Acceptor Fluorophores Research Articles

Development of Biocompatible Polymeric Nanoparticles for in Vivo NIR and FRET Imaging

The majority of near-infrared (NIR) fluorophores are organic molecules that show significant overlap between the excitation and emission spectra and therefore exhibit high fluorescence backgrounds during in vivo imaging. Recently, cyanine dyes with a large Stokes shift have shown great promise for NIR imaging but often undergo rapid photodegradation and nonspecific protein adsorption. Alternatively, fluorescence resonance energy transfer (FRET) is a promising technique to generate a larger gap between the excitation and emission maxima and thus can reduce the background signal. Here, we report the rational design of FRET-based polymeric nanoparticles for NIR and FRET imaging. The particles were assembled from diblock copolymers of poly(d,l-lactic-co-glycolic acid) and maleimide-activated poly(ethylene glycol), which were also encapsulated with both the donor (1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine) and acceptor (1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine) fluorophores. Because of their extreme hydrophobicity, thousands of fluorophores could be encapsulated inside a single particle without causing leakage. FRET resulted in a large Stokes shift (>100 nm) of the emission maxima, and the transfer efficiency could be fine-tuned by further adjusting the doping ratio of the donor and acceptor fluorophores. The optimized formulation was less than 100 nm in size, brighter than quantum dots, stable in biological media, and demonstrated similar biodistribution to most nanomaterials. Additional animal phantom studies demonstrated that the FRET imaging platform developed could have far-reaching applications in optical imaging.

Plasmon-Controlled Förster Resonance Energy Transfer

The localized plasmons of metal nanocrystals have been widely utilized to control a variety of optical signals, such as Raman, fluorescence, and circular dichroism, from proximal dye molecules. We show, on the single-particle level, that the Forster resonance energy transfer between two different fluorophores can be modulated by adjacent plasmonic nanocrystals. The donor and acceptor fluorophore molecules are embedded in a mesostructured silica shell that is uniformly coated on Au–Ag core–shell nanocrystals. The longitudinal plasmon wavelengths of the core–shell metal nanocrystals are synthetically tailored by varying the aspect ratio. Comparison of the scattering and fluorescence spectra taken from the different hybrid nanostructures indicates that the energy transfer efficiency can be controlled by the plasmon wavelength. When the plasmon peak overlaps with the emission peak of the donor, the energy transfer channel is turned off. When the plasmon peak is red-shifted to be in between the emission peak o...

Fluorescence-quenching-based homogeneous caspase-3 activity assay using photon upconversion

Caspase proteases are key mediators in apoptosis and thus of great interest in pharmaceutical industry. Enzyme-activity assays are commonly employed in the screening of protease inhibitors that are potential drug candidates. Conventional homogeneous fluorescence-based assays are susceptible to autofluorescence originating from biological material. This background autofluorescence can be eliminated by using upconverting phosphors (UCPs) that emit visible light upon excitation at near-infrared. In the assay energy was transferred from a UCP-donor to a conventional fluorophore acceptor that resided at one end of a caspase-3-specific substrate peptide. Attached to the other end was a quencher molecule that was used to attenuate the acceptor emission through intramolecular energy transfer in an intact peptide. In non-inhibitory conditions the enzyme reaction separated the fluorophore from the quencher and the emission of the fluorophore was recovered. The method was applied for the detection and characterization of a known caspase-3 inhibitor Z-DEVD-FMK, and the assay gave IC50 values of approximately 13nM for this inhibitor. We have demonstrated the applicability of UCPs on a fluorescence-quenching-based homogeneous enzyme-activity assay for the detection of caspase-3 inhibitors. The use of near-infrared excitable UCPs enables inexpensive instrumentation and total elimination of autofluorescence, while the use of an internally quenched substrate molecule diminishes the background resulting from radiatively excited acceptor molecules. The reduction of autofluorescence and radiative background result in high signal-to-background ratios (ratios of approximately 100 were obtained). By further utilizing assay miniaturization and signal enhancement in a white microtitration plate, a significant reduction in the reagent consumption can be achieved rendering the assay applicable for high-throughput screening.

