Live-cell FLIM-FRET using a commercially available system.

Förster resonance energy transfer (FRET)-based sensors have been powerful tools in cell biologists' toolkit for decades. Informed by fundamental understanding of fluorescent proteins, protein-protein interactions, and the structural biology of reporter components, researchers have been able to employ creative design approaches to build sensors that are uniquely capable of probing a wide range of phenomena in living cells including visualization of localized calcium signaling, sub-cellular activity gradients, and tension generation to name but a few. While FRET sensors have significantly impacted many fields, one must also be cognizant of the limitations to conventional, intensity-based FRET measurements stemming from variation in probe concentration, sensitivity to photobleaching, and bleed-through between the FRET fluorophores. Fluorescence lifetime imaging microscopy (FLIM) largely overcomes the limitations of intensity-based FRET measurements. In general terms, FLIM measures the time, which for the reporters described in this chapter is nanoseconds (ns), between photon absorption and emission by a fluorophore. When FLIM is applied to FRET sensors (FLIM-FRET), measurement of the donor fluorophore lifetime provides valuable information such as FRET efficiency and the percentage of reporters engaged in FRET. This chapter introduces fundamental principles of FLIM-FRET toward informing the practical application of the technique and, using two established FRET reporters as proofs of concept, outlines how to use a commercially available FLIM system.

Effects of consumer use practices on nanosilver release from commercially available food contact materials.

Migration evaluation involving nano-enabled food contact materials (FCMs) mostly focuses on potential nanoparticle release from new unused products. This may not represent consumer use practices encountered by the FCMs in their lifecycle. In order to determine if product use impacts the release of nanoparticles or other FCM components, it is necessary to perform migration evaluations under typical consumer use scenarios. A quantitative assessment of nanoparticle release from a commercially available nanosilver-enabled cutting board was performed under five conditions intended to simulate consumer use. Knife motion, washing and scratching scenarios were simulated by linear abrasion using knife blades, scrubbing pads and tungsten carbide burr attachments, respectively. Migration was evaluated using water and 3% acetic acid as food simulants. Low concentrations of silver (Ag) were detected in water simulants, a small portion (<4 ng dm-2) in the form of silver nanoparticles (AgNPs) with particle number concentrations on the order of 106 particles dm-2. Median particle diameter was 40 nm. Nanoparticle release into water was observed under all five consumer use scenarios studied, however there was no correlation with the different levels of stress simulated.

Solvation Dynamics and Proton Transfer in Diethylaminohydroxyflavone.

4'-N,N-Diethylamino-3-hydroxyflavone (DEAHF) exhibits dual fluorescence in most solvents as a result of a rapid excited-state intramolecular proton transfer reaction. The high sensitivity of its dual emission to solvent polarity and hydrogen bonding make DEAHF of interest as a ratiometric fluorescence sensor. In addition, prior work has suggested that the rate of this proton transfer should depend on solvent relaxation in an unusual manner. It has been proposed that rapid solvation of the initially excited reactant should retard reaction. The present work tests this idea by using femtosecond Kerr-gated emission spectroscopy to measure the reaction kinetics of DEAHF in mixtures of propylene carbonate (PC) + acetonitrile (ACN). This mixture was chosen to maintain constant solvent polarity and thereby constant reaction energies while varying solvation times ∼10-fold with composition. The reaction kinetics observed in these mixtures are multiexponential, consisting of resolvable components of ∼2 and ∼30 ps and a small fraction of reaction faster than detectable by the 400 fs resolution of the experiment. Average reaction times increase by approximately a factor of 2 as a function of ACN mole fraction, primarily as a result of changes to the slower kinetic component. This trend is opposite to the composition dependence of solvation times, thereby supporting the unusual role of polar solvation dynamics in this proton transfer. In n-alkane solvents, where electrostatic coupling is minimized, frictional properties of the solvent do not influence reaction rates.

Steady-State and Time-Resolved Studies into the Origin of the Intrinsic Fluorescence of G-Quadruplexes.

Stretches of guanines in DNA and RNA can fold into guanine quadruplex structures (GQSs). These structures protect telomeres in DNA and regulate gene expression in RNA. GQSs have an intrinsic fluorescence that is sensitive to different parameters, including loop sequence and length. However, the dependence of GQS fluorescence on solution and sequence parameters and the origin of this fluorescence are poorly understood. Herein we examine effects of dangling nucleotides and cosolute conditions on GQS fluorescence using both steady-state and time-resolved fluorescence spectroscopy. The quantum yield of dGGGTGGGTGGGTGGG, termed "dG3T", is found to be modest at ∼2 × 10(-3). Nevertheless, dG3T and its variants are significantly brighter than the common nucleic acid fluorophore 2-aminopurine (2AP) largely due to their sizable extinction coefficients. Dangling 5'-end nucleotides generally reduce emission and blue-shift the resultant spectrum, whereas dangling 3'-end nucleotides slightly enhance fluorescence, particularly on the red side of the emission band. Time-resolved fluorescence decays are broadly distributed in time and require three exponential components for accurate fits. Time-resolved emission spectra suggest the presence of two emitting populations centered at ∼330 and ∼390 nm, with the redder component being a well-defined long-lived (∼1 ns) entity. Insights into GQS fluorescence obtained here should be useful in designing brighter intrinsic RNA and DNA quadruplexes for use in label-free biotechnological applications.

