Polarized fluorescence resonance energy transfer microscopy

Current methods for fluorescence resonance energy transfer (FRET) microscopy of living cells involve taking a series of images with alternating excitation colors in separate camera exposures. Here we present a new FRET method based on polarization that requires only one camera exposure and thereby offers the possibility for better time resolution of dynamic associations among subcellular components. Polarized FRET (p-FRET) uses a simultaneous combination of excitation wavelengths from two orthogonally polarized sources, along with an emission channel tri-image splitter outfitted with appropriate polarizers, to concurrently excite and collect fluorescence from free donors, free acceptors, and FRET pairs. Based upon the throughput in each emission channel as premeasured on pure samples of each of the three species, decoupling of an unknown sample's three polarized fluorescence images can be performed to calculate the pixel-by-pixel concentrations of donor, acceptor, and FRET pairs. The theory of this approach is presented here, and its feasibility is experimentally confirmed by measurements on mixtures of cyan fluorescent protein (CFP), citrine ((Cit) a yellow fluorescent protein variant), and linked fusion proteins (CFP-L16-Cit, CFP-L7-Cit, CFP-L54-Cit) in living cells. The effects of shot noise, acceptor polarization, and FRET efficiency on the statistical accuracy of p-FRET experimental results are investigated by a noise-simulation program.     

Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy.

Fluorescence resonance energy transfer (FRET) is a technique used for quantifying the distance between two molecules conjugated to different fluorophores. By combining optical microscopy with FRET it is possible to obtain quantitative temporal and spatial information about the binding and interaction of proteins, lipids, enzymes, DNA, and RNA in vivo. In conjunction with the recent development of a variety of mutant green fluorescent proteins (mtGFPs), FRET microscopy provides the potential to measure the interaction of intracellular molecular species in intact living cells where the donor and acceptor fluorophores are actually part of the molecules themselves. However, steady-state FRET microscopy measurements can suffer from several sources of distortion, which need to be corrected. These include direct excitation of the acceptor at the donor excitation wavelengths and the dependence of FRET on the concentration of acceptor. We present a simple method for the analysis of FRET data obtained with standard filter sets in a fluorescence microscope. This method is corrected for cross talk (any detection of donor fluorescence with the acceptor emission filter and any detection of acceptor fluorescence with the donor emission filter), and for the dependence of FRET on the concentrations of the donor and acceptor. Measurements of the interaction of the proteins Bcl-2 and Beclin (a recently identified Bcl-2 interacting protein located on chromosome 17q21), are shown to document the accuracy of this approach for correction of donor and acceptor concentrations, and cross talk between the different filter units.     

Imaging protein-protein interactions using fluorescence resonance energy transfer microscopy

Fluorescence resonance energy transfer (FRET) detects the proximity of fluorescently labeled molecules over distances >100 A. When performed in a fluorescence microscope, FRET can be used to map protein-protein interactions in vivo. We here describe a FRET microscopy method that can be used to determine whether proteins that are colocalized at the level of light microscopy interact with one another. This method can be implemented using digital microscopy systems such as a confocal microscope or a wide-field fluorescence microscope coupled to a charge-coupled device (CCD) camera. It is readily applied to samples prepared with standard immunofluorescence techniques using antibodies labeled with fluorescent dyes that act as a donor and acceptor pair for FRET. Energy transfer efficiencies are quantified based on the release of quenching of donor fluorescence due to FRET, measured by comparing the intensity of donor fluorescence before and after complete photobleaching of the acceptor. As described, this method uses Cy3 and Cy5 as the donor and acceptor fluorophores, but can be adapted for other FRET pairs including cyan fluorescent protein and yellow fluorescent protein.     