Kinetics of Precursor Interactions with the Bacterial Tat Translocase Detected by Real-time FRET

The Escherichia coli twin-arginine translocation (Tat) system transports fully folded and assembled proteins across the inner membrane into the periplasmic space. Traditionally, in vitro protein translocation studies have been performed using gel-based transport assays. This technique suffers from low time resolution, and often, an inability to distinguish between different steps in a continuously occurring translocation process. To address these limitations, we have developed an in vitro FRET-based assay that reports on an early step in the Tat translocation process in real-time. The natural Tat substrate pre-SufI was labeled with Alexa532 (donor), and the fluorescent protein mCherry (acceptor) was fused to the C terminus of TatB or TatC. The colored Tat proteins were easily visible during purification, enabling identification of a highly active inverted membrane vesicle (IMV) fraction yielding transport rates with NADH almost an order of magnitude faster than previously reported. When pre-SufI was bound to the translocon, FRET was observed for both Tat proteins. FRET was diminished upon addition of nonfluorescent pre-SufI, indicating that the initial binding step is reversible. When the membranes were energized with NADH, the FRET signal was lost after a short delay. These data suggest a model in which a Tat cargo initially associates with the TatBC complex, and an electric field gradient is required for the cargo to proceed to the next stage of transport. This cargo migration away from the TatBC complex requires a significant fraction of the total transport time.

Pulse-shaping multiphoton FRET microscopy.

Fluorescence Resonance Energy Transfer (FRET) microscopy is a commonly-used technique to study problems in biophysics that range from uncovering cellular signaling pathways to detecting conformational changes in single biomolecules. Unfortunately, excitation and emission spectral overlap between the fluorophores create challenges in quantitative FRET studies. It has been shown previously that quantitative FRET stoichiometry can be performed by selective excitation of donor and acceptor fluorophores. Extending this approach to two-photon FRET applications is difficult when conventional femtosecond laser sources are used due to their limited bandwidth and slow tuning response time. Extremely broadband titanium:sapphire lasers enable the simultaneous excitation of both donor and acceptor for two-photon FRET, but do so without selectivity. Here we present a novel two-photon FRET microscopy technique that employs pulse-shaping to perform selective excitation of fluorophores in live cells and detect FRET between them. Pulse-shaping via multiphoton intrapulse interference can tailor the excitation pulses to achieve selective excitation. This technique overcomes the limitation of conventional femtosecond lasers to allow rapid switching between selective excitation of the donor and acceptor fluorophores. We apply the method to live cells expressing the fluorescent proteins mCerulean and mCherry, demonstrating selective excitation of fluorophores via pulse-shaping and the detection of two-photon FRET. This work paves the way for two-photon FRET stoichiometry.

A Flim-FRET Study of the Raft-Associated Proteins in Erythrocytes Expressing the Miltenberger Blood Group Antigen Subtype III (Mi.III) Phenotype

Lipid rafts are dynamic signaling hubs packed with ordered lipids on the cell membrane. Lipid rafts in erythrocytes can be classified into two types: flotillin-associated rafts and stomatin-associated rafts. We previously found by biochemical means that these two types of rafts coalesce on the erythrocytes expressing the Mi.III blood type. Mi.III is one of the most important blood groups in the field of transfusion medicine in Southeast Asia. Mi.III encompasses a characteristic hybrid structure of glycophorin A and glycophorin B, termed Gp.Mur, and is part of the band 3 macrocomplexes on the erythrocyte surface. Band 3 is known to be partially associated with erythrocyte rafts. The objective of this study was to examine if Gp.Mur was associated with rafts on Mi.III+ cells. We assessed the protein-protein interaction between Gp.Mur and band 3 by measurements of FRET using fluorescent lifetime imaging microscopy (FLIM). In these experiments, band 3 and GFP-Gp.Mur fusion constructs were co-transfected into HEK-293 cells, and band 3 was labeled with Alexa Fluor-568 prior to FLIM-FRET measurements. Here, GFP served as the donor and Alexa Fluor-568 as the acceptor fluorophore if FRET occurred. We found that the fluorescence lifetime of the donor was significantly shorter at some surface edges in the cells coexpressing Gp.Mur-GFP and band 3; this phenomenon however was not observed in the cells expressing Gp.Mur-GFP only. We thus conclude that Gp.Mur and band 3 interact within 10 nm on particular regions of the plasma membrane, some of which are likely lipid rafts. Because Mi.III+ erythrocytes are superior in CO2 metabolism, pH homeostasis and cell membrane resilience, as compared to the non-Mi.III cells, their unique raft organization is expected to have functional implications.

