# Advances in Engineering and Application of Optogenetic Indicators for Neuroscience
## 2 Calcium Indicators
NTnC is characterized by high brightness and pH stability, inherited from mNeonGreen, but low dynamic range (Table 1). Utilizing yellow fluorescent protein (YFP) as a sensing moiety helped to increase the dynamic range but at the expense of brightness and pH stability [39; 41]. While NTnC and YTnC are suitable for reporting neuronal activity at single cell resolution in behaving mice under both one- and two-photon microscopy, they exhibit lower overall performance than the GCaMP6 variants. However, we found the NTnC-like sensors significantly outperform GCaMP6s when targeted to organelles such as the mitochondria and endoplasmic reticulum [39]. Calcium homeostasis in mitochondria and the endoplasmic reticulum plays crucial roles in cell physiology in health and disease [49; 50; 51; 52]. However, currently available GECIs are not optimized for calcium visualization in these organelles. Therefore, the NTnC-like sensors represent promising templates for adjusting calcium affinity to match the range of calcium concentration in mitochondria and the endoplasmic reticulum. For example, truncation of the EF4-hand of the calcium-binding domain in YTnC2 indicator, which is capable of binding only one calcium ion, decreased affinity of the indicator to calcium ions by \(\sim\)10-fold. The generated sensor, dYTnC2, showed optimal biochemical characteristics for measuring large calcium transients in the endoplasmic reticulum (personal communication, Table 1). There is a great need for GECIs with red-shifted fluorescence, but engineering red-shifted variants has proven particularly challenging. Inserting calcium binding domains into red fluorescent proteins easily disrupts folding and chromophore maturation [43; 53]. Furthermore, red fluorescent proteins are known to exhibit inferior photophysical properties compared to green fluorescent proteins such as photoactivation under blue light illumination, leading to imaging artifacts [54; 55; 56]. Despite great efforts in the development of red-shifted GECIs [54; 56; 57; 58; 34], only the latest generation of red GECIs is suitable for imaging calcium dynamics in living organisms [9; 43]. However, further engineering and enhancement of the current red GECIs is certainly required before they can reach level of the best performing green sensors (Table 1). Brightness and dynamic range are perhaps the main properties requiring improvement. Another inherent drawback of red GECIs is their tendency to form bright fluorescence puncta in cell bodies, especially during in vivo expression. The puncta were shown to co-localize with a lysosomal marker LAMP-1. The aggregated proteins in the puncta do not show calcium sensitivity and contribute to background fluorescence, thus reducing the overall quality of the dynamic signal recordings. To reduce puncta formation, [PERSON] et al. exploited the novel red fluorescent protein FusionRed, known to exhibit good localization in neurons [43]. Replacing the fluorescent moiety in R-GECO with a circularly permutated FusionRed generated the K-GECO indicator, which indeed demonstrated no puncta-like localization in cultured neurons [43]. However, puncta formation was not completely resolved in vivo in mice. K-GECO could report single action potentials in neurons in zebrafish larva as well as detect calcium dynamics in the visual cortex of awake mice. However, K-GECO1 did not provide the same level of in vivo sensitivity as the highly optimized jRGECO1a (Table 1). Red-shifted GECIs enable facile application in conjugation with channelrhodopsins for all-optical interrogation of neuronal circuits [9; 42; 56; 43]. Due to the wide action spectra of the majority of channelrhodopsins, compared with the full width at half maximum of GFP-like fluorescent proteins ranging from 30 to 70 nm, spectrally multiplexed optogenetic control with red GECIs excited at \(\sim\)560 nm still remains challenging.