Förster Resonance Energy Transfer Measurements Research Articles

Myosin II tension sensors visualize force generation within the actin cytoskeleton in living cells.

Nonmuscle myosin II generates cytoskeletal forces that drive cell division, embryogenesis, muscle contraction, and many other cellular functions. However, at present there is no method that can directly measure the forces generated by myosins in living cells. Here we describe a Förster resonance energy transfer (FRET)-based tension sensor that can detect myosin associated force along the filamentous actin network. Fluorescence lifetime imaging microscopy (FLIM)-FRET measurements indicate that the forces generated by NMIIB exhibit significant spatial and temporal heterogeneity as a function of donor lifetime and fluorophore energy exchange. These measurements provide a proxy for inferred forces that vary widely along the actin cytoskeleton. This initial report highlights the potential utility of myosin-based tension sensors in elucidating the roles of cytoskeletal contractility in a wide variety of contexts.

Glyoxal-methyl-ethylene sulfonic acid fixative enhances the fixation of cytoskeletal structures for Förster resonance energy transfer measurements.

Förster resonance energy transfer (FRET) serves as a tool for measuring protein-protein interactions using various sensor molecules. The tension sensor module relies on FRET technology. In our study, this module was inserted within the actinin molecule to measure the surface tension of the cells. Given that the decay curve of FRET efficiency correlates with surface tension increase, precise and accurate efficiency measurement becomes crucial. Among the methods of FRET measurements, FRET efficiency remains the most accurate if sample fixation is successful. However, when cells were fixed with 4% paraformaldehyde (PFA), the actinin-FRET sensor diffused across the cytoplasm; this prompted us to explore fixation method enhancements. Glyoxal fixative has been reported to improve cytoskeletal morphologies compared to PFA. However, it was not known whether glyoxal fits FRET measurements. Glyoxal necessitates an acetic acid solution for fixation; however, acidic conditions could compromise fluorescence stability. We observed that the pH working range of glyoxal fixative aligns closely with MES (methyl-ethylene sulfonic acid) Good's buffer. Initially, we switched the acidic solution for MES buffer and optimized the fixation procedure for in vitro and in vivo FRET imaging. By comparing FRET measurements on hydrogels with known stiffness to tumor nodules in mouse lung, we estimated in vivo stiffness. The estimated stiffness of cancerous tissue was harder than the reported stiffness of smooth muscle. This discovery shed lights on how cancer cells perceive environmental stiffness during metastasis.

FRET-guided modeling of nucleic acids.

The functional diversity of RNAs is encoded in their innate conformational heterogeneity. The combination of single-molecule spectroscopy and computational modeling offers new attractive opportunities to map structural transitions within nucleic acid ensembles. Here, we describe a framework to harmonize single-molecule Förster resonance energy transfer (FRET) measurements with molecular dynamics simulations and de novo structure prediction. Using either all-atom or implicit fluorophore modeling, we recreate FRET experiments in silico, visualize the underlying structural dynamics and quantify the reaction coordinates. Using multiple accessible-contact volumes as a post hoc scoring method for fragment assembly in Rosetta, we demonstrate that FRET can be used to filter a de novo RNA structure prediction ensemble by refuting models that are not compatible with in vitro FRET measurement. We benchmark our FRET-assisted modeling approach on double-labeled DNA strands and validate it against an intrinsically dynamic manganese(II)-binding riboswitch. We show that a FRET coordinate describing the assembly of a four-way junction allows our pipeline to recapitulate the global fold of the riboswitch displayed by the crystal structure. We conclude that computational fluorescence spectroscopy facilitates the interpretability of dynamic structural ensembles and improves the mechanistic understanding of nucleic acid interactions.

Microwave analogy of Förster resonance energy transfer and effect of finite antenna length

The near-field interaction between quantum emitters, governed by Förster resonance energy transfer (FRET), plays a pivotal role in nanoscale energy transfer mechanisms. However, FRET measurements in the optical regime are challenging as they require nanoscale control of the position and orientation of the emitters. To overcome these challenges, microwave measurements were proposed for enhanced spatial resolution and precise orientation control. However, unlike in optical systems for which the dipole can be taken to be infinitesimal in size, the finite size of microwave antennas can affect energy transfer measurements, especially at short distances. This highlights the necessity to consider the finite antenna length to obtain accurate results. In this study, we advance the understanding of dipole–dipole energy transfer in the microwave regime by developing an analytical model that explicitly considers finite antennas. Unlike previous works, our model calculates the mutual impedance of finite-length thin-wire dipole antennas without assuming a uniform current distribution. We validate our analytical model through experiments investigating energy transfer between antennas placed adjacent to a perfect electric conductor mirror. This allows us to provide clear guidelines for designing microwave experiments, distinguishing conditions where finite-size effects can be neglected and where they must be taken into account. Our study not only contributes to the fundamental physics of energy transfer but also opens avenues for microwave antenna impedance-based measurements to complement optical FRET experiments and quantitatively explore dipole–dipole energy transfer in a wider range of conditions.

