Developing FRET Networks for Sensing.

Single-Chain Lanthanide Luminescence Biosensors for Cell-Based Imaging and Screening of Protein-Protein Interactions.

BackgroundFörster resonance energy transfer (FRET) is a mechanism where energy is transferred from an excited donor fluorophore to adjacent chromophores via non-radiative dipole-dipole interactions. FRET theory primarily considers the interactions of a single donor-acceptor pair. Unfortunately, it is rarely known if only a single acceptor is present in a molecular complex. Thus, the use of FRET as a tool for measuring protein-protein interactions inside living cells requires an understanding of how FRET changes with multiple acceptors. When multiple FRET acceptors are present it is assumed that a quantum of energy is either released from the donor, or transferred in toto to only one of the acceptors present. The rate of energy transfer between the donor and a specific acceptor (kD→A) can be measured in the absence of other acceptors, and these individual FRET transfer rates can be used to predict the ensemble FRET efficiency using a simple kinetic model where the sum of all FRET transfer rates is divided by the sum of all radiative and non-radiative transfer rates.Methodology/Principal FindingsThe generality of this approach was tested by measuring the ensemble FRET efficiency in two constructs, each containing a single fluorescent-protein donor (Cerulean) and either two or three FRET acceptors (Venus). FRET transfer rates between individual donor-acceptor pairs within these constructs were calculated from FRET efficiencies measured after systematically introducing point mutations to eliminate all other acceptors. We find that the amount of energy transfer observed in constructs having multiple acceptors is significantly greater than the FRET efficiency predicted from the sum of the individual donor to acceptor transfer rates.Conclusions/SignificanceWe conclude that either an additional energy transfer pathway exists when multiple acceptors are present, or that a theoretical assumption on which the kinetic model prediction is based is incorrect.

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

Herein, an ionic material (IM) with Förster Resonance Energy Transfer (FRET) characteristics is reported for the first time. The IM is designed by pairing a Nile Blue A cation (NBA+) with an anionic near-infrared (NIR) dye, IR820−, using a facile ion exchange reaction. These two dyes absorb at different wavelength regions. In addition, NBA+ fluorescence emission spectrum overlaps with IR820− absorption spectrum, which is one requirement for the occurrence of the FRET phenomenon. Therefore, the photophysical properties of the IM were studied in detail to investigate the FRET mechanism in IM for potential dye sensitized solar cell (DSSCs) application. Detailed examination of photophysical properties of parent compounds, a mixture of the parent compounds, and the IM revealed that the IM exhibits FRET characteristics, but not the mixture of two dyes. The presence of spectator counterion in the mixture hindered the FRET mechanism while in the IM, both dyes are in close proximity as an ion pair, thus exhibiting FRET. All FRET parameters such as spectral overlap integral, Förster distance, and FRET energy confirm the FRET characteristics of the IM. This article presents a simple synthesis of a compound with FRET properties which can be further used for a variety of applications.

The measurement of donor lifetime modification by Förster resonance energy transfer (FRET) is a widely used tool for detecting protein-protein interactions and protein conformation change. Such measurements can be compromised by the presence of a significant noninteracting fraction of molecules. Combining time-resolved intensity and anisotropy measurements gives access to both molecular distance and orientation. Fluorescent proteins frequently used to detect energy transfer in biological systems often exhibit decay characteristics indicative of more than one excited state. However, little attention has thus far been given to the specific modes of energy transfer, in particular, which states are predominantly coupled. Here, we use a previously characterized dimerization system to study energy transfer between EGFP and mCherry. Optically excited EGFP and mCherry both exhibit biexponential decays, and FRET should therefore involve dipole-dipole transfer between these four states. Analysis of the sensitized fluorescence anisotropy and intensity decays indicates that FRET transfer is predominantly from the shorter lived EGFP emitting state (2.43 ns) to the longer lived (ca. 2.77 ns) minority component (ca. 16%) of the optically excited mCherry emission. This high degree of state selection between these two widely used FRET pairs highlights the fundamental differences that can arise between direct optical excitation of an isotropic molecular population and dipole-dipole coupling in a far from isotropic interaction geometry and has consequences regarding the accurate interpretation of fluorescent protein FRET data.

