resolved fluorescence signal, generated by our two-photon bimodal endoscope from 46 fresh brain tissue samples, with a similar signal provided by a standard reference benchtop multiphoton microscope that has been validated for tissue diagnosis. The higher excitation efficiency and collection ability of an endogenous fluorescence signal were shown for the endoscope setup. Similar molecular ratios and fluorescence lifetime distributions were extracted from the two compared setups. Spectral discrimination ability of the bimodal endoscope was validated. As a preliminary step before tackling multimodality, the ability of the developed bimodal fibered endoscope to excite and to collect efficiently as well as to provide a fast exploitable high-quality signal that is reliable to discriminate different types of human brain tissues was validated.

Delving into Membrane Heterogeneity Utilizing Fluorescence Lifetime Distribution Analysis.

Lipid bilayer membranes are indispensable parts of cellular architecture. One of the integral properties of bilayer membranes is the environmental heterogeneity over a wide range of spatiotemporal scales. The environmental heterogeneity is a manifestation of the dynamic and compositional anisotropy in the plane of the membrane as well as along the bilayer normal. Fluorescence lifetime distribution analysis provides a spectroscopic tool to quantitatively characterize such heterogeneities. The review discusses recent applications of fluorescence lifetime distribution analysis utilizing the maximum entropy method to characterize horizontal and vertical heterogeneities in membranes.

Interaction of acridinedione dye with a globular protein in the presence of site selective and site specific binding drugs: Photophysical techniques assisted by molecular docking methods

Photophysical investigations and molecular docking studies of photoinduced electron transfer (PET) based fluorophores of acridine family with a globular protein, Bovine Serum Albumin (BSA) bound to non-narcotic drugs like phenylbutazone (PB) and flufenamic acid (FA) were carried out in aqueous solution. PB and FA are site specific and site selective drugs, wherein PB predominantly binds at the site (I) whereas FA selectively orients towards site (II) of BSA. Acridinedione (AD) dyes, both resorcinol and dimedone based are hydrophobic in nature and exhibits a combination of both hydrophobic and hydrogen-bonding interactions that are based on the binding sites in BSA. The extent of displacement of AD from the binding sites of BSA by PB and FA are elucidated and established from variation in the fluorescence lifetime and relative amplitude distribution of free and dye bound in site (I) and site (II). The extent of binding affinity of PB-BSA and FA-BSA in the presence of AD is minimal when compared to other site I and II drugs. This is attributed to AD dye bound to several amino acid residues present in BSA such that the dye prefers multiple binding sites in BSA even in the presence of FA and PB. Further, the dye bound to several amino acid residues of BSA ascertains the combination of hydrogen-bonding, hydrophobic interactions, pi-pi and pi-alkyl interaction apart from the binding through sites (I) and (II) from molecular docking methods. The combination of fluorescence tools with molecular modelling techniques provides an excellent approach in determining the stability of these complexes containing competitive guest molecules in the presence of a fluorescence probe and the binding characteristics of dye in a micro heterogeneous environment.

Label-free characterization of single extracellular vesicles using two-photon fluorescence lifetime imaging microscopy of NAD(P)H

The heterogeneous nature of extracellular vesicles (EVs) creates the need for single EV characterization techniques. However, many common biochemical and functional EV analysis techniques lack single EV resolution. Two-photon fluorescence lifetime imaging microscopy (FLIM) is widely used to functionally characterize the reduced form of nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate (NAD(P)H) in cells and tissues. Here, we demonstrate that FLIM can also be used to image and characterize NAD(P)H in single isolated EVs. EVs were isolated using standard differential ultracentrifugation techniques from multiple cell lines and imaged using a custom two-photon FLIM system. The presented data show that the NAD(P)H fluorescence lifetimes in isolated cell-derived EVs follow a wide Gaussian distribution, indicating the presence of a range of different protein-bound and free NAD(P)H species. EV NAD(P)H fluorescence lifetime distribution has a larger standard deviation than that of cells and a significantly different fluorescence lifetime distribution than the nuclei, mitochondria, and cytosol of cells. Additionally, changes in the metabolic conditions of cells were reflected in changes in the mean fluorescence lifetime of NAD(P)H in the produced EVs. These data suggest that FLIM of NAD(P)H could be a valuable tool for EV research.

