Green-to-Red Photoconversion of GCaMP

Abstract

Genetically encoded calcium indicators (GECIs) permit imaging intracellular calcium transients. Among GECIs, the GFP-based GCaMPs are the most widely used because of their high sensitivity and rapid response to changes in intracellular calcium concentrations. Here we report that the fluorescence of GCaMPs—including GCaMP3, GCaMP5 and GCaMP6—can be converted from green to red following exposure to blue-green light (450–500 nm). This photoconversion occurs in both insect and mammalian cells and is enhanced in a low oxygen environment. The red fluorescent GCaMPs retained calcium responsiveness, albeit with reduced sensitivity. We identified several amino acid residues in GCaMP important for photoconversion and generated a GCaMP variant with increased photoconversion efficiency in cell culture. This light-induced spectral shift allows the ready labeling of specific, targeted sets of GCaMP-expressing cells for functional imaging in the red channel. Together, these findings indicate the potential for greater utility of existing GCaMP reagents, including transgenic animals.

The Growing and Glowing Toolbox of Fluorescent and Photoactive Proteins

“…Interestingly, it has recently been reported that GCaMP6 itself can be “highlighted” by photoconversion to a red state that retains Ca 2+ responsiveness [117]. An exciting twist on the utility of photoconvertible Ca 2+ indicators is the recently reported CaMPARI variant [118].…”

Advances and current challenges in calcium signaling

“…An overview about GECIs has been published previously (P erez Koldenkova & Nagai, 2013). More recent developments include GECIs based on dimerization-dependent FP (ddFP) exchange, single-FP GECIs normalized by fusion to a reference FP (Waadt et al, 2017), and photoactivatable and photoconvertible GECIs (Hoi et al, 2013;Ai et al, 2015;Berlin et al, 2015;Ding et al, 2015).…”

Genetically encoded indicators of neuronal activity

“…GR-GECO permanently converts from green to red fluorescence upon 400-nm illumination while maintaining >500% calcium responses in both forms 109 . A recent study revealed that many GCaMPs are inherently photoconvertible to red emission by 400-nm light, with the red species retaining up to 41% of the responsiveness of the original green form 110 . These findings add to earlier observations of green-to-red photoconversion in GFP derivatives 111 , although the photoconversion may be less efficient than in fluorescent proteins evolved for photoconvertibility.…”