Comparative efficiency of energy transfer from CdSe-ZnS quantum dots or nanorods to organic dye molecules

Fluorescence resonance energy transfer (FRET) in conjugates of CdSe-ZnS semiconductor nanocrystals of different shapes (FRET donors) and an Alexa Fluor organic dye (FRET acceptors) is examined. The dye molecules are chemically conjugated with quantum dots (QDs) or nanorods (NRs) in dimethyl sulfoxide colloidal solutions, and FRET efficiency in the purified conjugates is measured. The FRET from NR to a single dye molecule is less efficient than that of the QD-dye conjugates and this effect is explained in terms of distance-limited energy-transfer rate in the case of a point-like acceptor and extended donor dipoles. However, the larger surface area of NRs allows for many more dye acceptors to be bound, and the total FRET efficiency in NR-dye conjugates approaches those of QD-dye conjugates.     

Ultrafast dynamics in a nanocage of enzymes: solvation and fluorescence resonance energy transfer in reverse micelles

In this contribution we report studies of the nature of solvation and resonance energy transfer processes in a reverse micelle (RM) upon encapsulation of a digestive enzyme, alpha-chymotrypsin (CHT). We have used one donor, Coumarin 500 (C500), and three acceptors Rhodamine 123 (R123, cationic), ethidium bromide (EtBr, cationic), and Merocyanine 540 (MC540, anionic). By selectively exciting the donor at the surface of the RM with a proper excitation wavelength we have examined solvation dynamics in the microenvironment. The solvation correlation function in the RM without CHT exhibits single-exponential decay with time constant approximately 660 ps, which is similar to that of the CHT-included RM. However, in the case of CHT-included RM (w(0)=10), the time-resolved anisotropy and spectral linewidth analysis of the surface-bound donor reveal the existence of an annular aqueous channel of thickness approximately 2.5 A between the enzyme surface and the inner surface of the RM. The aqueous channel is a potential host for the water-soluble substrate and also is involved in maintaining the proper functionality of RM encapsulated CHT. The studies use both steady-state and time-resolved fluorescence resonance energy transfer (FRET) techniques to measure donor-acceptor distances in the RM and also emphasize the danger of using steady-state fluorescence quenching as a method in careful estimation of the distances. The local geometrical restriction on the donor and acceptor molecules was estimated from time-resolved polarization (anisotropy) measurements. The time-resolved anisotropy of the donor and acceptor molecules also revealed significant randomization of the relative orientation of transition dipoles of the donor and acceptor, justifying the use of 2/3 as the value of the orientation factor kappa2. These studies attempt to elucidate the excellence of the RM as a nanohost of biological macromolecules.     

A photochromic acceptor as a reversible light-driven switch in fluorescence resonance energy transfer (FRET)

A method has been developed for the quantitative determination of fluorescence resonance energy transfer (FRET) based on the modulation of donor fluorescence upon the reversible photoconversion of a photochromic acceptor. A model system was devised, consisting of Lucifer Yellow cadaverine (LYC, donor) conjugated to the photochromic molecule, 6-nitroBIPS (1′,3′-dihydro-1′-(2-carboxyethyl)-3′,3′-dimethyl-6-nitrospiro[2H-1-benzopyran-2,2′-(2H)-indoline]). Near-ultraviolet irradiation catalyzes the conversion of the colorless spiropyran (SP) to the colored merocyanine (MC) form of 6-nitroBIPS. Only the MC form absorbs at the emission wavelengths of the donor, thereby potentiating FRET, as demonstrated by quenching of the donor. Subsequent irradiation in the visible MC absorption band reverts 6-nitroBIPS to the SP form and FRET is inactivated. The acceptor exhibited high photostability under repeated cycles of alternating UV–Vis irradiation. In this model system, the intramolecular FRET efficiency was close to 100%. The observed maximal donor quenching of 34±3% was indicative of an equilibrium determined by the high quantum efficiency of forward conversion (SP→MC) induced by near-UV irradiation and a low but finite quantum efficiency of the back reaction resulting from excitation of the MC form directly as well as indirectly (by FRET via the donor). A quantitative formalism for the photokinetic scheme was developed. Photochromic FRET (pcFRET) permits repeated, quantitative, and non-destructive FRET determinations for arbitrary relative concentrations of donor and acceptor and thus offers great potential for monitoring dynamic molecular interactions in living cells over extended observation times by fluorescence microscopy.     

