FRET-based fluorescent sensors for cytosolic and mitochondrial Ca2+ imaging in <i>Drosophila melanogaster</i>

Ca2+ imaging in the living tissues of experimental animals currently presents limitations due to motion artefacts, variable expression levels and the low basal fluorescence of available probes. These issues are even more troublesome when performing Ca2+ imaging in contractile tissues and moving organelles. Despite the well-known advantages of ratiometric sensors, their application in experimental animals is still limited. This study addresses these challenges in the fruit fly Drosophila melanogaster, introducing the development and characterization of novel transgenic fly lines expressing advanced FRET-based Cameleon probes for cytosolic and mitochondrial Ca2+ imaging. A series of Ca2+ imaging experiments in selected tissues demonstrates their functionality. These newly developed tools guarantee precise and quantitative measurement of Ca2+ in various cellular contexts, which will contribute to advancing Ca2+ research in Drosophila.