Fluorescence resonance energy transfer in ferritin labeled with multiple fluorescent dyes

We simultaneously labeled ferritin with two Alexa Fluor fluorophores (AF350 and AF430). When both fluorophores label the same ferritin subunit, fluorescence resonance energy transfer (FRET) takes place from the excited AF350 to the acceptor AF430. By varying the number and the ratio of labeling fluorophores, we can modulate FRET such that the ferritin particles can exhibit multiple colors under UV illumination. Labeling of the ferritin shell does not affect the properties of the metallic core. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Probing complexes with single fluorophores: factors contributing to dispersion of FRET in DNA/RNA duplexes

Single molecule fluorescent microscopy is a method for the analysis of the dynamics of biological macromolecules by detecting the fluorescence signal produced by fluorophores associated with the macromolecule. Two fluorophores located in a close proximity may result in Förster resonance energy transfer (FRET), which can be detected at the single molecule level and the efficiency of energy transfer calculated. In most cases, the experimentally observed distribution of FRET efficiency exhibits a significant width corresponding to 0.07–0.2 (on a scale of 0–1). Here, we present a general approach describing the analysis of experimental data for a DNA/RNA duplex. We have found that for a 15 bp duplex with Cy3 and Cy5 fluorophores attached to the opposite ends of the helix, the width of the energy transfer distribution is mainly determined by the photon shot noise and the orientation factor, whereas the variation of inter-dye distances plays a minor role.     

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.     

High-contrast imaging of fluorescent protein FRET by fluorescence polarization microscopy

Detection of Forster resonance energy transfer (FRET) between fluorescent protein labeled targets is a valuable strategy for measurement of protein-protein interactions and other intracellular processes. Despite the utility of FRET, widespread application of this technique to biological problems and high-throughput screening has been limited by low-contrast measurement strategies that rely on the detection of sensitized emission or photodestruction of the sample. Here we report a FRET detection strategy based on detecting depolarized sensitized emission. In the absence of FRET, we show that fluorescence emission from a donor fluorescent protein is highly polarized. Depolarization of fluorescence emission is observed only in the presence of energy transfer. A simple detection strategy was adapted for fluorescence microscopy using both laser scanning and wide-field approaches. This approach is able to distinguish FRET between linked and unlinked Cerulean and Venus fluorescent proteins in living cells with a larger dynamic range than other approaches.     

Fluorescence-based sensing of glucose using engineered glucose/galactose-binding protein: a comparison of fluorescence resonance energy transfer and environmentally sensitive dye labelling strategies

Fluorescence-based glucose sensors using glucose-binding protein (GBP) as the receptor have employed fluorescence resonance energy transfer (FRET) and environmentally sensitive dyes, but with widely varying sensitivity. We therefore compared signal changes in (a) a FRET system constructed by transglutaminase-mediated N-terminal attachment of Alexa Fluor 488/555 as donor and QSY 7 as acceptor at Cys 152 or 182 mutations with (b) GBP labelled with the environmentally sensitive dye badan at C152 or 182. Both FRET systems had a small maximal fluorescence change at saturating glucose (7% and 16%), badan attached at C152 was associated with a 300% maximal fluorescence increase with glucose, though with badan at C182 there was no change. We conclude that glucose sensing based on GBP and FRET does not produce a larger enough signal change for clinical use; both the nature of the environmentally sensitive dye and its site of conjugation seem important for maximum signal change; badan-GBP152C has a large glucose-induced fluorescence change, suitable for development as a glucose sensor.     

On the solution structure of the T4 sliding clamp (gp45)

Examination by time-resolved fluorescence spectroscopy of the trimeric bacteriophage T4 clamp protein labeled across its three subunit interfaces with a fluorescence resonance energy transfer (FRET) pair indicates that the clamp exists in just one state in solution, with one open and two closed interfaces. This is in contrast to what is observed in the X-ray crystal structure. The population distribution of the trFRET distance is bimodal, giving 67% as 17 A and 33% as 42 A. This leads to the conclusion that gp45 exists in an asymmetric open state in solution. The further increase in the separation of the FRET pair in the presence of the clamp loader and ATP may be ascribed to either further opening of the open interface or the opening of a closed interface. The ramifications for replisome remodeling by this pathway are discussed.     