Impact of Ion-Mixing Entropy on Orientational Preferences of DNA Helices: FRET Measurements and Computer Simulations.

Biological processes require DNA and RNA helices to pack together in specific interhelical orientations. While electrostatic repulsion between backbone charges is expected to be maximized when helices are in parallel alignment, such orientations are commonplace in nature. To better understand how the repulsion is overcome, we used experimental and computational approaches to investigate how the orientational preferences of DNA helices depend on the concentration and valence of mobile cations. We used Förster resonance energy transfer (FRET) to probe the relative orientations of two 24-bp helices held together via a freely rotating PEG linker. At low cation concentrations, the helices preferred more "cross"-like orientations over those closer to parallel, and this preference was reduced with increasing salt concentrations. The results were in good quantitative agreement with Poisson-Boltzmann (PB) calculations for monovalent salt (Na+). However, PB underestimated the ability of mixtures of monovalent and divalent ions (Mg2+) to reduce the conformational preference. As a complementary approach, we performed all-atom molecular dynamics (MD) simulations and found better agreement with the experimental results. While MD and PB predict similar electrostatic forces, MD predicts a greater accumulation of Mg2+ in the ion atmosphere surrounding the DNA. Mg2+ occupancy is predicted to be greater in conformations close to the parallel orientation than in conformations close to the crossed orientation, enabling a greater release of Na+ ions and providing an entropic gain (one bound ion for two released). MD predicts an entropy gain larger than that of PB because of the increased Mg2+ occupancy. The entropy changes have a negligible effect at low Mg2+ concentrations because the free energies are dominated by electrostatic repulsion. However, as the Mg2+ concentration increases, charge screening is more effective and the mixing entropy produces readily detectable changes in packing preferences. Our results underline the importance of mixing entropy of counterions in nucleic acid interactions and provide a new understanding on the impact of a mixed ion atmosphere on the packing of DNA helices.

Utility of FRET in studies of membrane protein oligomerization: The concept of the effective dissociation constant

The activity of many membrane receptors is controlled through their lateral association into dimers or higher-order oligomers. Although Förster resonance energy transfer (FRET) measurements have been used extensively to characterize the stability of receptor dimers, the utility of FRET in studies of larger oligomers has been limited. Here we introduce an effective equilibrium dissociation constant that can be extracted from FRET measurements for EphA2, a receptor tyrosine kinase (RTK) known to form active oligomers of heterogeneous distributions in response to its ligand ephrinA1-Fc. The newly introduced effective equilibrium dissociation constant has a well-defined physical meaning and biological significance. It denotes the receptor concentration for which half of the receptors are monomeric and inactive, and the other half are associated into oligomers and are active, irrespective of the exact oligomer size. This work introduces a new dimension to the utility of FRET in studies of membrane receptor association and signaling in the plasma membrane.

A FRET-Based Assay for the Identification of PCNA Inhibitors.

Proliferating cell nuclear antigen (PCNA) is the key regulator of human DNA metabolism. One important interaction partner is p15, involved in DNA replication and repair. Targeting the PCNA-p15 interaction is a promising therapeutic strategy against cancer. Here, a Förster resonance energy transfer (FRET)-based assay for the analysis of the PCNA-p15 interaction was developed. Next to the application as screening tool for the identification and characterization of PCNA-p15 interaction inhibitors, the assay is also suitable for the investigation of mutation-induced changes in their affinity. This is particularly useful for analyzing disease associated PCNA or p15 variants at the molecular level. Recently, the PCNA variant C148S has been associated with Ataxia-telangiectasia-like disorder type 2 (ATLD2). ATLD2 is a neurodegenerative disease based on defects in DNA repair due to an impaired PCNA. Incubation time dependent FRET measurements indicated no effect on PCNAC148S-p15 affinity, but on PCNA stability. The impaired stability and increased aggregation behavior of PCNAC148S was confirmed by intrinsic tryptophan fluorescence, differential scanning fluorimetry (DSF) and asymmetrical flow field-flow fractionation (AF4) measurements. The analysis of the disease associated PCNA variant demonstrated the versatility of the interaction assay as developed.