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.

Calculation of transition dipole moment in fluorescent proteins—towards efficient energy transfer

Förster Resonance Energy Transfer (FRET) between fluorescent proteins (FPs) is widely used to construct fluorescent sensor proteins, to study intracellular protein-protein interactions and to monitor conformational changes in multidomain proteins. Although FRET depends strongly on the orientation of the transition dipole moments (TDMs) of the donor and acceptor fluorophores, this orientation dependence is currently not taken into account in FRET sensor design. Similarly, studies that use FRET to derive structural constrains typically assume a κ(2) of 2/3 or use the TDM of green fluorescent protein, as this is the only FP for which the TDM has been determined experimentally. Here we used time-dependent density functional theory (TD-DFT) methods to calculate the TDM for a comprehensive list of commonly used fluorescent proteins. The method was validated against higher levels of calculation. Validation with model compounds and the experimentally determined TDM of GFP shows that the TDM is mostly determined by the structure of the π-conjugated fluorophore and is insensitive to non-conjugated side chains or the protein surrounding. Our calculations not only provide TDM for most of the currently used FPs, but also suggest an empirical rule that can be used to obtain the TDMs for newly developed fluorescent proteins in the future.

Mechanism of Activation in NMDA Receptors

The N-methyl D-aspartate receptors belong to the family of ionotropic glutamate receptors. They are obligate heteromeric receptors with two glycine binding subunits (GluN1) and two glutamate binding (GluN2A-D) subunits arranged as a dimer of dimers. We have used luminescence resonance energy transfer measurements to determine the specific arrangement of the GluN1 and GluN2 subunits within the tetramer. Based on this arrangement we have designed sites in the agonist binding domain that can probe changes in distances within the subunit and not across the subunits. By tagging these sites with donor and acceptor fluorophores we have investigated the conformational changes in the agonist binding domain of the GluN1 and GluN2A subunits using luminescence resonance energy transfer. The LRET based distances indicate a cleft closure conformational change at the GluN1 subunit upon binding agonists, however, no significant changes in the cleft closure are observed between partial and full agonists. Single molecule FRET investigations are being performed to determine if the partial agonist bound state exhibits more flexibility relative to the full agonist bound state, accounting for the lower activation. The LRET-based distances for the glutamate binding GluN2A subunit, on the other hand, shows a graded cleft closure upon binding agonists with varying efficacy, with the extent of cleft closure being proportional to the extent of activation. This graded cleft closure indicates that the mechanism of activation in this subunit is similar to that of the glutamate binding α-amino-5-methyl-3-hydroxy-4-isoxazole propionate and kainate subtypes of the ionotropic glutamate receptors.

Intermolecular approach to metal ion indicators based on polymer phase transitions coupled to fluorescence resonance energy transfer

An approach to ratiometric fluorescence detection of quenching metal ions was devised by copolymerizing N-isopropylacrylamide with small percentages of bipyridine and amine monomers. The copolymer was divided into two portions. The amine group on one portion was functionalized with AlexaFluor555 (donor fluorophore) and the other with AlexaFluor647 (acceptor fluorophore). The indicator consists of a mixture of these two portions. Aggregation above the lower critical solution temperature (LCST) of this copolymer brings about a large increase in fluorescence resonance energy transfer (FRET). Addition of Cu(II) and other complexing metal ions to the aggregated copolymer introduces charge onto the backbone, causing the copolymer to redissolve with a resulting decrease in FRET. The ratio of acceptor to donor fluorescence varies with Cu(II) concentration. A plot of intensity ratio vs. pCu is sigmoidal with a log K(f) of 6.1 for the Cu(II)-bipyridine complex. The data are consistent with the formation of a 1 : 1 complex. The copolymer responds to higher concentrations of other transition metal ions. The selectivity for Cu(II) is consistent with the literature values for 1 : 1 formation constants for bipyridine with metal ions.