Fluorescence as a method to reveal structures and membrane-interactions of amyloidogenic proteins

Amyloidogenesis is a characteristic feature of the 40 or so known protein deposition diseases, and accumulating evidence strongly suggests that self-association of misfolded proteins into either fibrils, protofibrils, or soluble oligomeric species is cytotoxic. The most likely mechanism for toxicity is through perturbation of membrane structure, leading to increased membrane permeability and eventual cell death. There have been a rather limited number of investigations of the interactions of amyloidogenic polypeptides and their aggregated states with membranes; these are briefly reviewed here. Amyloidogenic proteins discussed include A-beta from Alzheimer's disease, the prion protein, α-synuclein from Parkinson's disease, transthyretin (FAP, SSA amyloidosis), immunoglobulin light chains (primary (AL) amyloidosis), serum amyloid A (secondary (AA) amyloidosis), amylin or IAPP (Type 2 diabetes) and apolipoproteins. This review highlights the significant role played by fluorescence techniques in unraveling the nature of amyloid fibrils and their interactions and effects on membranes. Fluorescence spectroscopy is a valuable and versatile method for studying the complex mechanisms of protein aggregation, amyloid fibril formation and the interactions of amyloidogenic proteins with membranes. Commonly used fluorescent techniques include intrinsic and extrinsic fluorophores, fluorescent probes incorporated in the membrane, steady-state and lifetime measurements of fluorescence emission, fluorescence correlation spectroscopy, fluorescence anisotropy and polarization, fluorescence resonance energy transfer (FRET), fluorescence quenching, and fluorescence microscopy.     

Non fitting based FRET–FLIM analysis approaches applied to quantify protein–protein interactions in live cells

New imaging methodologies in quantitative fluorescence microscopy and nanoscopy have been developed in the last few years and are beginning to be extensively applied to biological problems, such as the localization and quantification of protein interactions. Fluorescence resonance energy transfer (FRET) detected by fluorescence lifetime imaging microscopy (FLIM) is currently employed not only in biophysics or chemistry but also in bio-medicine, thanks to new advancements in technology and also new developments in data treatment. FRET–FLIM can be a very useful tool to ascertain protein interactions occurring in single living cells. In this review, we stress the importance of increasing the acquisition speed when working in vivo employing Time-Domain FLIM. The development of the new mathematical-based non-fitting methods allows the determining of the fraction of interacting donor without the requirement of high count statistics, and thus allows the performing of high speed acquisitions in FRET–FLIM to still be quantitative.     

Fluorescent proteins for FRET microscopy: monitoring protein interactions in living cells

The discovery and engineering of novel fluorescent proteins (FPs) from diverse organisms is yielding fluorophores with exceptional characteristics for live-cell imaging. In particular, the development of FPs for fluorescence (or F?rster) resonance energy transfer (FRET) microscopy is providing important tools for monitoring dynamic protein interactions inside living cells. The increased interest in FRET microscopy has driven the development of many different methods to measure FRET. However, the interpretation of FRET measurements is complicated by several factors including the high fluorescence background, the potential for photoconversion artifacts and the relatively low dynamic range afforded by this technique. Here, we describe the advantages and disadvantages of four methods commonly used in FRET microscopy. We then discuss the selection of FPs for the different FRET methods, identifying the most useful FP candidates for FRET microscopy. The recent success in expanding the FP color palette offers the opportunity to explore new FRET pairs.     

Red-edge anisotropy microscopy enables dynamic imaging of homo-FRET between green fluorescent proteins in cells

Steady-state fluorescence anisotropy measurements can be used to detect fluorescence resonance energy transfer (FRET) between identical fluorophores (homo-FRET). However, the contribution of homo-FRET to the steady-state anisotropy must be discerned from those due to the orientational distribution and rotational diffusion, which so far has required photobleaching controls, largely precluding dynamic measurements in live cells. We describe a variation of steady-state anisotropy microscopy in which the contribution of homo-FRET is dynamically isolated from the total anisotropy by exploiting the loss of energy transfer that occurs at red-edge excitation. Excitation of enhanced green fluorescent protein (EGFP) at the red-edge of its absorption band shows the shift in the emission spectrum compared to main-band excitation that is characteristic for photo-selection of static low energy S(0)-S(1) transitions that fail to exhibit FRET. An experimental setup for steady-state fluorescent anisotropy microscopy is described that can be used to acquire anisotropy images in live cells at main-band and red-edge excitation of EGFP. We demonstrate in live cells homo-FRET suppression of protein fusion constructs that consist of two and three EGFP molecules connected by short linkers. This methodology represents a novel approach for the dynamic measurement of homo-FRET in live cells that will be of utility in the biological sciences to detect oligomerization and concentration dependent interactions between identically labeled molecules.     