Ionic Liquid-Based Dye for Highly Sensitive Optical Sensing Based on FRET and Ion Extraction System Using Plasticized PVC Membrane

The applicability of the three-step Förster resonance energy transfer (FRET) to detection of insulin amyloid fibrils was evaluated, using the chromophore system, containing Thioflavin T (ThT), 4-dimethylaminochalcone (DMC), and two squaraine dyes, referred to here as SQ1 and SQ4. The mediator chromophore DMC was found to enhance the fluorescence intensity of the terminal acceptor, SQ1, excited at 440 nm (at the absorption maximum of the principal donor, ThT), in fibrillar insulin compared to the system without DMC, providing the evidence for the cascade energy transfer in the chain ThT→DMC→SQ4→SQ1. Furthermore, the resulting Stokes shift in the four-chromophore system was 240 nm, as compared to 45 nm for the fibril-bound ThT, suggesting that higher signal-to-noise ratio is the advantage of amyloid fibril detection by multistep FRET. The maximum efficiencies of energy transfer in the insulin fibrils estimated from the quenching of the donor fluorescence in the presence of acceptor for the donor-acceptor pairs ThT-DMC, DMC-SQ4 and SQ4-SQ1 were 40%, 60% and 30% respectively, while negligible FRET occurred in the non-fibrillized protein. The most pronounced differences between fibrillar and non-fibrillized insulin were observed in the 3D fluorescence spectra. Specifically, two intensive spots centered at the emission wavelengths ~ 650 nm (SQ4) and ~ 685 nm (SQ1) were revealed at the excitation wavelength ~ 440 nm in the 3D patterns of insulin amyloid aggregates. In contrast, in the case of the non-fibrillized protein, the barely noticeable spots centered at the same wavelengths, as well as higher fluorescence intensities at the excitation above 550 nm were observed, suggesting the predominant impact of the direct excitation of SQ1 and SQ4 on their fluorescence responses. The inter-chromophore distances calculated from the experimental values of the energy transfer efficiency assuming the isotropic rotation of the dyes, were found to be 2.4, 4.5 and 4.3 nm for the ThT-DMC, DMC-SQ4 and SQ4-SQ1 pairs, respectively, revealing the different fibril binding sites for the examined dyes. The quantum-chemical calculations and simple docking studies provided evidence for the SQ1, SQ4 and ThT, DMC binding to the wet and dry interface of the insulin amyloid protofilament, respectively. The dye-protein complexes are likely to be stabilized by the hydrophobic, van der Waals, aromatic and electrostatic interactions. In summary, the above technique based on the multistep FRET can be employed for the identification and characterization of amyloid fibrils in vitro along with the classical ThT assay, allowing the increase of the amyloid detection sensitivity and lowering the probability of the pseudo-positive result. The applicability of the multistep FRET for amyloid visualization in vivo can be also tested by the involvement of the near-infrared fluorescent dyes to the cascade.

