In vivo imaging of intact Drosophila larvae at sub-cellular resolution.

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal lines have enabled biological processes to be studied in the context of a living, and in some instances even behaving, organism. In this protocol we will describe how to anesthetize intact Drosophila larvae, using the volatile anesthetic desflurane, to follow the development and plasticity of synaptic populations at sub-cellular resolution. While other useful methods to anesthetize Drosophila melanogaster larvae have been previously described, the protocol presented herein demonstrates significant improvements due to the following combined key features: (1) A very high degree of anesthetization; even the heart beat is arrested allowing for lateral resolution of up to 150 nm, (2) a high survival rate of >90% per anesthetization cycle, permitting the recording of more than five time-points over a period of hours to days and (3) a high sensitivity enabling us in 2 instances to study the dynamics of proteins expressed at physiological levels. In detail, we were able to visualize the postsynaptic glutamate receptor subunit GluR-IIA expressed via the endogenous promoter in stable transgenic lines and the exon trap line FasII-GFP. (4) In contrast to other methods the larvae can be imaged not only alive, but also intact (i.e. non-dissected) allowing observation to occur over a number of days. The accompanying video details the function of individual parts of the in vivo imaging chamber, the correct mounting of the larvae, the anesthetization procedure, how to re-identify specific positions within a larva and the safe removal of the larvae from the imaging chamber.