Spectral imaging and linear un-mixing enables improved FRET efficiency with a novel GFP2-YFP FRET pair

Spectral variants of the green fluorescent protein (GFP) have been extensively used as reporters to image molecular interactions in living cells by fluorescence resonance energy transfer (FRET). However, those GFP variants which are the most efficient donor acceptor pairs for FRET measurements show a high degree of spectral overlap which has hampered in the past their use in FRET applications. Here we use spectral imaging and subsequent un-mixing to quantitatively separate highly overlapping donor and acceptor emissions in FRET measurements. We demonstrate the method in fixed and living cells using a novel GFP based FRET pair (GFP2-YFP (yellow)), which has an increased FRET efficiency compared to the most commonly used FRET pair consisting of cyan fluorescent protein and YFP. Moreover, GFP2 has its excitation maximum at 396 nm at which the YFP acceptor is excited only below the detection level and thus this FRET pair is ideal for applications involving sensitized emission.     

Scanning near-field fluorescence resonance energy transfer microscopy

A new microscopic technique is demonstrated that combines attributes from both near-field scanning optical microscopy (NSOM) and fluorescence resonance energy transfer (FRET). The method relies on attaching the acceptor dye of a FRET pair to the end of a near-field fiber optic probe. Light exiting the NSOM probe, which is nonresonant with the acceptor dye, excites the donor dye introduced into a sample. As the tip approaches the sample containing the donor dye, energy transfer from the excited donor to the tip-bound acceptor produces a red-shifted fluorescence. By monitoring this red-shifted acceptor emission, a dramatic reduction in the sample volume probed by the uncoated NSOM tip is observed. This technique is demonstrated by imaging the fluorescence from a multilayer film created using the Langmuir-Blodgett (LB) technique. The film consists of L-alpha-dipalmitoylphosphatidylcholine (DPPC) monolayers containing the donor dye, fluorescein, separated by a spacer group of three arachidic acid layers. A DPPC monolayer containing the acceptor dye, rhodamine, was also transferred onto an NSOM tip using the LB technique. Using this modified probe, fluorescence images of the multilayer film reveal distinct differences between images collected monitoring either the donor or acceptor emission. The latter results from energy transfer from the sample to the NSOM probe. This method is shown to provide enhanced depth sensitivity in fluorescence measurements, which may be particularly informative in studies on thick specimens such as cells. The technique also provides a mechanism for obtaining high spatial resolution without the need for a metal coating around the NSOM probe and should work equally well with nonwaveguide probes such as atomic force microscopy tips. This may lead to dramatically improved spatial resolution in fluorescence imaging.     

FRET characterisation for cross-bridge dynamics in single-skinned rigor muscle fibres

In this work we demonstrate for the first time the use of Förster resonance energy transfer (FRET) as an assay to monitor the dynamics of cross-bridge conformational changes directly in single muscle fibres. The advantage of FRET imaging is its ability to measure distances in the nanometre range, relevant for structural changes in actomyosin cross-bridges. To reach this goal we have used several FRET couples to investigate different locations in the actomyosin complex. We exchanged the native essential light chain of myosin with a recombinant essential light chain labelled with various thiol-reactive chromophores. The second fluorophore of the FRET couple was introduced by three approaches: labelling actin, labelling SH1 cysteine and binding an adenosine triphosphate (ATP) analogue. We characterise FRET in rigor cross-bridges: in this condition muscle fibres are well described by a single FRET population model which allows us to evaluate the true FRET efficiency for a single couple and the consequent donor–acceptor distance. The results obtained are in good agreement with the distances expected from crystallographic data. The FRET characterisation presented herein is essential before moving onto dynamic measurements, as the FRET efficiency differences to be detected in an active muscle fibre are on the order of 10–15% of the FRET efficiencies evaluated here. This means that, to obtain reliable results to monitor the dynamics of cross-bridge conformational changes, we had to fully characterise the system in a steady-state condition, demonstrating firstly the possibility to detect FRET and secondly the viability of the present approach to distinguish small FRET variations.     

Serotonin transporter oligomerization documented in RN46A cells and neurons by sensitized acceptor emission FRET and fluorescence lifetime imaging microscopy

The serotonin transporter is a member of the monoamine transporter family that also includes transporters of dopamine and norepinephrine. We have used sensitized acceptor emission fluorescence resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM) to study the oligomerization of SERT in HEK-MSR-239 cells, RN46A cells and in cultured hippocampal neurons. We were able to show identical FRET efficiencies in cell lines as well as in primary cultured hippocampal neurons, demonstrating that the oligomerization is cell type independent. The results obtained with both FRET approaches are very similar and furthermore, in agreement with previous results obtained by donor bleaching FRET microscopy.     

