Neuronal communication relies on neurotransmitter release, which occurs through the regulated fusion of neurotransmitter-containing synaptic vesicles with the plasma membrane1. This process called exocytosis2,3,4,5,6 is highly dynamic and can be up- or down-regulated according to fluctuations in the rate of stimulation7. The proteins involved in these processes are subject to Brownian motion, and therefore display nanoscopic organization capable of sustaining a range of binding actions that underpin these physiological functions. Plasma membrane proteins are highly dynamic, allowing lateral trapping of molecules into nanoclusters on the plasma membrane7,8,9,10,11,12. As such, investigating the mobility of these proteins helps to elucidate their mode of action8. Synaptic proteins such as the soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs), for example Syntaxin-1A, are now known to exhibit lateral fast mobility, as well as lateral trapping within nanoclusters that may serve as molecular depots and sites for vesicle fusion9,13,14,15.

The recent development of photoconvertible fluorescent proteins, such as monomeric (m)Eos16,17 and Kaede18, has allowed the nanoscopic resolution of neuronal plasma membrane proteins through the use of approaches such as photo-activated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM)14,15,19. These developments have enabled investigations into the mobility and nanoclustering of biological proteins in cultured live cells and neurons. More recently, studies have been carried out to elucidate (at similar high resolutions) the dynamics and organization of these membrane proteins in the neurons of live intact organisms such Mus musculus, Caenorhabditis elegans, and Drosophila melanogaster14,20,21,22.

Investigating the dynamics and organization of a protein in vivo requires not only use of the recently developed super-resolution imaging tools (such as PALM, STORM, and photoconvertible fluorophores), but also the ability to overcome hindrances such as accessibility of the protein and the penetration depth of the imaging laser. Imaging intracellular proteins in an intact animal is inherently challenging as is imaging proteins on structures embedded deep within living tissues of the intact animal, such as the hippocampus of an intact mouse. Hence, proteins on or close to the surface of the tissue are more easily imaged. Another difficulty with imaging in vivo is in ensuring reproducibility across animals. As the anatomy might differ from one sample to the other, selecting a structure with ease of replication in different intact animal samples could also prove challenging. The use of stereotypical structures, such as synapses, help with overcoming some of these difficulties.

Recent studies have imaged protein mobility in animals with an optically transparent epidermis such as Caenorhabditis elegans22, while other investigations have imaged protein organization on the surface of a tissue/organ, for example actin imaging in cortical neurons through the skull of a live mouse21. In some cases, anesthetics have been used to keep the animal immobile; however, specific anesthetics may adversely affect the protein of interest. Alternative means to keep animals immobile during in vivo imaging should therefore be explored to minimize error.

Another caveat to super-resolution imaging in vivo is that the lasers used to illuminate the proteins of interest could adversely affect the surrounding tissue, and hence keeping the excitation intensity as low as possible is recommended. Illumination of the surrounding tissue by the laser also tends to increase the background signal. The use of low excitation intensity helps reduce the background, the caveat being it could also lead to a decrease in fluorescent protein photon yield. Background subtraction during analysis could be used as an alternative means to reduce the background signal prior to image analysis.

Drosophila presents an ideal organism for in vivo imaging as it provides well-established genetic tools for the investigation of specific protein function. The upstream activating sequence (UAS)-Gal4 system enables temporal and spatial expression of proteins and can therefore be used to manipulate neurotransmission23, such that thermo-genetic stimulation using Drosophila transient receptor potential sub-family A1 (dTRPA1)24 or opto-genetic stimulation using far-red-shifted CsChrimson25 can be combined with imaging14. We have overcome many of the complications of performing in vivo single-molecule imaging in this system and now describe how single-particle tracking can be carried out in Drosophila melanogaster third instar larvae.