Förster Resonance Energy Transfer (FRET): A Powerful Tool for Fluorescent Probe-Based Live-Cell Imaging

What Is Förster Resonance Energy Transfer (FRET) and How Does It Work?

Förster Resonance Energy Transfer, or Fluorescence Resonance Energy Transfer (FRET), is a non-radiative, distance-dependent phenomenon between a donor and an acceptor fluorophore. When the donor is excited by a specific wavelength of light, it can transfer energy to the acceptor if the two are close to each other. The acceptor then emits light as a result of this energy transfer. 

By using specific detection equipment, we can precisely measure both the reduction in fluorescence emission from the donor and the corresponding increase in the acceptor, providing a sensitive readout of molecular proximity or interaction.

FRET depends on three parameters: 

1. Spectral overlap between the donor’s emission and the acceptor’s excitation. 
2. The distance between the fluorophores (< 10 nm).
3. Relative orientation of their dipole moments towards each other (highest FRET for parallel dipole orientation).

Figure 1. Mechanism of FRET. Adapted from: Choi JH, Ha T, Shin M, Lee SN, Choi JW. Nanomaterial-Based Fluorescence Resonance Energy Transfer (FRET) and Metal-Enhanced Fluorescence (MEF) to Detect Nucleic Acid in Cancer Diagnosis. Biomedicines. 2021 Jul 31;9(8):928.

These properties make FRET an exceptionally sensitive method to probe molecular interactions under physiological conditions and in real time by live-cell imaging. Because the energy transfer process is radiationless and occurs over nanometer distances, FRET can detect subtle changes in molecular conformation or binding events without perturbing the system. When combined with time-lapse fluorescence microscopy or advanced imaging modalities like confocal, FRET enables the visualization of dynamic signaling events. 

In the field of GPCR research, FRET has revolutionized the ability to study dynamic processes such as receptor activation, ligand binding, G protein interaction, and downstream signaling. Fluorescent ligands are used in two types of fluorescence resonance energy transfer assays: 

• To study the interaction between the receptor and the ligand in the extracellular side of the GPCR.
• To detect oligomeric GPCR complexes by analyzing FRET between two different labeled receptor-bound fluorescent ligands.

Designing Fluorescent Probes for FRET Assays: Factors for Accuracy and Reproducibility

A successful FRET assay starts with the careful design of fluorescent probes, particularly when developing ligands labeled for GPCR studies. One of the most critical considerations is ensuring that the fluorophore conjugation does not interfere with the ligand’s pharmacological properties. The fluorophore must be attached in a way that preserves the binding affinity of the ligand for its target receptor.

In practical terms, the design process typically involves identifying ligand regions that are solvent-exposed or distal from the receptor-interacting core. These are preferred sites for linker and subsequent fluorophore attachment to minimize steric hindrance or disruption of key interaction points. 

Furthermore, the chemical nature of the linker between the fluorophore and the ligand can play a significant role both in keeping affinity and in favouring proximity between donor and acceptor fluorophore in FRET-based GPCR binding assays. Therefore, validating the labeled ligand is essential.

Figure 2 shows an excellent validation example. Navarro et al. published the comparison between reference compounds’ high-affinity data obtained through radioligand binding assays and a dual fluorescent ligand CELT-335 targeting cannabinoid receptors in a homogeneous time-resolved fluorescence assay. This assay is an evolution of FRET that will be further discussed in subsequent sections.

Figure 2. Schematic representation of the high correlation between reference compounds’ affinity data obtained through radioligand binding assays and the Tag-lite® binding assay developed in the reference article.  (a) CB1R binding affinities; (b) CB2R binding affinities. Adapted from: Navarro G, Sotelo E, Raïch I, Loza MI, Brea J, Majellaro M. A Robust and Efficient FRET-Based Assay for Cannabinoid Receptor Ligands Discovery. Molecules. 2023 Dec 15;28(24):8107.  

In addition to preserving ligand function, the selected fluorophore pair must exhibit strong spectral overlap and photostability for effective energy transfer and signal detection. Genetically encoded fluorescent proteins such as CFP/YFP or newer variants like mTurquoise are widely used due to their compatibility with live-cell imaging. Alternatively, small-molecule dyes such as Alexa Fluor, BODIPY, or fluorescein derivatives can be chemically conjugated as acceptors in applications that require more compact probes or involve time-resolved FRET (TR-FRET). As donors, one can use fluorescent proteins or lanthanides like europium and terbium, which are particularly well-suited for TR-FRET due to their long-lived emission, enabling signal detection beyond the initial excitation window.