Simultaneous binding of minor groove binder and intercalator to dodecamer DNA: importance of relative orientation of donor and acceptor in FRET

In the present study, steady-state, picosecond time-resolved fluorescence and polarization gated anisotropy have been used to establish simultaneous binding of an intercalator (ethidium bromide, EtBr) and a minor groove binder (Hoeschst 33258, H258) to a dodecamer DNA of specific sequence. The F?rster resonance energy transfer (FRET) studies between the dyes H258 (donor) and EtBr (acceptor) in the dodecamer, where the ligands have a particular relative orientation of the transition dipoles, in contrast to the cases in sodium dodecyl sulfate (SDS) micelles and larger genomic DNA, where the orientations are random, reveal the effect of the binding geometry of the ligands in the constrained environment. Our study establishes that reconsideration of the value of the orientation factor (kappa2) is crucial for correct estimation of the donor-acceptor distance when the ligands are simultaneously bound to a specific region of biological macromolecules.     

Ultrafast fluorescence resonance energy transfer in a reverse micelle: excitation wavelength dependence

Fluorescence resonance energy transfer (FRET) from coumarin 480 (C480) to fluorescein 548 (F548) in a sodium dioctyl sulfosuccinate (AOT) reverse micelle is studied by picosecond and femtosecond emission spectroscopy. In bulk water, at the low concentration of the donor (C480) and the acceptor (F548), no FRET is observed. However, when the donor (C480) and the acceptor (F548) are confined in a AOT reverse micelle very fast FRET is observed. The time constants of FRET were obtained from the rise time of the emission of the acceptor (F548). In a AOT microemulsion, FRET is found to occur in multiple time scales--3, 200, and 2700 ps. The 3 ps component is assigned to FRET in the water pool of the reverse micelle with a donor-acceptor distance, 16 A. The 200 ps component corresponds to a donor-acceptor distance of 30 A and is ascribed to the negatively charged acceptor inside the water pool and the neutral donor inside the alkyl chains of AOT. The very long 2700 ps component may arise due to FRET from a donor outside the micelle to an acceptor inside the water pool and also from diffusion of the donor from bulk heptane to the reverse micelle. With increase in the excitation wavelength from 375 to 405 nm the relative contribution of the FRET due to C480 in the AOT reverse micelle (the 3 and 200 ps components) increases.     

General and reliable quantitative measurement of fluorescence resonance energy transfer using three fluorescence channels

In this paper, we describe a comprehensive general system adapted for quantitative fluorescence resonance energy transfer (FRET) measurement using signals from three channels of a fluorescence instrument. The general FRET measurement system involves two established methods, as well as two novel approaches. Unlike the previous measurements, which can be taken correctly only when the quantity of the acceptor is greater than or equal to that of the donor, one of our novel methods can overcome this obstacle and take quantitative FRET measurements when the donor is in excess of the acceptor. Hence the general FRET measurement system allowed one to determine the exact distance when the donor and acceptor were present in different quantities, and integrated the methods for quantitative FRET measurements. The uniformity of measured values and utility of each method were validated using molecular standards based on DNA oligonucleotide rulers. We also discussed and validated the use of a novel method for estimating the relative quantities of the donor and acceptor fluorophores when they were not known before an appropriate method of this system can be selected.     

Diheteroarylethenes as thermally stable photoswitchable acceptors in photochromic fluorescence resonance energy transfer (pcFRET)

We have employed diheteroarylethenes as acceptors for photochromic FRET (pcFRET), a technique introduced for the quantitative determination of fluorescence resonance energy transfer (FRET). In pcFRET, the fluorescent emission of the donor is modulated by cyclical transformations of a photochromic acceptor. Light induces a reversible change in the structure and, concomitantly, in the absorption properties of the acceptor. Only the closed forms of the selected diheteroarylethenes 2a and 2b have an absorption band overlapping the emission band of the donor, 1. The corresponding variation in the overlap integral (and thus critical transfer distance R(o)) between the two states provides the means for reversibly switching the process of FRET on and off, allowing direct and repeated evaluation of the relative changes in the donor fluorescence quantum yield. The diheteroarylethenes demonstrate excellent stability in aqueous media, an absence of thermal back reactions, and negligible fatigue. The equilibration of these systems after exposure to near-UV or visible light follows simple monoexponential kinetics. We developed a general conceptual scheme for such coupled photochromic-FRET reactions, allowing quantitative interpretations of the photostationary and kinetic data, from which the quantum yields for the cyclization and cycloreversion reactions of the photochromic acceptor were calculated.     