Dynamic Coupling at the Ångström Scale.

While momentum transfer from active particles to their immediate surroundings has been studied for both synthetic and biological micron-scale systems, a similar phenomenon was presumed unlikely to exist at smaller length scales due to the dominance of viscosity in the ultralow Reynolds number regime. Using diffusion NMR spectroscopy, we studied the motion of two passive tracers--tetramethylsilane and benzene--dissolved in an organic solution of active Grubbs catalyst. Significant enhancements in diffusion were observed for both the tracers and the catalyst as a function of reaction rate. A similar behavior was also observed for the enzyme urease in aqueous solution. Surprisingly, momentum transfer at the molecular scale closely resembles that reported for microscale systems and appears to be independent of swimming mechanism. Our work provides new insight into the role of active particles on advection and mixing at the Ångström scale.

Observations of probe dependence of the solvation dynamics in ionic liquids.

Solvation and rotational dynamics of 4-aminophthalimide (4AP) in four ionic liquids (ILs) are measured using a combination of fluorescence upconversion spectroscopy and time-correlated single photon counting. These data are compared with previously reported data for coumarin 153 (C153) to investigate the probe dependence of solvation dynamics. No fast component (<15 ps) in the fluorescence anisotropy is observed with 4AP. The differences between the solvation response functions of 4AP and C153 are significant in all four ILs, but these differences can be reduced by applying a correction for solute rotation using measured emission anisotropies. Response functions of other probes available in the literature are used to further examine the validity of this correction. The corrected data are also compared to predictions of dielectric continuum models of solvation. By replacing the measured static conductivity of the ILs with an estimated value, such predictions show good agreement with the observed spectral response functions, especially when the anion size is small.

The Photophysics of Three Naphthylmethylene Malononitriles.

The solvent dependence of the photophysical properties of three naphthylmethylene malononitriles, 1-(1-naphthalenylmethylene)-propanedinitrile (1-MN), 2-(2-naphthalenylmethylene)-propanedinitrile (2-MN), and 2-(3,4-dihydro-1(2H)-phenanthrenylidene)-propanedinitile (r2-MN), was studied in order to determine their potential utility as fluidity probes and to make comparisons to the better studied benzylidene malononitriles. Density functional calculations were used to understand the possible conformational states related to rotation about the vinyl-aromatic bond ("τ"). Absorption and emission frequencies, extinction coefficients, fluorescence quantum yields, and fluorescence lifetimes were measured in 11 representative solvents. Both the computational and experimental results indicate that the S0 → S1 transitions of these molecules have substantial charge-transfer character and produce highly polar excited states. Emission appears to result from relaxed S1 states which do not differ qualitatively from the Franck-Condon states reached by absorption. In 2-MN, time-resolved emission reveals the presence of two ground-state conformers ("a" and "b" differing by ∼180° rotation about τ) coexisting in low-polarity solvents. In contrast, 1-MN appears to exist primarily as a single dominant ground-state conformer. Fluorescence lifetimes vary from ∼1 ps in 1-MN to ∼200 ps in 2-MN(a) at room temperature. With the exception of 2-MN(a), the lifetimes vary systematically with solvent in a manner similar to what is observed in the benzylidene malononitriles. Both solvent polarity and fluidity appear to be important determinants of lifetime. The primary mechanism of fluorescence decay in naphthylmethylene malononitriles is likely to be the same as that of the benzylidene malononitriles-twisting about the double bond in S1, which leads to rapid internal conversion via a conical intersection with S0.

Sub-picosecond fluorescence evolution of amino-cyano-stilbenes in methanol: polar solvation obeys continuum theory without evidence of twisting.

The spectral evolution of fluorescence from 4-(dimethylamino)-4'-cyanostilbene (DCS) in methanol, and of two derivatives bearing either the anilino (ACS) or the julolidino (JCS) moiety, was measured by optical Kerr-gating with a time resolution of 0.35 ps. A special thin Glan polariser in the Kerr shutter allows high contrast without unnecessarily increasing the group delay dispersion. The emission band may thus be gated and observed even with highly fluorescent samples. The spectral dynamics consists of a continuous red-shift and narrowing with similar relaxation behavior throughout, i.e. between these two observables and the three compounds. This suggests that polar solvation is the common cause for spectral relaxation after 0.2 ps. The continuum model describes the dynamic Stokes shift quantitatively. Contrary to previous reports we do not find a temporary isosbestic point in the fluorescence of JCS, nor is there evidence for a dependence on anilino substituents. The crystal structures of DCS and JCS are provided.