Fiber laser based two‐photon fret measurement of calmodulin and mcherry‐E0GFP proteins

The speed and accuracy of Förster resonance energy transfer (FRET) measurements can be improved by rapidly alternating excitation wavelengths between the donor and acceptor fluorophore. We demonstrate FRET efficiency measurements based on a fiber laser and photonic crystal fiber as the source for two-photon excitation (TPE). This system offers the potential for rapid wavelength switching with the benefits of axial optical sectioning and improved penetration depth provided by TPE. Correction of FRET signals for cross excitation and cross emission was achieved by switching the excitation wavelength with an electrically controlled modulator. Measurement speed was primarily limited by integration times required to measure fluorescence. Using this system, we measured the FRET efficiency of calmodulin labeled with Alexa Fluor 488 and Texas Red dyes. In addition, we measured two-photon induced FRET in an E(0)GFP-mCherry protein construct. Results from one-photon and two-photon excitation are compared to validate the rapid wavelength switched two-photon measurements.

Quantification of protein interaction in living cells by two‐photon spectral imaging with fluorescent protein fluorescence resonance energy transfer pair devoid of acceptor bleed‐through

Fluorescence resonance energy transfer (FRET) between fluorescent proteins (FPs) is a powerful method to visualize and quantify protein-protein interaction in living cells. Unfortunately, the emission bleed-through of FPs limits the usage of this complex technique. To circumvent undesirable excitation of the acceptor fluorophore, using two-photon excitation, we searched for FRET pairs that show selective excitation of the donor but not of the acceptor fluorescent molecule. We found this property in the fluorescent cyan fluorescent protein (CFP)/yellow fluorescent protein (YFP) and YFP/mCherry FRET pairs and performed two-photon excited FRET spectral imaging to quantify protein interactions on the later pair that shows better spectral discrimination. Applying non-negative matrix factorization to unmix two-photon excited spectral imaging data, we were able to eliminate the donor bleed-through as well as the autofluorescence. As a result, we achieved FRET quantification by means of a single spectral acquisition, making the FRET approach not only easy and straightforward but also less prone to calculation artifacts. As an application of our approach, the intermolecular interaction of amyloid precursor protein and the adaptor protein Fe65 associated with Alzheimer's disease was quantified. We believe that the FRET approach using two-photon and fluorescent YFP/mCherry pair is a promising method to monitor protein interaction in living cells.

FRET Detection of Calmodulin Binding to the Cardiac RyR2 Calcium Release Channel

Calmodulin (CaM) binding to the type 2 ryanodine receptor (RyR2) regulates Ca release from the cardiac sarcoplasmic reticulum (SR). However, the structural basis of CaM regulation of the RyR2 is poorly defined, and the presence of other potential CaM binding partners in cardiac myocytes complicates resolution of CaM's regulatory interactions with RyR2. Here, we show that a fluorescence-resonance-energy-transfer (FRET)-based approach can effectively resolve RyR2 CaM binding, both in isolated SR membrane vesicles and in permeabilized ventricular myocytes. A small FRET donor was targeted to the RyR2 cytoplasmic assembly via fluorescent labeling of the FKBP12.6 subunit. Acceptor fluorophore was attached at discrete positions within either the N- or the C-lobe of CaM. FRET between FKBP12.6 and CaM bound to SR vesicles indicated CaM binding at a single high-affinity site within 60 Å of FKBP12.6. Micromolar Ca increased the apparent affinity of CaM binding and slowed CaM dissociation, but did not significantly affect maximal FRET efficiency at saturating CaM. FRET was strongest when the acceptor was attached at either of two positions within CaM's N-lobe versus sites in CaM's C-lobe, providing CaM orientation information. In permeabilized ventricular myocytes, FKBP12.6 and CaM colocalized to Z-lines, and the efficiency of energy transfer to both the N- and C-lobes of CaM was comparable to that observed in SR vesicle experiments. Results also indicate that both the location and orientation of CaM binding on the RyR2 are very similar to the skeletal muscle RyR1 isoform. Specific binding of CaM to functional RyR2 channels in the cardiac myocyte environment can be monitored using FKBP biosensors and FRET.

