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 F�rster 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.