Ultrafast fluorescence resonance energy transfer in a micelle

Ultrafast fluorescence resonance energy transfer (FRET) from coumarin 153 (C153) to rhodamine 6G (R6G) is studied in a neutral PEO(20)-PPO(70)-PEO(20) triblock copolymer (P123) micelle and an anionic micelle (sodium dodecyl sulfate, SDS) using a femtosecond up-conversion setup. Time constants of FRET were determined from the rise time of the acceptor emission. It is shown that a micelle increases efficiency of FRET by holding the donor and the acceptor at a close distance (intramicellar FRET) and also by tuning the donor and acceptor energies. It is demonstrated that in the P123 micelle, intramicellar FRET (i.e., donor and acceptor in same micelle) occurs in 1.2 and 24 ps. In SDS micelle, there are two ultrafast components (0.7 and 13 ps) corresponding to intramicellar FRET. The role of diffusion is found to be minor in the ultrafast components of FRET. We also detected a much longer component (1000 ps) for intramicellar FRET in the larger P123 micelle.     

Self-assembled pi-nanotapes as donor scaffolds for selective and thermally gated fluorescence resonance energy transfer (FRET)

Self-assembled nanotapes of a few tailor-made oligo(p-phenylenevinylene)s (OPVs) have been prepared and used as supramolecular donor scaffold to study the fluorescence resonance energy transfer (FRET) to a suitable acceptor. In nonpolar solvents, FRET occurs with nearly 63-81% efficiency, exclusively from the self-assembled OPVs to entrapped Rhodamine B, resulting in the quenching of the donor emission with concomitant formation of the acceptor emission at 625 nm. The efficiency of FRET is considerably influenced by the ability of the OPVs to form the self-assembled aggregates and hence could be controlled by structural variation of the molecules, and polarity of the solvent. Most importantly, FRET could be controlled by temperature as a result of the thermally reversible self-assembly process. The FRET efficiency was significantly enhanced (ca. 90%) in a xerogel film of the OPV1 which is dispersed with relatively less amount of the acceptor (33 mol %), when compared to that of the aggregates in dodecane gel. FRET is not efficient in polar solvents due to weak self-organization of the chromophores. These results indicate that energy transfer occurs exclusively from the self-assembled donor and not directly from the individual donor molecules. The present study illustrates that the self-assembly of chromophores facilitates temperature and solvent controlled FRET within pi-conjugated nanostructures.     

Dipole Effects on Electron Transfer are Enormous

Molecular dipoles present important, but underutilized, methods for guiding electron transfer (ET) processes. While dipoles generate fields of Gigavolts per meter in their vicinity, reported differences between rates of ET along versus against dipoles are often small or undetectable. Herein we show unprecedentedly large dipole effects on ET. Depending on their orientation, dipoles either ensure picosecond ET, or turn ET completely off. Furthermore, favorable dipole orientation makes ET possible even in lipophilic medium, which appears counterintuitive for non‐charged donor–acceptor systems. Our analysis reveals that dipoles can substantially alter the ET driving force for low solvent polarity, which accounts for these unique trends. This discovery opens doors for guiding forward ET processes while suppressing undesired backward electron transduction, which is one of the holy grails of photophysics and energy science.     