Characterization of one- and two-photon excitation fluorescence resonance energy transfer microscopy

Advances in molecular biology provide various methods to define the structure and function of the individual proteins that form the component parts of subcellular structures. The ability to see the dynamic behavior of a specific protein inside the living cell became possible through the application of advanced fluorescence resonance energy transfer (FRET) microscope techniques. The fluorophore molecule used for FRET imaging has a characteristic absorption and emission spectrum that should be considered for characterizing the FRET signal. In this article we describe the system development for the image acquisition for one- and two-photon excitation FRET microscopy. We also describe the precision FRET (PFRET) data analysis algorithm that we developed to remove spectral bleed-through and variation in the fluorophore expression level (or concentration) for the donor and acceptor molecules. The acquired images have been processed using a PFRET algorithm to calculate the energy transfer efficiency and the distance between donor and acceptor molecules. We implemented the software correction to study the organization of the apical endosome in epithelial polarized MDCK cells and dimerization of the CAATT/enhancer binding protein alpha (C/EBPalpha). For these proteins, the results revealed that the extent of correction affects the conventionally calculated energy transfer efficiency (E) and the distance (r) between donor and acceptor molecules by 38 and 9%, respectively.     

Imaging cellulose fibre interfaces with fluorescence microscopy and resonance energy transfer

Future developments in cellulosic materials are predicated by the need to understand the fundamental interactions that occur at cellulose fibre interfaces. These interfaces strongly influence the material properties of fibre networks and fibre reinforced composites. This study takes advantage of fluorescence resonance energy transfer (FRET) and fluorescence microscopy to image cellulose interfaces. Steady-state epi-fluorescence microscopy suggests that energy transfer from coumarin dyed fibres to fluorescein dyed fibres is occurring at the fibre–fibre interface. The FRET response for natural spruce fibre interfaces is distinctly different from that observed in synthetic viscose fibres. This approach constitutes a novel methodology for the characterization of soft material interfaces on the molecular scale, and represents a major opportunity for advancing the understanding of fibrous network structures.     

Single-molecule fluorescence resonance energy transfer

Fluorescent resonance energy transfer (FRET) is a powerful technique for studying conformational distribution and dynamics of biological molecules. Some conformational changes are difficult to synchronize or too rare to detect using ensemble FRET. FRET, detected at the single-molecule level, opens up new opportunities to probe the detailed kinetics of structural changes without the need for synchronization. Here, we discuss practical considerations for its implementation including experimental apparatus, fluorescent probe selection, surface immobilization, single-molecule FRET analysis schemes, and interpretation.     

Imaging spatiotemporal dynamics of neuronal signaling using fluorescence resonance energy transfer and fluorescence lifetime imaging microscopy

The spatiotemporal localization of neuronal signaling is important for triggering neuronal responses in specific locations at precise times. Fluorescence resonance energy transfer imaging enables measurement of spatiotemporal dynamics of signaling activity in live neurons. Although the usefulness of fluorescence resonance energy transfer is well recognized, there are many difficulties in applying it, particularly when imaging in neuronal micro-compartments in light-scattering brain tissue. Fluorescence resonance energy transfer has been imaged using several techniques including intensity-based methods, fluorescence lifetime imaging and fluorescence anisotropy imaging. These methods have different advantages and disadvantages, and thus are suitable in different applications.     

Oligomerization of dopamine transporters visualized in living cells by fluorescence resonance energy transfer microscopy

To examine the oligomeric state and trafficking of the dopamine transporter (DAT) in different compartments of living cells, human DAT was fused to yellow (YFP) or cyan fluorescent protein (CFP). YFP-DAT and CFP-DAT were transiently and stably expressed in porcine aortic endothelial (PAE) cells, human embryonic kidney (HEK) 293 cells, and an immortalized dopaminergic cell line 1RB3AN27. Fluorescence microscopic imaging of cells co-expressing YFP-DAT and CFP-DAT revealed fluorescence resonance energy transfer (FRET) between CFP and YFP, which is consistent with an intermolecular interaction of DAT fusion proteins. FRET signals were detected between CFP- and YFP-DAT located at the plasma membrane and in intracellular membrane compartments. Phorbol esters or amphetamine induced the endocytosis of YFP/CFP-DAT to early and recycling endosomes, identified by Rab5, Rab11, Hrs and EEA.1 proteins. Interestingly, however, DAT was mainly excluded from Rab5- and Hrs-containing microdomains within the endosomes. The strongest FRET signals were measured in endosomes, indicative of efficient oligomerization of internalized DAT. The intermolecular DAT interactions were confirmed by co-immunoprecipitation. A DAT mutant that was retained in the endoplasmic reticulum (ER) after biosynthesis was used to show that DAT is oligomeric in the ER. Moreover, co-expression of an ER-retained DAT mutant and wild-type DAT resulted in the retention of wild-type DAT in the ER. These data suggest that DAT oligomers are formed in the ER and then are constitutively maintained both at the cell surface and during trafficking between the plasma membrane and endosomes.     