FRET Microscopy for Live-Cell Imaging: Applications and Advantages

FRET microscopy enables direct observation of molecular interactions inside living cells with nanometer precision. It has become a foundational tool for analyzing GPCR signaling, where ligand-induced changes in receptor conformation or protein–protein interactions can be visualized in real time. This includes dissociation of G protein subunits, recruitment of β-arrestins, or receptor internalization.

One of the key strengths of FRET microscopy is its adaptability. Confocal and wide-field microscopy systems support sensitized emission FRET, while time-resolved methods like fluorescence lifetime imaging microscopy (FLIM) increase robustness against fluorophore concentration variability. These imaging techniques reveal not just if an interaction occurs but when and where in the cell, allowing researchers to study spatially compartmentalized signaling events.

FRET is also increasingly used to evaluate ligand-receptor binding kinetics using fluorescent ligands. For example, using labeled peptide ligands with receptors tagged at the N-terminus provides real-time insights into binding affinity and receptor conformational states.

Challenges and Considerations in FRET-Based Live-Cell Imaging

Despite its strengths, FRET possesses some challenges. Photobleaching and autofluorescence can compromise signal quality. Moreover, the large size of fluorescent proteins (~27 kDa) may interfere with protein folding or localization, particularly in sensitive systems like GPCRs. Newer labeling systems and smaller fluorophores help mitigate these concerns.

A more subtle but critical consideration is the accurate quantification of FRET efficiency. In sensitized emission FRET, rigorous correction for donor bleedthrough and acceptor cross-excitation is necessary. Fluorescence lifetime imaging microscopy (FLIM)-based FRET offers an advantage here, as it directly measures changes in donor lifetime and is less sensitive to fluorophore concentration or excitation intensity.

Expanding the Toolbox: Time-Resolved FRET (TR-FRET)

A particularly powerful evolution of FRET is TR-FRET or homogeneous time-resolved fluorescence (HTRF). This technique uses lanthanide complexes, such as europium or terbium, as donors, which exhibit long-lived luminescence. Their millisecond-range lifetimes allow for a delay in detection, effectively eliminating background fluorescence from the sample and significantly improving the signal-to-noise ratio. 

TR-FRET is less dependent on dipole orientation than conventional FRET, making it a more robust choice for applications where precise fluorophore alignment is difficult to achieve. This has made it especially popular for high-throughput screening (HTS) platforms, where reproducibility and sensitivity are crucial.

Building on the power of FRET and its applications in live-cell imaging, having access to high-quality fluorescent ligands becomes essential for reliable and reproducible results. 

At Celtarys, we bring deep expertise in ligand–receptor pharmacology and advanced fluorescence techniques to support researchers at every step. We create tailored fluorescent ligands with strong affinity and selectivity for a wide range of GPCR targets, ensuring that each ligand is optimized not only for binding performance but also for compatibility with your specific assay conditions.

If you’re looking to enhance your FRET-based studies or accelerate GPCR-targeted drug discovery, we’re here to collaborate.

References

Choi JH, Ha T, Shin M, Lee SN, Choi JW. Nanomaterial-Based Fluorescence Resonance Energy Transfer (FRET) and Metal-Enhanced Fluorescence (MEF) to Detect Nucleic Acid in Cancer Diagnosis. Biomedicines. 2021 Jul 31;9(8):928. doi: 10.3390/biomedicines9080928. 

Lohse MJ, Nuber S, Hoffmann C. Fluorescence/bioluminescence resonance energy transfer techniques to study G-protein-coupled receptor activation and signaling. Pharmacol Rev. 2012 Apr;64(2):299-336. doi: 10.1124/pr.110.004309.

Navarro G, Sotelo E, Raïch I, Loza MI, Brea J, Majellaro M. A Robust and Efficient FRET-Based Assay for Cannabinoid Receptor Ligands Discovery. Molecules. 2023 Dec 15;28(24):8107. doi: 10.3390/molecules28248107.Xu X, Brzostowski JA, Jin T. Monitoring dynamic GPCR signaling events using fluorescence microscopy, FRET imaging, and single-molecule imaging. Methods Mol Biol. 2009;571:371-83. doi: 10.1007/978-1-60761-198-1_25.