Membrane Interactions of α-Synuclein Probed by Neutrons and Photons.

α-Synuclein (α-syn) is a key protein in the etiology of Parkinson's disease. In a disease state, α-syn accumulates as insoluble amyloid fibrils enriched in β-sheet structure. However, in its functional state, α-syn adopts an amphipathic helix upon membrane association and plays a role in synaptic vesicle docking, fusion, and clustering. In this Account, we describe our contributions made in the past decade toward developing a molecular understanding of α-syn membrane interactions, which are crucial for function and have pathological implications. Three topics are covered: α-syn membrane binding probed by neutron reflectometry (NR), the effects of membrane on α-syn amyloid formation, and interactions of α-syn with cellular membranes.NR offers a unique perspective by providing direct measurements of protein penetration depth. By the use of segmentally deuterated α-syn generated through native chemical ligation, the spatial resolution of specific membrane-bound polypeptide regions was determined by NR. Additionally, we used NR to characterize the membrane-bound complex of α-syn and glucocerebrosidase, a lysosomal hydrolase whose mutations are a common genetic risk factor for Parkinson's disease. Although phosphatidylcholine (PC) is the most abundant lipid species in mammalian cells, interactions of PC with α-syn have been largely ignored because they are substantially weaker compared with the electrostatically driven binding of negatively charged lipids. We discovered that α-syn tubulates zwitterionic PC membranes, which is likely related to its involvement in synaptic vesicle fusion by stabilization of membrane curvature. Interestingly, PC lipid tubules inhibit amyloid formation, in contrast to anionic phosphatidylglycerol lipid tubules, which stimulate protein aggregation. We also found that membrane fluidity influences the propensity of α-synuclein amyloid formation. Most recently, we obtained direct evidence of binding of α-syn to exocytic sites on intact cellular membranes using a method called cellular unroofing. This method provides direct access to the cytosolic plasma membrane. Importantly, measurements of fluorescence lifetime distributions revealed that α-syn is more conformationally dynamic at the membrane interface than previously appreciated. This exquisite responsiveness to specific lipid composition and membrane topology is important for both its physiological and pathological functions. Collectively, our work has provided insights into the effects of the chemical nature of phospholipid headgroups on the interplay among membrane remodeling, protein structure, and α-syn amyloid formation.

Morphological Evolution of Strongly Fluorescent Water Soluble AIEEgen-Triblock Copolymer Mixed Aggregates with Shape-Dependent Cell Permeability.

Dimethyl-2,5-bis(4-methoxyphenylamino)terephthalate (DBMPT) is a water-insoluble fluorogenic molecule, which has been rendered water-soluble in physiological conditions, by the addition of triblock copolymers (TBPs), P123 (PEO19PPO69PEO19), and F127 (PEO100PPO65PEO100). DBMPT-TBP mixed aggregates, formed in the process, exhibit significant aggregation-induced enhancement of emission, with nanosecond fluorescence lifetimes. Dynamics involved in suppression of nonradiative pathways and consequent enhancement of fluorescence are followed by femtosecond transient absorption and time-resolved fluorescence spectroscopic techniques. Interestingly, shapes of the aggregates formed with the two TBPs are found to be very different, even though they differ only in the length of hydrophilic blocks. DBMPT-P123 aggregates are micrometer-sized and spherical, while DBMPT-F127 aggregates form nanorods. Evolution of their morphologies, as a function of TBP concentration, is monitored using cryo-TEM, FESEM, and fluorescence lifetime imaging microscopy. Fluorescence lifetime distribution provides useful insight into microheterogeneity in these mixed aggregates. Excellent cell permeability is observed for DBMPT-F127 nanorods, in contrast to DBMPT-P123 microspheres. These fluorescent nanorods exhibit the ability to mark lipid droplets within the cell and hence bear the promise for application in intracellular imaging.

