Hyperspectral imaging of FRET-based cGMP probes

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.