Quantitative FRET analysis with the EGFP-mCherry fluorescent protein pair

Fluorescence resonance energy transfer (FRET) between fluorescent proteins (FPs) is a powerful tool to investigate protein–protein interaction and even protein modifications in living cells. Here, we analyze the E⁰GFP-mCherry pair and show that it can yield a reproducible quantitative determination of the energy transfer efficiency both in vivo and in vitro . The photophysics of the two proteins is reported and shows good spectral overlap (Förster radius R ₀ = 51 Å), low crosstalk between acceptor and donor channels, and independence of the emission spectra from pH and halide ion concentration. Acceptor photobleaching (APB) and one- and two-photon fluorescence lifetime imaging microscopy (FLIM) are used to quantitatively determine FRET efficiency values. A FRET standard is introduced based on a tandem construct comprising donor and acceptor together with a 20 amino acid long cleavable peptidic linker. Reference values are obtained via enzymatic cleavage of the linker and are used as benchmarks for APB and FLIM data. E⁰GFP-mCherry shows ideal properties for FLIM detection of FRET and yields high accuracy both in vitro and in vivo . Furthermore, the recently introduced phasor approach to FLIM is shown to yield straightforward and accurate two-photon FRET efficiency data even in suboptimal experimental conditions. The consistence of these results with the reference method (both in vitro and in vivo ) reveals that this new pair can be used for very effective quantitative FRET imaging.     

CdS量子点与曙红Y间的荧光共振能量转移研究

在水溶液中,量子点与有机荧光染料之间可能发生荧光共振能量转移(FRET)。本文以发射波长470nm的Cd S量子点为供体,曙红Y为受体,建立了Cd S量子点-曙红Y的FRET体系,研究了该体系的FRET参数。该体系受体供体数目比为8,猝灭效率为45.6%,增强效率为20.1%;供体-受体间的距离为4.4nm;临界能量转移距离为2.4nm。     

Fluorescence energy transfer-sensitized photobleaching of a fluorescent label as a tool to study donor-acceptor distance distributions and dynamics in protein assemblies: studies of a complex of biotinylated IgM with streptavidin and aggregates of concanavalin A

A photokinetic method of detection of fluorescence resonance energy transfer (FRET) between special fluorescent labels is applied to study time-averaged spatial distribution of labeled proteins in protein assemblies. Prolonged irradiation of a sample at the absorption maximum of the energy donor initiates FRET-sensitized fluorescence photobleaching of the energy acceptor label, which was monitored by steady-state fluorimetric measurements. Kinetics of the acceptor photobleaching and kinetics of decreasing the efficiency of FRET from donors to unbleached acceptors were determined. The FRET efficiency was found from measuring sensitization of acceptor fluorescence. Analysis of the photokinetic data permits to estimate the time-averaged distribution of acceptors on donor-acceptor distances in the range of characteristic distances of FRET. Dynamic processes influencing donor-acceptor distances can be also investigated by the method. Application of the method is demonstrated by the studies of a complex of biotinylated IgM with streptavidin and aggregates composed of concanavalin A and sodium dodecyl sulphate. A new thiadicarbocyanine dye was used as the acceptor label. R-phycoerythrin and tetramethylrhodamine isothiocyanate were the donor labels. In the IgM-streptavidin complex, 16% of acceptors most contributed to FRET provided 90% of FRET efficiency, whereas acceptors made about the same time-averaged contribution to FRET in the concanavalin A aggregates.     

Origin of simultaneous donor-acceptor emission in single molecules of peryleneimide-terrylenediimide labeled polyphenylene dendrimers

F?rster type resonance energy transfer (FRET) in donor-acceptor peryleneimide-terrylenediimide dendrimers has been examined at the single molecule level. Very efficient energy transfer between the donor and the acceptor prevent the detection of donor emission before photobleaching of the acceptor. Indeed, in solution, on exciting the donor, only acceptor emission is detected. However, at the single molecule level, an important fraction of the investigated individual molecules (about 10-15%) show simultaneous emission from both donor and acceptor chromophores. The effect becomes apparent mostly after photobleaching of the majority of donors. Single molecule photon flux correlation measurements in combination with computer simulations and a variety of excitation conditions were used to determine the contribution of an exciton blockade to this two-color emission. Two-color defocused wide-field imaging showed that the two-color emission goes hand in hand with an unfavorable orientation between one of the donors and the acceptor chromophore.     

