# Advances in Engineering and Application of Optogenetic Indicators for Neuroscience
## 2 Calcium Indicators
Even one of the most blue-shifted channelrhodopsins, CheRiff [59], with acceptable in vivo performance [60], generates a sustained inward current under 560 nm illumination at moderate light power of \(\sim\)15 mW/mm\({}^{2}\) that can cause substantial alternations in membrane potentials during extended imaging [61; 62]. Shifting fluorescence of GECIs into the near-infrared range will provide completely crosstalk-free coupling with optogenetic tools. Near-infrared fluorescent proteins derived from phytochromes [63] have shown to be useful for labeling neurons in vivo in mammals [64], zebrafish [65], and flies [66]. Near-infrared light is also beneficial for the noninvasive whole-bodyimaging of small mammals [67; 68] due to reduced autofluorescence, low light scattering, and minimal absorbance at longer wavelengths. Interestingly, the first attempts engineering calcium sensors based on the phytochrome-derived fluorescence proteins were unsuccessful. Obtaining functional circularly permutated variants was impossible due to the phytochrome's topology, with the N- and C-termini far apart from each other, unlike in GFP-like proteins (personal communications). Instead, the first near-infrared GECIs were engineered by inserting the calcium binding domain into phytochrome-derived fluorescent proteins. In this respect, their design appears similar to that of the NTrC-like family (Figure 1b). The GAF-FP [69] and mIFP [66] proteins were used as fluorescent scaffolds producing GAF-CaMP [44] and NIR-GECO1 [45] indicators containing cGMP phosphodiesterase/adenylate cyclase/FhIA transcriptional activator (GAF) and Per-ARNT-Sim-GAF (PAS-GAF) domains from the phytochromes, respectively [63]. While GAF-CaMP has not been yet tested in neurons, NIR-GECO1 was shown to detect single action potentials in cultured neurons and intact mouse brain tissue. NIR-GECO1 is efficiently excited by far-red light in the range from 630 to 680 nm, enabling crosstalk free spectral multiplexing with the majority of channelrhodopsins and other fluorescence sensors. In addition, NIR-GECO1 allowed mesoscale fluorescence imaging of somatosensory cortex through the intact skin and skull of anaesthetized mice, although reliable detection of fluorescent changes required averaging of multiple trials. As a first-generation indicator, NIR-GECO1 falls short of the most extensively optimized fluorescent protein-based GECIs in several critical performance parameters but is an exciting progression in the development of far red-shifted calcium sensors. An alternative method to record neuronal activity of a large number of neurons with single cell resolution is the permanent labeling of neurons, as demonstrated by the CaMPARI calcium sensor [70]. The CaMPARI family of light-inducible calcium sensors is based on EosFP green-to-red photoconvertable fluorescent protein, allowing ex vivo visualization of integrated over time calcium transients (Figure 1). The CaMPARI integrators are irreversibly and efficiently photoconverted from a green fluorescent state to a red state by ultraviolet light (\(\sim\)405 nm) irradiation only in the presence of elevated concentrations of calcium ions. The second generation of this family, CaMPARI2, is characterized by improved dynamic range of photoconversion, brightness, faster calcium dissociation kinetics, decreased photoconversion rate in inactive neurons, and compatibility with paraformaldehyde fixation [40]. In addition, the authors introduced the anti-CaMPARI-red antibody that specifically recognizes the photoconverted red form and thus allows immunohistochemical amplification of calcium signal in fixed cells or tissue. The CaMPARI2 indicator enables acquisitions of snapshots of calcium as a proxy for neuronal activity integrated during 30 seconds in the brain of zebrafish from up to 6000 individual neurons. In this respect CaMPARI integrators are comparable with CaMP and GECO indicators which allow simultaneous two-photon imaging of calcium activity over 10,000 cells in visual cortex of mice with 2.5 Hz rate [71].