Summary form only given. Forster resonance energy transfer (FRET) is the dominant nonradiative energy transfer mechanism between a donor and acceptor fluorophore in nanometer proximity. FRET plays a pivotal role in the photosynthetic apparatus of plants and bacteria and many applications, ranging from photovoltaics and lighting, to probing molecular distances and interactions.It is an intriguing open question whether the FRET rate γFRET and the energy transfer efficiency ηFRET can also be controlled by the nanoscale optical environment, characterized by the local density of optical states (LDOS) [1]. Pioneering work suggested that the transfer rate depends linearly on the LDOS at the donor emission frequency [2], while later work suggested a dependence on the LDOS squared [3], or even a transfer rate independent of the LDOS [4]. We study the influence of the LDOS on Forster transfer, using precisely-defined, isolated, and efficient donor-acceptor pairs. The FRET pairs are dye molecules that covalently bound to the opposite ends of a 15 basepair long double-stranded with a precisely defined distance of 6.8 nm. Control over the LDOS is realized by positioning the FRET systems at well-defined distances (ranging from 60 nm to 270 nm) from a metallic mirror. The energy transfer rate γFRET and efficiency ηFRET are obtained by measuring the donor emission rate γDA in presence and the rate γD in absence of the acceptor using time-correlated single-photon counting based lifetime imaging. Our data unequivocally show that the FRET rate is independent of the LDOS at donor emission frequencies, consistent with quantum-optical theory. The FRET efficiency clearly changes with LDOS [5], since the LDOS alters the competition between the different decay processes. By controlling the radiative decay rate of the energy donor by the LDOS, the energy transfer efficiency can be enhanced or reduced. If a donor with unit quantum efficiency is placed in a 3D photonic bandgap, the energy transfer efficiency will approach 100 %, independent of the acceptor, and of the distances and orientations between the FRET partners.

Fӧrster resonance energy transfer (FRET) based cAMP sensors have become a standard approach to visualize cAMP signals in cells. However, FRET sensors inherently offer low signal-to-noise ratios, making cAMP measurements challenging, especially in 3 spatial dimensions. In previous studies, we used 4-dimensional (x, y, z, and λ) hyperspectral imaging and analysis approaches to measure agonist – induced distribution of cAMP signals in cultured pulmonary microvascular endothelial cells. In many cell types including PMVECs, transient transfection of FRET sensors results in a low transfection efficiency (1-5%), which limits the possibilities of studying spatial distribution of FRET signals in many cells simultaneously. We developed a transgenic rat model that expresses the H187 FRET sensor. In current studies, we utilize hyperspectral imaging and analysis approaches to characterize the distribution of FRET signals in different tissues and vessels harvested from transgenic rats. Hyperspectral z-stacks of tissues and vessels were acquired using a Nikon A1R confocal microscope equipped with 20X multi-immersion objective and a 32 channel PMT detector. Samples were excited using 405 nm and 561 nm lasers and emission was collected from 424 nm -724 nm at 10 nm intervals. Tissues including lungs, heart, mesentery, kidney, expressed the H187 FRET sensor. In conclusion, transgenic rats provide a platform to visualize cAMP signals in-vitro and in-situ.

We propose a linear measure of the Forster resonant energy transfer (FRET) efficiency derived in joint, donor and acceptor, intensity space and compare it quantitatively with the conventional ratiometric FRET measure normally used for FRET fluorescence microscopy images. Because of the low signal-to-noise characteristic of these images, particularly time-lapse images, we determine a quantitative model of shot noise and combine it with our measure. This provides a single measure of the certainty of FRET events that can be used as a pixel-wise classifier of FRET images. We evaluate our measures quantitatively by simulating photon emission under realistic conditions and demonstrate: linearity with FRET efficiency; less bias to shot noise than the ratio; and high correlation (r = 0.96) between our FRET certainty measure and SNR.

Förster Resonant Energy Transfer (FRET) is a near-field interaction between pairs of quantum emitters that can be modified by their electromagnetic environment. The different approaches to control FRET have been studied analytically and experimentally in the optical frequency range, although, such experiments are challenging. An effective way to investigate this effect is to directly measure an impedances of dipole antennas in the microwave frequency range, which is an analog of the FRET. In this work, we perform an analytical and experimental analysis of the energy transfer between two dipoles near a conductive plane in the microwave range for two dipole orientations: parallel and perpendicular. We analytically investigate the influence of the dipole size on the energy transfer and compare it with the solution for FRET between ideal point dipoles. We show theoretical and experimental evidences of quenching and enhancement of the energy transfer for respectively parallel and perpendicular orientations of the dipoles.

