One of the primary goal of neuroscience research is to dissect the role of different cell-types in neuronal circuits. Thanks to the development of optogenetic reporters and control tools in recent years, researchers can now optically control and monitor the activity of genetically defined neuronal populations. Going deeper into the understanding of neuronal circuit requires to follow and control the activity of individual cells within these genetically-defined populations. Imaging the activity of individual neurons in vitro and in vivo can be achieved with classical microscopy techniques but labelling and following the activity of arbitrarily defined subsets of cells in a given population remains hard to implement. Optical highlighters which can be photoactivated or photoconverted are routinely used to label cell subpopulations but have only been used so far for anatomical tracing or cell migration studies. In a recent study published in JACS, Campbell and coworkers introduce a photoconvertible genetically-encoded calcium indicator, providing the possibility to easily monitor the activity of subsets of cells of a given cell type.
This dual-function Ca2+ indicator was made by combining two of the most powerful implementations of fluorescent protein (FP) technology: the “highlighters” that can be converted from non-fluorescent to fluorescent or from one color to another by near-UV illumination, and the Ca2+ indicators. In an effort to create such a hybrid tool, the authors explored three different protein design strategies. The strategy that ultimately proved successful involved the creation of a circularly permuted version of a green-to-red photoconvertible FP and its introduction into a G-CaMP-type single FP-based Ca2+ indicator (Fig 1). Optimization by directed evolution led to the identification of two promising variants, named as GR-GECO1.1 and GR-GECO1.2 respectively, that exhibit excellent photoconversion properties and have an up to 4.6-fold increase in red fluorescence intensity upon binding Ca2+. Characterization and validation in mammalian cells was described. The primary advantage of such a highlightable Ca2+ indicator was demonstrated by highlighting a single cell in a population of primary neurons transfected with GR-GECO1.2, followed by imaging of spontaneous Ca2+ oscillations (Fig 2). As expected, only the highlighted cells were observed in the red emission channel.
With the newly developed highlightable Ca2+ indicators presented in this work, researchers have a ‘universal’ method of selective Ca2+ imaging in single cells, or collections of cells, in a transfected tissue with ubiquitous expression. Similarly, irreversible highlighting will facilitate the ability of researchers to return to, and image the activity of, the same cell in a chronic experiment.
Both GR-GECOs will be soon be available on Addgene.