Improved estimation of the ratio of detection efficiencies of excited acceptors and donors for FRET measurements.

Förster resonance energy transfer (FRET) is a radiationless interaction between a donor and an acceptor whose distance dependence makes it a sensitive tool for studying the oligomerization and the structure of proteins. When FRET is determined by measuring the sensitized emission of the acceptor, a parameter characterizing the ratio of detection efficiencies of an excited acceptor versus an excited donor is invariably involved in the formalism. For FRET measurements involving fluorescent antibodies or other external labels, this parameter, designated by α, is usually determined by comparing the intensity of a known number of donors and acceptors in two independent samples leading to a large statistical variability if the sample size is small. Here, we present a method that improves precision by applying microbeads with a calibrated number of antibody binding sites and a donor-acceptor mixture in which donors and acceptors are present in a certain, experimentally determined ratio. A formalism is developed for determining α and the superior reproducibility of the proposed method compared to the conventional approach is demonstrated. Since the novel methodology does not require sophisticated calibration samples or special instrumentation, it can be widely applied for the quantification of FRET experiments in biological research.

Pixel-by-pixel autofluorescence corrected FRET in fluorescence microscopy improves accuracy for samples with spatially varied autofluorescence to signal ratio

The actual interaction between signaling species in cellular processes is often more important than their expression levels. Förster resonance energy transfer (FRET) is a popular tool for studying molecular interactions, since it is highly sensitive to proximity in the range of 2–10 nm. Spectral spillover-corrected quantitative (3-cube) FRET is a cost effective and versatile approach, which can be applied in flow cytometry and various modalities of fluorescence microscopy, but may be hampered by varying levels of autofluorescence. Here, we have implemented pixel-by-pixel autofluorescence correction in microscopy FRET measurements, exploiting cell-free calibration standards void of autofluorescence that allow the correct determination of all spectral spillover factors. We also present an ImageJ/Fiji plugin for interactive analysis of single images as well as automatic creation of quantitative FRET efficiency maps from large image sets. For validation, we used bead and cell based FRET models covering a range of signal to autofluorescence ratios and FRET efficiencies and compared the approach with conventional average autofluorescence/background correction. Pixel-by-pixel autofluorescence correction proved to be superior in the accuracy of results, particularly for samples with spatially varying autofluorescence and low fluorescence to autofluorescence ratios, the latter often being the case for physiological expression levels.

Detecting RNA–Protein Interactions With EGFP‐Cy3 FRET by Acceptor Photobleaching

Förster Resonance Energy Transfer (FRET) is a great tool for cell biologists to investigate molecular interactions in live specimens. FRET is a distance-dependent phenomenon which can detect molecular interactions at distances between 1-10 nm. Several FRET approaches are reported in the literature for live and fixed cells to study protein-protein interactions; this protocol provides details of acceptor photobleaching as a FRET method to study RNA-Protein interactions. Cy3-labeled RNA foci (FRET acceptors) are photobleached at the intra-cellular site of interest (the nuclei) and the intensity of the EGFP-tagged proteins (FRET donors) at that same site are measured pre- and post- photobleaching. In principle, FRET is detected if the intensity of EGFP increases after photobleaching of Cy3. This protocol describes necessary steps and appropriate controls to conduct FRET measurements by the acceptor photobleaching method. Successful applications of this protocol will provide data to support the conclusion that EGFP-labeled proteins directly interact with Cy3-labeled RNA at the site of photobleaching. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: FRET in fixed cells Alternate Protocol: FRET in live cells.

Mechanics of divalent ion sensitivity of the manganese-sensing riboswitch measured by combined high-resolution optical tweezers and single-molecule fluorescence.

