In a recent study published in in ACS Chemical Neuroscience, Campbell, Li, Nagai and coworkers report the development of a new series of orange and red genetically encoded Ca2+ indicators with improved sensitivity. To expand the color palette of genetically encoded Ca2+ indicators, semi-rational design and directed evolution were used to explore different chromophore structures and to modulate the environment adjacent to the chromophore of a previously reported red Ca2+ indicator, R-GECO1. These efforts lead to the identification of O-GECO (blue shifted), R-GECO1.2 and CAR-GECO1 (red shifted emission) with Ca2+ dependent intensiometric signal changes of 14600%, 3300% and 2700%, respectively (see figure 1 below). The authors go on to describe a troublesome photoactivation phenomenon that was discovered when these new indicators were used in conjunction with ChR2. Specifically, the fluorescence signals of these orange and red Ca2+ sensors exhibit a reversible increase with the intense blue light illumination used for ChR2 activation, even when there is no change in the Ca2+ concentration (see figure 2 below). By carrying out extensive in vitro and tissue-based characterizations, the authors showed that using an appropriate intensity of blue light could minimize this photoactivation problem and allowed these new orange and red Ca2+ indicators to be used in combination with ChR2 in INS-1 cells and mouse neocortical slice culture.
With the development of these new Ca2+ indicators, there is a greater selection than ever before of genetically encoded Ca2+ indicators available for monitoring the activities of neurons and other cell types. However, this work also serves as cautionary tale about how the complex photophysics of genetically encoded fluorophores can give rise to experimental artifacts. One of the take away messages from this work is that workers using multiple optogenetic tools in the same tissue should always perform the key control experiments of expressing each construct alone and subjecting it the experimental illumination conditions used in the experiment. The authors of this study saw a strong dependence on light intensity and wavelength, so it is important to repeat these controls for each new set of experimental conditions used.