Abstract
Fluorescent protein-based indicators are invaluable tools for functional imaging of living cells and organisms. Genetically encoded calcium indicators (GECIs) such as derivatives of yellow cameleons (YCs) and GCaMPs/pericams (Miyawaki et al., 1997; Nakai et al., 2001; Nagai et al., 2001) are a highly advanced class of indicators. Continued efforts for improvement of the performance of GECIs have resulted in brighter indicators with better photo-stability and expanded dynamic range, thus improving the sensitivity of detection. Fine-tuning of other properties, including Ca2+ affinity and Hill constant, have also contributed to increase the detectability of Ca2+ dynamics. Emerging optogenetic technology has forced the spectrally compatible GECI color variants. In this opinion, we highlight the recent development of GECIs including photo-switchable Ca2+ indicators and bioluminescence-based Ca2+ indicator, mainly invented in our group, focusing especially on the parameters determining their performance in order to provide a guideline for the selection of appropriate GECI for a given experiment.
Highlights
Fluorescent protein-based indicators are invaluable tools for functional imaging of living cells and organisms
Compatibility of Genetically encoded calcium indicators (GECIs) color variants with optogenetic control was demonstrated by using CA3 pyramidal neuron co-expressing ChR2 and RCaMP1.07, which was in turn developed by the site-directed mutagenesis of R-GECO1 (Ohkura et al, 2012)
BRAC is the cameleon like fluorescence resonance energy transfer (FRET)-based indicator harboring CaMM13 moiety fused with an improved luciferase (RLuc8) derived from Renilla reniformis, which acts as a donor, and Venus, which acts as an acceptor
Summary
Fluorescent protein-based indicators are invaluable tools for functional imaging of living cells and organisms. AFFINITY VARIANT After the first reports regarding design concept of YCs and GCaMPs/pericams (Miyawaki et al, 1997; Nagai et al, 2001; Nakai et al, 2001), their properties have been modified in term of dynamic range of signal change, pH sensitivity and color hue, and so on. These affinity variants of YCs called YC-Nano showed increased sensitivity and could detect subtle changes in [Ca2+] in pyramidal neurons (Horikawa et al, 2010; Yamada et al, 2011) (Table 1), becoming an ideal toolbox to efficiently monitor the novel Ca2+ dynamics in cases where the concentration range of Ca2+ is poorly described (Table 1).
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