Riboswitches are structured non-coding RNA typically found in the 5′ untranslated regions of bacterial mRNAs. They modulate gene expression through the binding of specific ligands that induce changes in the riboswitch structure, most often at the level of transcription and translation. The Mn2+-sensing yybP-ykoY RNA motif is one of the most widespread riboswitches across bacteria, including human and plant pathogens. It contains two divalent metal ion binding sites: one is specific for Mn2+ whereas the other is nonspecific for a divalent metal ion, including Mg2+. Previous crystal structures have shown that Mn2+ binds in between the two distal helical legs of a four-way junction and induces a large “docking” conformational change. It is unclear how competition between Mn2+ and Mg2+ for the two binding sites and the associated RNA conformational changes give rise to Mn2+ detection function and sensitivity. Therefore, we explored the global structural dynamics of the L. lactis Mn2+-sensing riboswitch in the presence of competing divalent cations using high-resolution optical tweezers simultaneous with confocal Förster resonance energy transfer (FRET) spectroscopy. Initial non-equilibrium force ramp measurements revealed force-dependent docking distribution, suggesting a highly selective docked state at suboptimal Mn2+ ion concentration. Follow-up high-resolution equilibrium fixed-force measurements directly measured docking and undocking rate constants versus force to determine docking and undocking mechanics, including transition states and pathways, at varying Mn2+ vs Mg2+ concentrations. Simultaneous FRET measurements confirmed specific conformational transitions. Docking and undocking were symmetric processes with nearly equal force sensitivity and a transition state located nearly midway between the docked and undocked states. Mn2+ sensitivity appears to optimize at a concentration where further increasing Mn2+ competes with Mg2+ ions for the non-specific divalent metal ion binding site.

Diffusion-Enhanced Förster Resonance Energy Transfer in Flexible Peptides: From the Haas-Steinberg Partial Differential Equation to a Closed Analytical Expression.

In the huge field of polymer structure and dynamics, including intrinsically disordered peptides, protein folding, and enzyme activity, many questions remain that cannot be answered by methodology based on artificial intelligence, X-ray, or NMR spectroscopy but maybe by fluorescence spectroscopy. The theory of Förster resonance energy transfer (FRET) describes how an optically excited fluorophore transfers its excitation energy through space to an acceptor moiety-with a rate that depends on the distance between donor and acceptor. When the donor and acceptor moiety are conjugated to different sites of a flexible peptide chain or any other linear polymer, the pair could in principle report on chain structure and dynamics, on the site-to-site distance distribution, and on the diffusion coefficient of mutual site-to-site motion of the peptide chain. However, the dependence of FRET on distance distribution and diffusion is not defined by a closed analytical expression but by a partial differential equation (PDE), by the Haas-Steinberg equation (HSE), which can only be solved by time-consuming numerical methods. As a second complication, time-resolved FRET measurements have thus far been deemed necessary. As a third complication, the evaluation requires a computationally demanding but indispensable global analysis of an extended experimental data set. These requirements have made the method accessible to only a few experts. Here, we show how the Haas-Steinberg equation leads to a closed analytical expression (CAE), the Haas-Steinberg-Jacob equation (HSJE), which relates a diffusion-diagnosing parameter, the effective donor-acceptor distance, to the augmented diffusion coefficient, J, composed of the diffusion coefficient, D, and the photophysical parameters that characterize the used FRET method. The effective donor-acceptor distance is easily retrieved either through time-resolved or steady-state fluorescence measurements. Any global fit can now be performed in seconds and minimizes the sum-of-square difference between the experimental values of the effective distance and the values obtained from the HSJE. In summary, the HSJE can give a decisive advantage in applying the speed and sensitivity of FRET spectroscopy to standing questions of polymer structure and dynamics.

Principles of Resonance Energy Transfer

This unit describes the basic principles of Förster resonance energy transfer (FRET). Beginning with a brief summary of the history of FRET applications, the theory of FRET is introduced in detail using figures to explain all the important parameters of the FRET process. After listing various approaches for measuring FRET efficiency, several pieces of advice are given on choosing the appropriate instrumentation. The unit concludes with a discussion of the limitations of FRET measurements followed by a few examples of the latest FRET applications, including new developments such as spectral flow cytometric FRET, single-molecule FRET, and combinations of FRET with super-resolution or lifetime imaging microscopy and with molecular dynamics simulations. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC.

