Improved Orange and Red Ca²⁺ Indicators and Photophysical Considerations for Optogenetic Applications

Abstract

We have used protein engineering to expand the palette of genetically encoded calcium ion (Ca(2+)) indicators to include orange and improved red fluorescent variants, and validated the latter for combined use with optogenetic activation by channelrhodopsin-2 (ChR2). These indicators feature intensiometric signal changes that are 1.7- to 9.7-fold improved relatively to the progenitor Ca(2+) indicator, R-GECO1. In the course of this work, we discovered a photoactivation phenomenon in red fluorescent Ca(2+) indicators that, if not appreciated and accounted for, can cause false-positive artifacts in Ca(2+) imaging traces during optogenetic activation with ChR2. We demonstrate, in both a beta cell line and slice culture of developing mouse neocortex, that these artifacts can be avoided by using an appropriately low intensity of blue light for ChR2 activation.

Red fluorescent proteins (RFPs) and RFP-based biosensors for neuronal imaging applications

“…R-GECO1 was further optimized and engineered into spectrally diversified and low-affinity variants, including an improved R-GECO1.2, a blue-shifted O-GECO, a red-shifted CAR-GECO, 102 a highlightable GR-GECO, 103 an LSS REX-GECO 104 and low-affinity red-GECO variants. 105 RCaMP, a similar single RFP-based Ca 2þ biosensor, was engineered from the cpmRuby template.…”

“…109 CAR-GECO1, a red-shifted variant based on R-GECO1, was used for optogenetic activation and Ca 2þ imaging concurrently in combination with channelrhodopsin-2(T159C)-EGFP in mouse neocortical slice culture. 102 RCaMP, along with green glutamate sensor, was used for imaging synaptic input and output in Caenorhabditis elegans neurons. 93 The LSS REX-GECO1 was used in the eye and optic tectum of albino Xenopus laevis tadpoles for two-photon fluorescence imaging of Ca 2þ dynamics in vivo.…”

“…102 In comparison, this photoactivation effect was not observed from mRuby-based RCaMP series indicators. Therefore, RCaMP should be a better-suited Ca 2þ indicator for use with ChR2 or other violet/blue light activatable optogenetic tools.…”

Genetically Encoded Ca 2+ Indicators in Cardiac Myocytes

“…The indicators mentioned and the references referring to the indicators are listed in Figure 2, except for DXcpv 94 and the GECOs (genetically encoded Ca 2+ indicators for optical imaging). [125][126][127] GCaMPX refers to the GCaMP series of recently introduced and tested GECIs, ranging from GCaMP3 63 (this article) to GCaMP6f.…”

Genetically encoded molecular probes to visualize and perturb signaling dynamics in living biological systems

“…), which makes it difficult to monitor Ca 2+ without also activating the channel (Akerboom et al, 2013). Thus, numerous protein engineering efforts are underway to produce spectrally shifted variants of both channelrhodopsins (Prigge et al, 2012) and GECIs (Akerboom et al, 2013;Ohkura et al, 2012;Wu et al, 2013) in order to facilitate their simultaneous use. For example, a recently published study describes the construction of multiple color variants of GCaMP, including the red-shifted RCaMP (Akerboom et al, 2013).…”