Red Photoactivatable Genetic Optical-Indicators

Emerging genetically-encoded Ca²⁺-indicators (GECIs) are intensiometric reporters that increase in fluorescence when bound to Ca²⁺; highly suited for studying calcium-signaling in many cell types, notably neurons. Today, major efforts are devoted toward optimizing red-emitting [red fluorescent protein (RFP)-based] GECIs (R-GECI), as these provide several advantages over GFP-based reporters, for instance, increased imaging depth, reduced photodamage by longer imaging wavelengths and, in principle, are better suited for use with prevalent blue-absorbing optogenetic tools (e.g., channelrhodopsin). However, excessive fluorescence from intersecting neighboring cells in very dense tissues, notably the brain, hinders the ability to collect signals from single cells and their processes. This challenge can be addressed by photoactivatable (PA) fluorescent proteins that can be rendered fluorescent on demand by user-defined targeted light. This allows activation and, thereby, collection of fluorescent signals exclusively from desired cells and their processes, while leaving all neighboring cells in the dark (i.e., non-fluorescent). Nevertheless, there are no PA R-GECIs. Here, we sought to develop PA-R-GECIs. To do so, we initially explored a recently discovered phenomenon of Ca²⁺-independent increases in fluorescence (i.e., artifacts) in an emerging R-GECI, which has led us to rationally engineer several functional PA-R-GECIs. We also take advantage of our findings to quickly engineer a novel PA-RFP, namely, PA-mRuby3.