Deciphering the conformational dynamics of gephyrin-mediated collybistin activation

Efficient neuronal signaling depends on the proper assembly of the postsynaptic neurotransmitter machinery. The majority of inhibitory synapses feature γ-aminobutyric acid type A (GABAA) receptors. The function of these GABAergic synapses is controlled by the scaffolding protein gephyrin and collybistin, a Dbl family guanine nucleotide exchange factor and neuronal adaptor protein. Specifically, collybistin interacts with small GTPases, cell adhesion proteins, and phosphoinositides to recruit gephyrin and GABAA receptors to postsynaptic membrane specializations. Collybistin usually contains an N-terminal SH3 domain and exists in closed/inactive or open/active states. Here, we elucidate the molecular basis of the gephyrin-collybistin interaction with newly designed collybistin Förster resonance energy transfer (FRET) sensors. Using fluorescence lifetime-based FRET measurements, we deduce the affinity of the gephyrin-collybistin complex, thereby confirming that the C-terminal dimer-forming E domain binds collybistin, an interaction that does not require E domain dimerization. Simulations based on fluorescence lifetime and sensor distance distributions reveal at least a two-state equilibrium of the SH3 domain already in the free/unbound collybistin, thereby illustrating the accessible volume of the SH3 domain. Finally, our data provide strong evidence for a tightly regulated collybistin-gephyrin interplay, where, unexpectedly, switching of collybistin from closed/inactive to open/active states is efficiently triggered by gephyrin.

Hyperspectral imaging and adaptive thresholding to identify agonist-induced cAMP signals in pulmonary microvascular endothelial cells.

Cyclic AMP (cAMP) is a second messenger that regulates a wide variety of cellular functions. There is increasing evidence suggesting that signaling specificity is due in part to cAMP compartmentalization. In the last 15 years, development of cAMP-specific Förster resonance energy transfer (FRET) probes have allowed us to visualize spatial distributions of intracellular cAMP signals. The use of FRET-based sensors is not without its limitations, as FRET probes display low signal to noise ratio (SNR). Hyperspectral imaging and analysis approaches have, in part, allowed us to overcome these limitations by improving the SNR of FRET measurements. Here we demonstrate that the combination of hyperspectral imaging approaches, linear unmixing, and adaptive thresholding allow us to visualize regions of elevated cAMP (regions of interest - ROIs) in an unbiased manner. We transfected cDNA encoding the H188 FRET-based cAMP probe into pulmonary microvascular endothelial cells. Application of isoproterenol and prostaglandin E1 (PGE1) triggered complex cAMP responses. Spatial and temporal aspects of cAMP responses were quantified using an adaptive thresholding approach and compared between agonist treatment groups. Our data indicate that both the origination sites and spatial/temporal distributions of cAMP signals are agonist dependent in PMVECs. We are currently analyzing the data in order to better quantify the distribution of cAMP signals triggered by different agonists.

A FRET Approach to Detect Paraoxon among Organophosphate Pesticides Using a Fluorescent Biosensor.

The development of faster, sensitive and real-time methods for detecting organophosphate (OP) pesticides is of utmost priority in the in situ monitoring of these widespread compounds. Research on enzyme-based biosensors is increasing, and a promising candidate as a bioreceptor is the thermostable enzyme esterase-2 from Alicyclobacillus acidocaldarius (EST2), with a lipase-like Ser–His–Asp catalytic triad with a high affinity for OPs. This study aimed to evaluate the applicability of Förster resonance energy transfer (FRET) as a sensitive and reliable method to quantify OPs at environmentally relevant concentrations. For this purpose, the previously developed IAEDANS-labelled EST2-S35C mutant was used, in which tryptophan and IAEDANS fluorophores are the donor and the acceptor, respectively. Fluorometric measurements showed linearity with increased EST2-S35C concentrations. No significant interference was observed in the FRET measurements due to changes in the pH of the medium or the addition of other organic components (glucose, ascorbic acid or yeast extract). Fluorescence quenching due to the presence of paraoxon was observed at concentrations as low as 2 nM, which are considered harmful for the ecosystem. These results pave the way for further experiments encompassing more complex matrices.

Single-molecule Detection of Ultrafast Biomolecular Dynamics with Nanophotonics.

Single-molecule Förster resonance energy transfer (FRET) is a versatile technique for probing the structure and dynamics of biomolecules even in heterogeneous ensembles. However, because of the limited fluorescence brightness per molecule and the relatively long fluorescence lifetimes, probing ultrafast structural dynamics in the nanosecond time scale has thus far been very challenging. Here, we demonstrate that nanophotonic fluorescence enhancement in zero-mode waveguides enables measurements of previously inaccessible low-nanosecond dynamics by dramatically improving time resolution and reduces data acquisition times by more than an order of magnitude. As a prototypical example, we use this approach to probe the dynamics of a short intrinsically disordered peptide that were previously inaccessible with single-molecule FRET measurements. We show that we are now able to detect the low-nanosecond correlations in this peptide, and we obtain a detailed interpretation of the underlying distance distributions and dynamics in conjunction with all-atom molecular dynamics simulations, which agree remarkably well with the experiments. We expect this combined approach to be widely applicable to the investigation of very rapid biomolecular dynamics.

