Two-Photon Imaging of Population Activity with Genetically Encoded Calcium Indicators in Living Flies

Genetically encoded calcium indicators make it possible to track neural activity on a population-wide level. Here we describe a preparation that enables two-photon imaging of neural activity in an essentially intact fly. We present strategies to minimize motion of the brain, both in preparation technique and in apparatus design. We discuss key variables for reducing the problems of photobleaching and phototoxicity in order to collect high quality imaging data. Finally, we discuss approaches to analyze the large quantities of data that can now be readily acquired using the latest generation of genetically encoded calcium indicators.