Unroofing site-specific α-synuclein–lipid interactions at the plasma membrane

Parkinson's disease is associated with α-synuclein (α-syn), a cytosolic protein enriched in presynaptic terminals. The biological function of α-syn remains elusive; however, increasing evidence suggests that the protein is involved in the regulation of synaptic vesicle fusion, signifying the importance of α-syn-lipid interactions. We show that α-syn preferentially binds to GM1-rich, liquid-ordered lipid domains on cytoplasmic membranes by using unroofed cells, which encapsulates lipid complexity and cellular topology. Moreover, proteins (Rab3a, syntaxin-1A, and VAMP2) involved in exocytosis also localize with α-syn, supporting its proposed functional role in exocytosis. To investigate how these lipid/protein interactions influence α-syn at the residue level, α-syn was derivatized with an environmentally sensitive fluorophore (7-nitrobenz-2-oxa-1,3-diazol-4-yl [NBD]) at different N- and C-terminal sites. Measurements of NBD fluorescence lifetime distributions reveal that α-syn adopts a multitude of membrane-bound conformations, which were not recapitulated in simple micelle or vesicle models, indicating an exquisite sensitivity of the protein to the complex lipid environment. Interestingly, these data also suggest the participation of the C terminus in membrane localization, which is generally overlooked and thus emphasize the need to use cellularly derived and biologically relevant membranes for biophysical characterization. Collectively, our results demonstrate that α-syn is more conformationally dynamic at the membrane interface than previously appreciated, which may be important for both its physiological and pathological functions.

Role of Cholesterol and Its Immediate Biosynthetic Precursors in Membrane Dynamics and Heterogeneity: Implications for Health and Disease.

Cholesterol is an indispensible component of cellular membranes in higher eukaryotes and plays a vital role in many cellular functions. 7-Dehydrocholesterol (7-DHC) and desmosterol represent two immediate biosynthetic precursors of cholesterol in the Kandutsch-Russell and Bloch pathways of cholesterol biosynthesis, respectively. Although 7-DHC and desmosterol differ from cholesterol merely by a double bond, accumulation of these two immediate biosynthetic precursors due to defective cholesterol biosynthesis leads to severe developmental and neurological disorders. In this context, we explored the role of cholesterol and its immediate biosynthetic precursors (7-DHC and desmosterol) on the dynamics and heterogeneity of fluid phase POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and gel phase DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) membranes, using fluorescence lifetime distribution analysis of Nile Red (9-diethylamino-5H-benzo[α]phenoxazine-5-one) using the maximum entropy method (MEM). We show here that the membrane interfacial dynamic heterogeneity, manifested as the width of the fluorescence lifetime distribution of Nile Red, exhibited by 7-DHC and desmosterol vastly differ from that displayed by cholesterol, particularly in fluid phase membranes. We conclude that a subtle alteration in sterol structure could considerably alter dynamic membrane heterogeneity, which could have implications in pathogenicity associated with defective cholesterol biosynthesis.

Digital scanned laser light-sheet fluorescence lifetime microscopy with wide-field time-gated imaging.

We develop a multidimensional fluorescence imaging technique by implementing a wide-field time-gated fluorescence lifetime imaging into digital scanned laser light-sheet microscopy (FLIM-DSLM) to measure 3D fluorescence lifetime distribution in mesoscopic specimens with high resolution. This is achieved by acquiring a series of time-gated images at different relative time delays with respect of excitation pulses at different depths. The lifetime is determined for each voxel by iteratively fitting to single exponential decay. The performance of the developed system is evaluated with the measurements of a lifetime reference Rhodamine 6G solution and a subresolution fluorescent bead phantom. We also demonstrate the application performances of this system to ex vivo and in vivo imaging of Tg(kdrl:EGFP) transgenic zebrafish embryos, illustrating the lifetime differences between the GFP signal and the autofluorescence signal. The results show that FLIM-DSLM can be used for sample size up to a few millimetres and can be utilised as a powerful and robust method for biomedical research, for example as a readout of protein-protein interactions via Förster resonance energy transfer.