Förster Resonance Energy Transfer beyond 10 nm: Exploiting the Triplet State Kinetics of Organic Fluorophores

Inter- or intramolecular distances of biomolecules can be studied by Förster resonance energy transfer (FRET). For most FRET methods, the observable range of distances is limited to 1-10 nm, and the labeling efficiency has to be controlled carefully to obtain accurate distance determinations, especially for intensity-based methods. In this study, we exploit the triplet state of the acceptor fluorophore as a FRET readout using fluorescence correlation spectroscopy and transient state monitoring. The influence of donor fluorescence leaking into the acceptor channel is minimized by a novel suppression algorithm for spectral bleed-through, thereby tolerating a high excess (up to 100-fold) of donor-only labeled samples. The suppression algorithm and the high sensitivity of the triplet state to small changes in the fluorophore excitation rate make it possible to extend the observable range of FRET efficiencies by up to 50% in the presence of large donor-only populations. Given this increased range of FRET efficiencies, its compatibility with organic fluorophores, and the low requirements on the labeling efficiency and instrumentation, we foresee that this approach will be attractive for in vitro and in vivo FRET-based spectroscopy and imaging.

A single copy of SecYEG is sufficient for preprotein translocation

The heterotrimeric SecYEG complex comprises a protein-conducting channel in the bacterial cytoplasmic membrane. SecYEG functions together with the motor protein SecA in preprotein translocation. Here, we have addressed the functional oligomeric state of SecYEG when actively engaged in preprotein translocation. We reconstituted functional SecYEG complexes labelled with fluorescent markers into giant unilamellar vesicles at a natively low density. Förster's resonance energy transfer and fluorescence (cross-) correlation spectroscopy with single-molecule sensitivity allowed for independent observations of the SecYEG and preprotein dynamics, as well as complex formation. In the presence of ATP and SecA up to 80% of the SecYEG complexes were loaded with a preprotein translocation intermediate. Neither the interaction with SecA nor preprotein translocation resulted in the formation of SecYEG oligomers, whereas such oligomers can be detected when enforced by crosslinking. These data imply that the SecYEG monomer is sufficient to form a functional translocon in the lipid membrane.

Decrease in Luminescence Lifetime Indicating Nonradiative Energy Transfer from Upconverting Phosphors to Fluorescent Acceptors in Aqueous Suspensions

Utilization of energy transfer from anti-Stokes photoluminescent upconverting phosphors (UCPs) to acceptor fluorophores has been successfully described in various homogeneous bioanalytical assays with subnanomolar detection limits. However, only limited evidence has been shown concerning the nature of the energy transfer mechanism involved, especially in aqueous environment. Energy may be transferred in a photon-mediated way (e.g., photon reabsorption) or through strongly distance-dependent dipole–dipole interactions between resonant energy levels (resonance energy transfer). To study the mechanism, we determined the luminescence lifetimes of the UCP donor in aqueous suspension by using the frequency-domain measurement and examined the lifetime changes in systems involving acceptor molecules in close proximity or within undefined distance. A decreased lifetime component in the presence of close-proximity acceptors was confirmed (64–72% reduction), and strong support for the contribution of the nonradiative resonance energy transfer in the process was obtained, although photon reabsorption is always present to some extent.

Single-Molecule Nanopositioning: Structural Transitions of a Helicase-DNA Complex during ATP Hydrolysis

The conformational states of Escherichia coli Rep helicase undergoing ATP hydrolysis while bound to a partial-duplex DNA (pdDNA) were studied using single-molecule FRET. Crystallographic studies showed that Rep bound to single-stranded DNA can exist in open and closed conformations that differ in the orientation of the 2B subdomain. FRET measurements between eight Rep mutants donor-labeled at different residues and pdDNA acceptor-labeled at the junction were conducted at each of the four nucleotide states. The positions of donor-labeled residues, based on crystal structure, and FRET measurements between these donor molecules and the acceptor fluorophore at the DNA junction were used to predict the most likely position for the DNA junction using a triangulation algorithm. These predicted junction positions are compared with the crystal structure to determine whether the open or closed conformation is more consistent with the FRET data. Our data revealed that there are two distinct Rep-pdDNA conformations in the ATPγS and ADP states, an unexpected finding. The primary conformation is similar to that observed in nucleotide-free and ADP.Pi states, and the secondary conformation is a novel conformation where the duplex DNA and 2B subdomain moved as a unit by 13 Å relative to the rest of the protein. The primary conformation found in all nucleotide states is consistent with the closed conformation of the crystal structure however; the secondary conformation is a new conformation that has not been observed before. We discuss the possible implications of this newly observed conformation.