单分子荧光共振能量转移技术

单分子荧光共振能量转移技术（single molecule fluorescence resonance energy transfer,smFRET）通过检测单个分子内的荧光供体及受体间荧光能量转移的效率,来研究分子构象的变化。在单分子探测技术发展之前,大多数的分子实验是探测分子的综合平均效应（ensemble averages）,这一平均效应掩盖了许多特殊的信息。单分子探测可以对体系中的单个分子进行研究,得到某一分子特性的分布状况,也可研究生物分子的动力学反应。介绍了近来单分子荧光共振能量转移技术的进展。     

The renaissance of fluorescence resonance energy transfer

Recent advances in fluorescence resonance energy transfer have led to qualitative and quantitative improvements in the technique, including increased spatial resolution, distance range, and sensitivity. These advances, due largely to new fluorescent dyes, but also to new optical methods and instrumentation, have opened up new biological applications.     

Calculation on fluorescence resonance energy transfer on surfaces.

A general method for estimating fluorescence resonance energy transfer between distributions of donors and acceptors on surfaces is presented. Continued fraction approximants are obtained from equivalent power series expansions of the change in quantum yield in terms of the fluorescent lifetimes or the steady-state fluorescence. These approximants provide analytic equations for the analysis of energy transfer and error bounds for the approximants. Specific approximants are derived for five models of interest for membrane biochemistry: (a) an infinite plane, (b) parallel infinite planes, (c) the surface of a sphere, (d) the surfaces of concentric spheres, and (e) the surfaces of two separated spheres. Recent experimental results in the literature are analyzed with the equations obtained.     

Reliable and global measurement of fluorescence resonance energy transfer using fluorescence microscopes

Green fluorescence protein (GFP)-based fluorescence resonance energy transfer (FRET) is increasingly used in investigation of inter- and intramolecular interactions in living cells. In this report, we present a modified method for FRET quantification in cultured cells using conventional fluorescence microscopy. To reliably measure FRET, three positive control constructs in which a cyan fluorescence protein and a yellow fluorescence protein were linked by peptides of 15, 24, or 37 amino acid residues were prepared. FRET was detected using a spectrofluorometer, a laser scanning confocal microscope, and an inverted fluorescence microscope. Three calculation methods for FRET quantification using fluorescence microscopes were compared. By normalization against expression levels of GFP fusion proteins, the modified method gave consistent FRET values that could be compared among different cells with varying protein expression levels. Whole-cell global analysis using this method allowed FRET measurement with high spatial resolutions. Using such a procedure, the interaction of synaptic proteins syntaxin and the synaptosomal associated protein of 25 kDa (SNAP-25) was examined in PC12 cells, which showed strong FRET on plasma membranes. These results demonstrate the effectiveness of the modified method for FRET measurement in live cell systems.     

Application of spectral imaging microscopy in cytomics and fluorescence resonance energy transfer (FRET) analysis.