Treatment of cotton with an alkaline Bacillus spp cellulase: activity towards crystalline cellulose

We analysed the influence of several enzymatic treatment processes using an alkaline cellulase enzyme from Bacillus spp. on the sorption properties of cotton fabrics. Although cellulases are commonly applied in detergent formulations due to their anti-redeposition and depilling benefits, determining the mechanism of action of alkaline cellulases on cotton fibres requires a deeper understanding of the morphology and structure of cotton fibres in terms of fibre cleaning. The accessibility of cellulose fibres was studied by evaluating the iodine sorption value and by fluorescent-labelled enzyme microscopy; the surface morphology of fabrics was analysed by scanning microscopy. The action of enzyme hydrolysis over short time periods can produce fibrillation on cotton fibre surface without any release of cellulosic material. The results indicate that several short consecutive treatments were more effective in increasing the fibre accessibility than one long treatment. In addition, no detectable hydrolytic activity, in terms of reducing sugar production, was found.     

Optical techniques for imaging membrane topography

In recent years three powerful optical imaging techniques have emerged that provide nanometer-scale information about the topography of membrane surfaces, whether cellular or artificial: intermembrane fluorescence resonance energy transfer (FRET), fluorescence interference contrast microscopy (FLIC), and reflection interference contrast microscopy (RICM). In intermembrane FRET, the sharp distance dependence of resonant energy transfer between fluorophores allows topographic measurements in the Angstrom to few-nanometer range. In FLIC and RICM, interference between light from a membrane (either from fluorescent probes, or reflected illumination) and light reflected by a planar substrate provide spatial sensitivity in the few to hundreds of nanometer range, with few-nanometer resolution. All of these techniques are fairly easy to implement. We discuss the physics and optics behind each of these tools, as well as practical concerns regarding their uses. We also provide examples of their application in imaging molecular-scale structures at intermembrane junctions.     

Microspectroscopic fluorescence analysis with prism-based imaging spectrometers: review and current studies.

BACKGROUND: Fluorescence imaging spectroscopy is a powerful but under-utilized tool. This article gives perspective on the use of imaging spectroscopy, and provides two examples of imaging spectroscopy done with a prism-based system. The intent is to give insight into the power of imaging spectroscopy when used in combination with other imaging techniques. In particular, studies of intact coral photobleaching and beads designed to show energy transfer are reported. In the bead study, spectroscopic lifetime imaging was performed at each photobleaching step. RESULTS: Spectroscopic photobleaching of the hard coral, Montastrea annularis, revealed two spectral regions. A region in the red portion of the spectrum bleached rapidly while progressively increasing fluorescence was observed over a wide portion of the spectrum. This behavior is consistent with current theories for the role of fluorescent proteins in corals.Following a photobleaching study of beads designed to exhibit energy transfer with imaging spectroscopic fluorescence lifetime imaging microscopy (ISFLIM) allowed unambiguous assignment of fluorescence resonance energy transfer (FRET). The data in this experiment indicated that most of the commonly used markers of FRET would have been inconclusive. The ability of the ISFLIM to look at all regions of the spectrum, particularly the acceptor region, allowed FRET to be assigned. CONCLUSIONS: Fluorescence imaging spectroscopy is a rapidly advancing technology, uniquely suited to the flexible detection of dyes over a wide range of wavelengths.     

A homogeneous, high-throughput fluorescence resonance energy transfer-based DNA polymerase assay

A homogeneous, fluorescence resonance energy transfer (FRET)-based DNA polymerase assay that is suitable for high-throughput screening for inhibitors, and can also be used for steady-state kinetic investigations, is described. The activity, kinetic mechanism, and processivity of the isolated alpha subunit of DNA polymerase III, the product of the dnaE gene, from the gram-negative pathogen Haemophilus influenzae were investigated using the FRET assay.     

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.     

Surface activation of dyed fabric for cellulase treatment

Surface activation of fabric made from cellulose fibres, such as viscose, lyocell, modal fibres and cotton, can be achieved by printing of a concentrated NaOH-containing paste. From the concentration of reducing sugars formed in solution, an increase in intensity of the cellulase hydrolysis by a factor of six to eight was observed, which was mainly concentrated at the activated parts of the fabric surface. This method of local activation is of particular interest for modification of materials that have been dyed with special processes to attain an uneven distribution of dyestuff within the yarn cross-section, e.g., indigo ring-dyed denim yarn for jeans production. Fabrics made from regenerated cellulose fibres were used as model substrate to express the effects of surface activation on indigo-dyed material. Wash-down experiments on indigo-dyed denim demonstrated significant colour removal from the activated surface at low overall weight loss of 4-5%. The method is of relevance for a more eco-friendly processing of jeans in the garment industry.