FRET-FCS Detection of Intralobe Dynamics in Calmodulin

Fluorescence correlation spectroscopy (FCS) can be coupled with Förster resonance energy transfer (FRET) to detect intramolecular dynamics of proteins on the microsecond time scale. Here we describe application of FRET-FCS to detect fluctuations within the N-terminal and C-terminal domains of the Ca(2+)-signaling protein calmodulin. Intramolecular fluctuations were resolved by global fitting of the two fluorescence autocorrelation functions (green-green and red-red) together with the two cross-correlation functions (green-red and red-green). To match the Förster radius for FRET to the dimensions of the N-terminal and C-terminal domains, a near-infrared acceptor fluorophore (Atto 740) was coupled with a green-emitting donor (Alexa Fluor 488). Fluctuations were detected in both domains on the time scale of 30 to 40 μs. In the N-terminal domain, the amplitude of the fluctuations was dependent on occupancy of Ca(2+) binding sites. A high amplitude of dynamics in apo-calmodulin (in the absence of Ca(2+)) was nearly abolished at a high Ca(2+) concentration. For the C-terminal domain, the dynamic amplitude changed little with Ca(2+) concentration. The Ca(2+) dependence of dynamics for the N-terminal domain suggests that the fluctuations detected by FCS in the N-terminal domain are coupled to the opening and closing of the EF-hand Ca(2+)-binding loops.

Retroviral Integrases Promote Fraying of Viral DNA Ends

In the initial step of integration, retroviral integrase (IN) introduces precise nicks in the degenerate, short inverted repeats at the ends of linear viral DNA. The scissile phosphodiester bond is located immediately 3' of a highly conserved CA/GT dinucleotide, usually 2 bp from the ends. These nicks create new recessed 3'-OH viral DNA ends that are required for joining to host cell DNA. Previous studies have indicated that unpairing, "fraying," of the viral DNA ends by IN contributes to end recognition or catalysis. Here, we report that end fraying can be detected independently of catalysis with both avian sarcoma virus (ASV) and human immunodeficiency virus type 1 (HIV-1) IN proteins by use of fluorescence resonance energy transfer (FRET). The results were indicative of an IN-induced intramolecular conformational change in the viral DNA ends (cis FRET). Fraying activity is tightly coupled to the DNA binding capabilities of these enzymes, as follows: an inhibitor effective against both IN proteins was shown to block ASV IN DNA binding and end fraying, with similar dose responses; ASV IN substitutions that reduced DNA binding also reduced end fraying activity; and HIV-1 IN DNA binding and end fraying were both undetectable in the absence of a metal cofactor. Consistent with our previous results, end fraying is sequence-independent, suggesting that the DNA terminus per se is a major structural determinant for recognition. We conclude that frayed ends represent a functional intermediate in which DNA termini can be sampled for suitability for endonucleolytic processing.

An Ion-insensitive cAMP Biosensor for Long Term Quantitative Ratiometric Fluorescence Resonance Energy Transfer (FRET) Measurements under Variable Physiological Conditions

Ratiometric measurements with FRET-based biosensors in living cells using a single fluorescence excitation wavelength are often affected by a significant ion sensitivity and the aggregation behavior of the FRET pair. This is an important problem for quantitative approaches. Here we report on the influence of physiological ion concentration changes on quantitative ratiometric measurements by comparing different FRET pairs for a cAMP-detecting biosensor. We exchanged the enhanced CFP/enhanced YFP FRET pair of an established Epac1-based biosensor by the fluorophores mCerulean/mCitrine. In the case of enhanced CFP/enhanced YFP, we showed that changes in proton, and (to a lesser extent) chloride ion concentrations result in incorrect ratiometric FRET signals, which may exceed the dynamic range of the biosensor. Calcium ions have no direct, but an indirect pH-driven effect by mobilizing protons. These ion dependences were greatly eliminated when mCerulean/mCitrine fluorophores were used. For such advanced FRET pairs the biosensor is less sensitive to changes in ion concentration and allows consistent cAMP concentration measurements under different physiological conditions, as occur in metabolically active cells. In addition, we verified that the described FRET pair exchange increased the dynamic range of the FRET efficiency response. The time window for stable experimental conditions was also prolonged by a faster biosensor expression rate in transfected cells and a greatly reduced tendency to aggregate, which reduces cytotoxicity. These properties were verified in functional tests in single cells co-expressing the biosensor and the 5-HT(1A) receptor.