We demonstrate the surface-plasmon-induced enhancement of Förster resonance energy transfer (FRET)using a model multilayer core-shell nanostructure consisting of an Au core and surrounding FRET pairs, i.e., CdSe quantum dot donors and S101 dye acceptors. The multilayer configuration was demonstrated to exhibit synergistic effects of surface plasmon energy transfer from the metal to the CdSe and plasmon-enhanced FRET from the quantum dots to the dye. With precise control over the distance between the components in the nanostructure, significant improvement in the emission of CdSe was achieved by combined resonance energy transfer and near-field enhancement by the metal, as well as subsequent improvement in the emission of dye induced by the enhanced emission of CdSe. Consequently, the Förster radius was increased to 7.92 nm and the FRET efficiency was improved to 86.57% in the tailored plasmonic FRET nanostructure compared to the conventional FRET system (22.46%) without plasmonic metals.

We demonstrate optofluidic microlasers using highly efficient non-radiative Förster resonance energy transfer (FRET) for pumping of gain medium placed within liquid microdroplets situated on a superhydrophobic surface. Microdroplets generated from a mixture of ethylene glycol, glycerol, and water and stained with the FRET donor–acceptor dye pair Rhodamine 6G-Rhodamine 700 serve as active optical resonant cavities hosting high-quality whispering gallery modes. Upon direct optical pumping of the donor with a pulsed laser, lasing is observed in the emission band of the acceptor as a result of efficient FRET coupling between the acceptor and donor molecules. FRET lasing is characterized for different acceptor and donor concentrations, and threshold pump fluences of acceptor lasing as low as 6.3 mJ cm−2 are demonstrated. We also verify the dominance of the non-radiative FRET over cavity-assisted radiative energy transfer for the range of parameters studied in the experiments.

Förster resonance energy transfer (FRET) spectroscopy and microscopy are constantly expanding sensing techniques for analyzing biomolecular interactions. In addition to the biological recognition molecules and biological or chemical analytes, the most important components for designing FRET biosensing systems are the materials that constitute the FRET donor-acceptor pair. These FRET materials consist of small molecules, biological or chemical nanoscaffolds, or nanomaterials that function in the ultraviolet, visible, or infrared spectral range. They can absorb light, fluoresce or phosphoresce with lifetimes ranging from picoseconds to milliseconds, and can be applied for sensing in situ, in vitro, and in vivo. Organic dyes and quenchers, fluorescent or light harvesting proteins, or quantum dots are only some examples from the ever growing FRET material toolbox. A particular example are gold nanoparticles, whose strong localized surface plasmon resonance makes them frequently used as nanosurface energy transfer (NSET) acceptors. After a short recapitulation of FRET and NSET theory, we review a wide variety of FRET and NSET materials, provide representative examples of FRET/NSET systems and applications for each material, and critically discuss the benefits and drawbacks of their properties.

The self-assembled systems of surfactants/polymers, which are capable of supporting energy funneling between fluorophores, have recently gained significant attraction. Surfactant and polymeric micelles form nanoscale structures spanning a radius of 2-10 nm are generally suitable for the transduction of energy among fluorophores. These systems have shown great potential in Förster resonance energy transfer (FRET) due to their unique characteristics of being aqueous based, tendency to remain self-assembled, spontaneous formation, tunable nature, and responsiveness to different external stimuli. This review presents current developments in the field of energy transfer, particularly the multi-step FRET processes in the self-assembled nanostructures of surfactants/polymers. The part one of this review presents a background and brief overview of soft systems and discusses certain aspects of the self-assemblies of surfactants/polymers and their co-solubilization property to bring fluorophores to close proximity to transduce energy. The second part of this review deals with single-step and multi-step FRET in the self-assemblies of surfactants/polymers and links FRET systems with advanced smart technologies including multicolor formation, data encryption, and artificial antenna systems. This review also discusses the diverse examples in the literature to present the emerging applications of FRET. Finally, the prospects regarding further improvement of FRET in self-assembled soft systems are outlined.