title: Hyperspectral imaging of FRET-based cGMP probes.
abstract: In recent years a variety of fluorescent probes for measurement of cGMP signals have been developed (Nikolaev et al., Nat. Methods 3:23-25, 2006; Honda et al., Proc Natl Acad Sci USA 98:2437-42, 2001; Nausch et al., Proc Natl Acad Sci USA 105:365-70, 2008). The probes are comprised of known cGMP binding sites-e.g., from phosphodiesterase type 5 (PDE5) or protein kinase G (PKG)-attached to fluorescent proteins. Binding of cGMP triggers conformational changes that alter the emitted fluorescence. In the case of Förster resonance energy transfer (FRET)-based probes, binding of cGMP alters the distance between the donor and acceptor fluorophores and thus alters FRET. However, FRET-based probes inherently have low signal-to-noise ratios, limiting the utility of these probes. Here we describe the use of hyperspectral imaging and analysis approaches to increase the signal-to-noise ratio of FRET-based cGMP measurements. These approaches are appropriate for monitoring changes in cGMP signals either in cell populations using a spectrofluorimeter or in single cells using spectral microscope systems with appropriate spectral filtering capabilities.
authors.affiliation: Department of Pharmacology, University of South Alabama, Mobile, USA.
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Rich
authors.forename: Andrea L
authors.initials: AL
authors.lastname: Britain
authors.forename: Tiffany
authors.initials: T
authors.lastname: Stedman
authors.forename: Silas J
authors.initials: SJ
authors.lastname: Leavesley
mesh_terms: D006152:Cyclic GMP / Q000032:analysis / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000032:analysis / Q000737:chemistry*; D008856:Microscopy, Fluorescence / Q000379:methods
chemical_list: D005456:Fluorescent Dyes; D006152:Cyclic GMP