Genetically encoded calcium indicators for multi-color neural activity imaging and combination with optogenetics

Abstract

Genetically encoded calcium indicators (GECIs) are powerful tools for systems neuroscience. Here we describe red, single-wavelength GECIs, “RCaMPs,” engineered from circular permutation of the thermostable red fluorescent protein mRuby. High-resolution crystal structures of mRuby, the red sensor RCaMP, and the recently published red GECI R-GECO1 give insight into the chromophore environments of the Ca2+-bound state of the sensors and the engineered protein domain interfaces of the different indicators. We characterized the biophysical properties and performance of RCaMP sensors in vitro and in vivo in Caenorhabditis elegans, Drosophila larvae, and larval zebrafish. Further, we demonstrate 2-color calcium imaging both within the same cell (registering mitochondrial and somatic [Ca2+]) and between two populations of cells: neurons and astrocytes. Finally, we perform integrated optogenetics experiments, wherein neural activation via channelrhodopsin-2 (ChR2) or a red-shifted variant, and activity imaging via RCaMP or GCaMP, are conducted simultaneously, with the ChR2/RCaMP pair providing independently addressable spectral channels. Using this paradigm, we measure calcium responses of naturalistic and ChR2-evoked muscle contractions in vivo in crawling C. elegans. We systematically compare the RCaMP sensors to R-GECO1, in terms of action potential-evoked fluorescence increases in neurons, photobleaching, and photoswitching. R-GECO1 displays higher Ca2+ affinity and larger dynamic range than RCaMP, but exhibits significant photoactivation with blue and green light, suggesting that integrated channelrhodopsin-based optogenetics using R-GECO1 may be subject to artifact. Finally, we create and test blue, cyan, and yellow variants engineered from GCaMP by rational design. This engineered set of chromatic variants facilitates new experiments in functional imaging and optogenetics.

Excited-state structural dynamics of a dual-emission calmodulin-green fluorescent protein sensor for calcium ion imaging

“…Recently, the color palette of genetically encoded Ca 2+ sensors for optical imaging (the GECO series) has been expanded to include blue, improved green, red intensiometric, and emission ratiometric sensors (4)(5)(6)(7). The GECO proteins belong to the GCaMP family of Ca 2+ sensors that are chimeras of a circularly permutated (cp)GFP, calmodulin (CaM), and a peptide derived from myosin light chain kinase (M13) (8).…”

Regulation of Cellular Molecular Signaling by Light (Invited Paper)

“…This idea was thus named as optogenetics, and now it has been developed as the most powerful tool to stimulate cells, especially in neuroscience to control neural excitability. dyes such as Fluo-5F [57], or genetically encoded calcium sensors like G-CaMP [58][59][60] for real-time Ca 2+ dynamics detection with a temporal resolution of around 10 ms. Generally, this technique has been developing very fast and contributing greatly to neuroscience [61][62][63], especially to in vivo research [64][65][66]. Recently, it was found that proteins from optogenetics could be activated by two-photon excitation and thus combined with two-photon microscopy system [67,68].…”

Optical Tools to Investigate Cellular Activity in the Intestinal Wall

“…Red shifted variants have also been developed and compared in terms of spectral and kinetic properties with other GECIs. 53 Here again, these proteins are not yet comparable with respect to the signal to noise properties and the ease of use of GCaMP3. For a comprehensive overview of the various probes and techniques to perform intracellular Ca 2+ imaging, we refer to an excellent recent neuron-focused review by Grienberger and Konnerth.…”