Sensing metabolites using donor-acceptor nanodistributions in fluorescence resonance energy transfer

Before fluorescence sensing techniques can be applied to media as delicate and complicated as human tissue, an adequate interpretation of the measured observables is required, i.e., an inverse problem needs to be solved. Recently we have solved the inverse problem relating to the kinetics of fluorescence resonance energy transfer (FRET), which clears the way for the determination of the donor-acceptor distribution function in FRET assays. In this letter this approach to monitoring metabolic processes is highlighted and the application to glucose sensing demonstrated

Detecting beta-amyloid aggregation from the time-resolved emission spectra

Aggregation of beta-amyloids is one of key processes responsible for the development of Alzheimer's desease. Early molecular-level detection of beta-amyloid oligomers may help in early diagnosis and in the development of new intervention therapies. Our previous studies on changes in beta-amyloid's single tyrosine intrinsic fluorescence response during aggregation demonstrated a four-exponential fluorescence intensity decay, and that the ratio of the pre-exponential factors indicated the extent of aggregation in the early stages of the process before the beta-sheets are formed. Here we present a complementary approach based on time-resolved emission spectra (TRES) of amyloid's tyrosine excited at 279 nm and fluorescent in the window 240-450 nm. TRES has been used to demonstrate sturctural changes occuring on the nanosecond time scale after excitation which has significant advantages over using steady-state spectra. We demonstrate this by resolving the fluorescent species and revealing that beta-amyloid's monomers show very fast dielectric relaxation and its oligomers display a substantial spectral shift due to dielectric relaxation, which gradually decreases when oligomers become larger

Time-resolved FRET fluorescence spectroscopy of visible fluorescent protein pairs

Förster resonance energy transfer (FRET) is a powerful method for obtaining information about small-scale lengths between biomacromolecules. Visible fluorescent proteins (VFPs) are widely used as spectrally different FRET pairs, where one VFP acts as a donor and another VFP as an acceptor. The VFPs are usually fused to the proteins of interest, and this fusion product is genetically encoded in cells. FRET between VFPs can be determined by analysis of either the fluorescence decay properties of the donor molecule or the rise time of acceptor fluorescence. Time-resolved fluorescence spectroscopy is the technique of choice to perform these measurements. FRET can be measured not only in solution, but also in living cells by the technique of fluorescence lifetime imaging microscopy (FLIM), where fluorescence lifetimes are determined with the spatial resolution of an optical microscope. Here we focus attention on time-resolved fluorescence spectroscopy of purified, selected VFPs (both single VFPs and FRET pairs of VFPs) in cuvette-type experiments. For quantitative interpretation of FRET–FLIM experiments in cellular systems, details of the molecular fluorescence are needed that can be obtained from experiments with isolated VFPs. For analysis of the time-resolved fluorescence experiments of VFPs, we have utilised the maximum entropy method procedure to obtain a distribution of fluorescence lifetimes. Distributed lifetime patterns turn out to have diagnostic value, for instance, in observing populations of VFP pairs that are FRET-inactiv

Nonextensive kinetics of fluorescence resonance energy transfer

Some fluorescence dyes in complex media, such as those found in biology, demonstrate nonextensive kinetics, which implies representing their fluorescence decays in terms of lifetime distributions rather than simple exponentials. Complex kinetics usually discourage application to lifetime sensors, as it is believed, that additional molecular mechanisms employed for detection of an analyte will make the resulting kinetics ambiguous and the sensor response inconclusive. In this paper we investigate theoretically the applicability of complex dye kinetics as a fluorescence resonance energy transfer based lifetime sensor and demonstrate that the nonextensive nature of its kinetics does not decrease the sensing performance, and indeed even provides richer structural information than a simple exponential behavior. (Abstract is from AIP web site: http://jcp.aip.org/jcpsa6/v129/i14

Fluorescence resonance energy transfer sensors

We describe the various implementations of fluorescence resonance energy transfer with respect to the kinetic design principles involved in fluorescence lifetime sensors. Applications to metal ion and glucose detection are discussed. The versatility and key developments for using timecorrelated single-photon counting in fluorescence lifetime based sensing are illustrated

Determination of acceptor distribution from fluorescence resonance energy transfer: theory and simulation

A new method for determining the donor-acceptor distribution function in fluorescence resonance energy transfer (FRET) systems is presented. The approach is based on time-resolved fluorescence experiments with nanosecond resolution. Potential applications of this method include: determination of the morphology of porous materials (e.g., polymers, sol-gels, resins, etc.), monitoring processes occurring on the nanometer scale including biomolecules labeled with the donor/acceptor species, and FRET sensors based on competitive binding. In this paper a theoretical derivation of the method is presented and the method is tested in a series of numerical simulations. The experimental conditions regarding this approach are discussed and its applicability to real measurement systems is demonstrate

Fluorescence nanotomography: a structural tool in biomedical sensing

Fluorescence nanotomography (FN) is a newly developed method for determining molecular distributions on a nanometre scale in soft solids, biological macromolecules and medically important systems. FN uses fluorescence resonance energy transfer (FRET) for the recognition of the separations between molecules. By using a fluorescence lifetime measurement of sub-nanosecond time resolution, the spatial resolution of the resulting distribution function can be better than 1 �. In this paper the theoretical background of the method is outlined and the results of simulations on model molecular distributions presented. This is followed by demonstration of several applications of FN to real molecular systems, including bulk solutions of molecules of different sizes, complexes, porous polymers, phospholipids and sugar-protein competitive binding sensors glucose. The experimental requirements of FN as a structural tool for wide class of biomedical systems are discussed

Fluorescence lifetime sensor of copper ions in water

We demonstrate an optical method for the selective detection of Cu(II) ions in water using time‐resolved fluorescence resonance energy transfer from the dye rhodamine 800 encapsulated in a sensor. In comparison to copper, quenching by other metal ions such as cobalt, nickel, and chromium is shown to be negligible. The experimental arrangement incorporates picosecond diode laser excitation and time‐correlated single‐photon counting for detection. Down to 5 mM of copper deposited on the sensor can be measured and a linear response is obtained up to at least 50 mM. A lower limit of detection for the sensor in the region of 10 ppb is shown to be readily achievable with good resolution

Sol-gel nanometrology: gated sampling can reveal initial sol formation kinetics

This report describes a gated sampling approach for studying the initial formation of sol-gel glasses prepared from sodium silicate solution (water glass) and sulphuric acid. Previously described were how changes in particle size and subsequently how sol-gel formation dynamics can be tracked using time-resolved fluorescence anisotropy, by labeling growing silica nanoparticles with suitable fluorescence probes. One limiting factor of this approach was the 2 minute measurement time, which limits this technique for studying the initial sol formation dynamics and limits the measurement precision. Using a continuous flow system and delaying sol flow through different tubing lengths overcomes this problem and allows monitoring of the very early stages of sol formation, second by second after sol preparation, irrespective of the anisotropy measurement time. This technique was applied to studying the initial formation dynamics, within the first 30 seconds, of a 12.01% SiO2 (w/w), pH 0.66 sol-gel, finding that silica particles of 1.5 nm mean radius are formed within 10 seconds of mixing the sol-gel