Applications of Fluorescent Probes in Confocal Imaging of GPCRs: From Live to Fixed Cells
G protein-coupled receptors (GPCRs) are pivotal in transmitting signals across cell membranes, making them essential targets in biomedical research and drug development. Confocal fluorescence microscopy imaging has emerged as one of the most versatile tools to visualize the spatial and temporal dynamics of GPCRs. Central to this approach are fluorescent probes, which have evolved to provide high specificity, minimal phototoxicity, and compatibility with both live and fixed cell imaging.
How Fluorescent Probes Enable High-Resolution Confocal Imaging of GPCRs
The structural complexity and dynamic behaviour of GPCRs require imaging methods that offer both spatial precision and real-time monitoring capabilities. Fluorescent probes, ranging from small-molecule ligands to genetically encoded tags, allow for visualizing GPCRs in a way that retains their functional integrity. When combined with confocal microscopy, these probes enable the selective excitation and collection of fluorescence from a specific focal plane, reducing background noise and increasing resolution.
The versatility of confocal platforms, compatible with a broad range of fluorescent probes and labelling strategies, makes them particularly well-suited for GPCR research workflows. Moreover, the ability to acquire three-dimensional image stacks enables the reconstruction of GPCR distribution in complex cellular environments, offering both qualitative and quantitative insights into receptor biology.
Figure 1. Nikon inverted fluorescence microscope. Source: ArthurLau1997, via Wikimedia Commons.
Optimizing Fluorescent Probe Selection for GPCR Imaging
Choosing the right fluorescent probe is crucial for obtaining meaningful results. Probe selection depends on several factors, including the biological context (live or fixed cells), the desired spatial and temporal resolution, and the compatibility with other experimental components. For live-cell imaging, probes must be minimally toxic and resistant to photobleaching. Fluorescent ligands offer the advantage of maintaining receptor activity while allowing for labelling without genetic modification, making them ideal for endogenous GPCR studies.
Recent advances include ligand-directed labelling strategies, which enable the covalent attachment of fluorescent tags to GPCRs through ligand binding. This approach preserves receptor functionality while allowing the fluorescent ligand to be washed out after binding, leaving the receptor permanently labelled. Crucially, the covalent nature of the attachment ensures that the signal remains stable after fixation, making it suitable for high-resolution imaging in both live and fixed cells.
Self-labelling tags such as SNAP-tag and HaloTag involve the genetic fusion of the GPCR to an engineered enzyme that catalyses the covalent attachment of a synthetic fluorescent substrate. They provide flexibility in probe colour and enable dual-labelling strategies for colocalization or interaction studies. They are relatively large, which can interfere with protein function in certain contexts. Nevertheless, SNAP-tags have demonstrated compatibility with GPCR labeling with high yields, since this tag is relatively small, which helps preserve GPCR expression and activity. When combined with fluorescent ligands, these tags are especially useful in Time-Resolved Förster Energy Resonance Transfer (TR-FRET) assays.
Exploring the Applications of Confocal Fluorescence Microscopy in GPCR Research
Confocal fluorescence microscopy has become a cornerstone in the study of GPCRs due to its ability to generate high-resolution, optically sectioned images that reveal receptor trafficking, localization, and signalling dynamics with precision. By selectively capturing fluorescence from a single focal plane while rejecting out-of-focus light, confocal imaging allows for detailed analysis of GPCR distribution at the plasma membrane, within endocytic vesicles, or across subcellular compartments.
In live-cell experiments, confocal microscopy is especially valuable for tracking real-time receptor internalization, monitoring ligand-induced clustering, and evaluating protein–protein interactions using Fluorescence Resonance Energy Transfer (FRET) or colocalization analyses. In fixed cells, it provides the structural context necessary to map receptor localization in defined states, whether under pharmacological stimulation, genetic modification, or disease models.
Selecting the Right Fluorescent Probe for Confocal Imaging Workflows
Fluorescent ligands have been extensively validated as useful tools to visualize GPCRs in native and transfected cells, both in living cells and cells after fixation.
At Celtarys, our GPCR fluorescent ligands have been designed to fill current gaps in the commercially available tools. Their fluorophores are stable, guarantee their affinity, and display a variety of photo-physical properties that meet the market demand for several applications, from competitive binding assays to microscopy.
Figure 2 is an example of how Celtarys ligands, in this case, CELT-327 A2B/A3 receptor fluorescent ligand, are suitable to visualize these Adenosine receptors in native HTC-116 colon cancer cells.
Figure 2. Left panel, confocal microscopy of living cells incubated for 1h at 37ºC with CELT-327. Right panel, the fluorescent signal is displaced when, after incubating with CELT-327, cells are incubated with an excess of the A2B/A3 antagonist, MRS1220. Fluorescence Microscopy validation performed in the ONCOMET laboratory (Health Research Institute of Santiago de Compostela).
We have recently launched CELT-503 and CELT-505, fluorescent GTPγS emitting in the red spectrum (589/616) and green spectrum (503/506) to support binding affinity data with valuable insights into cellular mechanisms, such as compound-induced G protein modulation. Their performance has been validated and proven effective on Arcoscreen’s platform for GPCR functional screening.
References
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