BACKGROUND: Specific signal detection has been a fundamental issue in fluorescence microscopy. In the context of tissue samples, this problem has been even more pronounced, with respect to spectral overlap and autofluorescence. METHODS: Recent improvements in confocal laser scanning microscopy combine sophisticated hardware to obtain fluorescence emission spectra on a single-pixel basis and a mathematical procedure called "linear unmixing" of fluorescence signals. By improving both the specificity of fluorescence acquisition and the number of simultaneously detectable fluorochromes, this technique of spectral imaging (SI) allows complex interrelations in cells and tissues to be addressed. RESULTS: In a comparative approach, SI microscopy on a quantitative basis was compared to conventional bandpass (BP) filter detection, demonstrating substantial superiority of SI with respect to detection accuracy and dye combination. An eight-color immunofluorescence protocol for tissue sections was successfully established. Moreover, advanced use of SI in fluorescence resonance energy transfer (FRET) applications using enhanced green fluorescence protein (EGFP) and enhanced yellow fluorescence protein (EYFP) in a confocal set up could be demonstrated. CONCLUSIONS: This novel technology will help to perform complex multiparameter investigations at the cellular level by increasing the detection specificity and permitting simultaneous use of more fluorochromes than with classical techniques based on emission filters. Moreover, SI significantly extends the possibilities for specialized microscopy applications, such as the visualization of macromolecular interactions or conformational changes, by detecting FRET.     

Homomultimerization of the coxsackievirus 2B protein in living cells visualized by fluorescence resonance energy transfer microscopy

The 2B protein of enteroviruses is the viral membrane-active protein that is responsible for the modifications in host cell membrane permeability that take place in enterovirus-infected cells. The 2B protein shows structural similarities to the group of lytic polypeptides, polypeptides that permeate membranes either by forming multimeric membrane-integral pores or, alternatively, by lying parallel to the lipid bilayer and disturbing the curvature and symmetry of the membrane. Our aim is to gain more insight into the molecular architecture of the 2B protein in vivo. In this study, the possible existence of multimers of the coxsackie B3 virus 2B protein in single living cells was explored by fluorescence resonance energy transfer (FRET) microscopy. FRET between fusion proteins 2B-ECFP and 2B-EYFP (enhanced cyan and yellow fluorescent variants of green fluorescent protein) was monitored by using spectral imaging microscopy (SPIM) and fluorescence lifetime imaging microscopy (FLIM). Both techniques revealed the occurrence of intermolecular FRET between 2B-ECFP and 2B-EYFP, providing evidence for the formation of protein 2B homomultimers. Putative models for the mode of action of the membrane-active 2B protein and the formation of membrane-integral pores by 2B multimers are discussed.     

DNA tetraplex formation studied with fluorescence resonance energy transfer.

It is emerging that DNA tetraplexes are pivotal for many major cellular processes, and techniques that assess their structure and nature to the point are under development. Here we show how the structural conversion of largely unstructured single-stranded DNA molecules into compact intrastrand DNA tetraplexes can be monitored by fluorescence resonance energy transfer. We recently reported that intrastrand tetraplex formation takes place in a nuclease hypersensitive element upstream of the human c-myc proto-oncogene. Despite the highly repetitive guanine-rich sequence of the hypersensitive element, fluorescence resonance energy transfer measurements indicate that only one well defined tetraplex structure forms therein. The proposed structure, which is specifically stabilized by potassium ions in vitro, has a core of three stacked guanine tetrads that is capped by two intrastrand A-T base pairs.     

Use of fluorescence resonance energy transfer to study serpin-proteinase interactions

One of the important questions in the serpin mechanism of inhibition of serine and cysteine proteinases of different specificities and structural classes is whether a common "crushing" mechanism of proteinase inactivation is used in all cases. This mechanism was seen in an X-ray structure of the complex between alpha(1)-proteinase inhibitor and trypsin and required the full insertion of the reactive center loop into beta-sheet A and translocation of the proteinase from one pole of the serpin to the other. However, it has yet to be shown to be general for serine proteinases of structural classes other than the trypsin-fold or for cysteine proteinases with the papain-fold or for the caspases. Fluorescence resonance energy transfer offers a potential means of obtaining an answer to this question for each of these classes, without the concern for the effect that increasing size has on the observed signal that applies to NMR spectroscopy. However, care must be taken to ensure that measurements made represent sufficient overdetermination that the answer obtained is unambiguous.