FRET measurement of cytochrome bc1 and reaction centre complex proximity in live Rhodobacter sphaeroides cells

In the model purple phototrophic bacterium Rhodobacter (Rba.) sphaeroides, solar energy is converted via coupled electron and proton transfer reactions within the intracytoplasmic membranes (ICMs), infoldings of the cytoplasmic membrane that form spherical ‘chromatophore’ vesicles. These bacterial ‘organelles’ are ideal model systems for studying how the organisation of the photosynthetic complexes therein shape membrane architecture. In Rba. sphaeroides, light-harvesting 2 (LH2) complexes transfer absorbed excitation energy to dimeric reaction centre (RC)-LH1-PufX complexes. The PufX polypeptide creates a channel that allows the lipid soluble electron carrier quinol, produced by RC photochemistry, to diffuse to the cytochrome bc1 complex, where quinols are oxidised to quinones, with the liberated protons used to generate a transmembrane proton gradient and the electrons returned to the RC via cytochrome c2. Proximity between cytochrome bc1 and RC-LH1-PufX minimises quinone/quinol/cytochrome c2 diffusion distances within this protein-crowded membrane, however this distance has not yet been measured. Here, we tag the RC and cytochrome bc1 with yellow or cyan fluorescent proteins (YFP/CFP) and record the lifetimes of YFP/CFP Förster resonance energy transfer (FRET) pairs in whole cells. FRET analysis shows that that these complexes lie on average within 6 nm of each other. Complementary high-resolution atomic force microscopy (AFM) of intact, purified chromatophores verifies the close association of cytochrome bc1 complexes with RC-LH1-PufX dimers. Our results provide a structural basis for the close kinetic coupling between RC-LH1-PufX and cytochrome bc1 observed by spectroscopy, and explain how quinols/quinones and cytochrome c2 shuttle on a millisecond timescale between these complexes, sustaining efficient photosynthetic electron flow.

FRETboard: Semisupervised classification of FRET traces

Förster resonance energy transfer (FRET) is a useful phenomenon in biomolecular investigations, as it can be leveraged for nanoscale measurements. The optical signals produced by such experiments can be analyzed by fitting a statistical model. Several software tools exist to fit such models in an unsupervised manner but lack the flexibility to adapt to different experimental setups and require local installations. Here, we propose to fit models to optical signals more intuitively by adopting a semisupervised approach, in which the user interactively guides the model to fit a given data set, and introduce FRETboard, a web tool that allows users to provide such guidance. We show that our approach is able to closely reproduce ground truth FRET statistics in a wide range of simulated single-molecule scenarios and correctly estimate parameters for up to 11 states. On in vitro data, we retrieve parameters identical to those obtained by laborious manual classification in a fraction of the required time. Moreover, we designed FRETboard to be easily extendable to other models, allowing it to adapt to future developments in FRET measurement and analysis.

Förster Resonance Energy Transfer Measurements in Living Plant Cells.

Sensitized emission-based Förster resonance energy transfer (FRET) experiments are easily done but depend on the microscopic setup. Confocal laser scanning microscopes have become a workhorse for biologists. Commercial systems offer high flexibility in laser power adjustment and detector sensitivity and often combine different detectors to obtain the perfect image. However, the comparison of intensity-based data from different experiments and setups is often impossible due to this flexibility. Biologist-friendly procedures are of advantage and allow for simple and reliable adjustment of laser and detector settings. Furthermore, as FRET experiments in living cells are affected by the variability in protein expression and donor-acceptor ratios, protein expression levels must be considered for data evaluation. Described here is a simple protocol for reliable and reproducible FRET measurements, including routines for the estimation of protein expression and adjustment of laser intensity and detector settings. Data evaluation will be performed by calibration with a fluorophore fusion of known FRET efficiency. To improve simplicity, correction factors have been compared that have been obtained in cells and by measuring recombinant fluorescent proteins.