Imaging the Drosophila neuromuscular junction (NMJ): basic optical principles and equipment

Electrophysiological studies of synaptic function cannot directly reveal the internal workings of the nerve terminal and do not robustly report release of neuropeptides and neurotrophins. These limitations can now be overcome with the presynaptic expression of green fluorescent protein (GFP) indicators of vesicle motion, release, and signaling. This article describes how to image single wavelength and ratiometric fluorescence resonance energy transfer (FRET)-based GFP indicators with fluorescence microscopy in living synaptic boutons of the Drosophila neuromuscular junction (NMJ). The basic optical principles and equipment required for epifluorescence microscopy are described in detail.