A novel FRET approach for in situ investigation of cellulase–cellulose interaction

A novel real-time in situ detection method for the investigation of cellulase–cellulose interactions based on fluorescence resonance energy transfer (FRET) has been developed. FRET has been widely used in biological and biophysical fields for studies related to proteins, nucleic acids, and small biological molecules. Here, we report the efficient labeling of carboxymethyl cellulose (CMC) with donor dye 5-(aminomethyl)fluorescein and its use as a donor in a FRET assay together with an Alexa Fluor 594 (AF594, acceptor)–cellulase conjugate as acceptor. This methodology was successfully employed to investigate the temperature dependency of cellulase binding to cellulose at a molecular level by monitoring the fluorescence emission change of donor (or acceptor) in a homogeneous liquid environment. It also provides a sound base for ongoing cellulase–cellulose study using cellulosic fiber.     

Joint statistical analysis of multichannel time series from single quantum dot-(Cy5)n constructs

One of the major challenges in single-molecule studies is how to extract reliable information from the inevitably noisy data. Here, we demonstrate the unique capabilities of multichannel joint statistical analysis of multispectral time series using F?ster resonance energy transfer (FRET) in single quantum dot (QD)-organic dye hybrids as a model system. The multispectral photon-by-photon registration allows model-free determination of intensity change points of the donor and acceptor channels independently. The subsequent joint analysis of these change points gives high-confidence assignments of acceptor photobleaching events despite the interference from background noise and from intermittent blinking of the QD donors and acceptors themselves. Finally, the excited-state lifetimes of donors and acceptors are calculated using the joint maximum likelihood estimation (MLE) method on the donor and acceptor decay profiles, guided by a four-state kinetics model.     

Single lanthanide-doped oxide nanoparticles as donors in fluorescence resonance energy transfer experiments

We used lanthanide-ion doped oxide nanoparticles, Y(0.6)Eu(0.4)VO(4), as donors in fluorescent resonance energy transfer (FRET) experiments. The choice of these nanoparticles allows us to combine the advantages of the lanthanide-ion emission, in particular the long lifetime and the large Stokes shift between absorption and emission, with the detectability of the nanoparticles at the single-particle level. Using cyanine 5 (Cy5) organic molecules as acceptors, we demonstrated FRET down to the single-nanoparticle level. We showed that, due to the long donor lifetime, unambiguous and precise FRET measurements can be performed in solution even in the presence of large free acceptor concentrations. Highly efficient energy transfer was obtained for a large number of acceptor molecules per donor nanoparticle. We determined FRET efficiencies as a function of Cy5 concentration which are in good agreement with a multiple acceptor-multiple donor calculation. On the basis of the donor emission recovery due to acceptor photobleaching, we demonstrated energy transfer from single-nanoparticle donors in fluorescence microscopy experiments.     

Two-step FRET as a structural tool

The power of FRET to study molecular complexes is expanded by the use of two or more donor/acceptor pairs. A general theoretical framework for distance measurements in three-chromophore systems is presented. Three energy transfer schemes applicable to many diverse situations are considered: (I) two-step FRET relay with FRET between the first and second chromophores and between the second and third, (II) FRET from a single donor to two different acceptors, and (III) two-step FRET relay with FRET also between the first and third chromophores. Equations for the efficiencies involving multiple energy transfer steps are derived for both donor quenching and sensitized emission measurements. The theory is supported by experimental data on model systems of known structure using steady-state donor quenching, lifetime quenching, and sensitized emission. The distances measured in the three-chromophore systems agree with those in two-chromophore systems and molecular models. Finally, labeling requirements for diagnosis of the energy transfer scheme and subsequent distance measurements are discussed.     

基于钙黄绿素－臧红T荧光共振能量转移检测六偏磷酸钠

在pH8．5的Tris－HCl缓冲溶液中,钙黄绿素作为能量供体（D）可以与藏红T受体（A）发生有效的荧光共振能量转移（FRET）,但加入六偏磷酸钠（SHMP）后,因其与受体发生静电作用破坏了该能量转移体系,使得荧光供体钙黄绿素荧光强度的增加（△FD）与受体藏红T荧光强度的降低（△FA）的比值（△FD／△R）-9SHMP浓度（csHMP）呈良好的线性关系．基于此,建立了一种检测六偏磷酸盐的新方法．在优化条件下,该方法的检测范围为3．0×10^-6-1．0×10^-5mol／L,对6．0×10拍mol／L的六偏磷酸盐连续平行测定11次,其相对标准偏差（RSD）为3．1％．该方法具有选择性好、操作简单和检测速度快等优点,已成功应用于饮料中六偏磷酸钠的分析检测．