Mitochondria are highly dynamic organelles and their correct distribution is crucial to support many cellular functions. The remarkably long processes of neurons mean that these cells are particularly dependent on mechanisms for long-range cytoskeletal transport of mitochondria. Due to their peculiar architecture, and the stereotypical directionality of the transport in the axons, neurons are a particularly good model to understand the logic of intracellular trafficking.
The fundamental importance of axonal transport for achieving maturation and maintaining the functionality of neurons is well documented  and many models have been developed to visualise this process. High throughput, ease of gene manipulation and accessibility to pharmacological treatments make cultured neuronal cells, for example primary neurons or neurons derived from neuroblastoma and stem cells, arguably the mainstay of neuronal trafficking studies and have significantly contributed towards the mechanistic understanding of the transport process [2–13]. Ex vivo models, where a whole tissue is dissected and maintained in culture or imaged directly after dissection, are valid alternatives [14–20] with the advantage of potentially preserving a near-native environment while affording accessibility for pharmacological and electrophysiological studies.
In vivo animal models to study the transport process in neurons are now also widely used [6, 21–29]. A clear advantage of an in vivo system is the possibility of studying the transport process while retaining the full complexity of an organismal setting, which may be preferable when studying axonal transport during ageing and in animal models of age-dependent neurodegenerative disorders . However, because the neurons of interest are often buried deep into the tissue that is being imaged, this advantage tends to be offset by the need of complex surgery required to visualize the neurons of interests. This is the case, for example, for the implantation of cranial windows for the observation of thalamo-cortical projections in mice  or for the exposure of the mouse sciatic nerve for trafficking studies . In addition, the resolution at which dynamic subcellular components are resolved during in vivo imaging is often modest compared to the quality that can be obtained from in vitro cultured cells. Despite the current rapid development of super-resolution techniques, only few in vitro studies have focused on long-range intracellular transport captured by live-cell super-resolution imaging [33, 34]. While challenging, achieving super-resolution of trafficked organelles in vivo would be desirable, and a tangible step forward, to better understand the intricacies of intracellular trafficking in this context.
Performing gene manipulations to interfere with the transport or function of specific cargoes might not be straightforward in vivo, especially in mammalian systems. Knockdown or knockout studies are time-consuming; acute modulation of transport, for instance by the addition of drugs, also presents challenges in an in vivo setting, limiting the possibility of gaining in-depth mechanistic insight of this process. This is problematic when studying mitochondrial dynamics which often relies on pharmacological manipulation to interfere with different stages of the oxidative phosphorylation.
We developed a system for detailed imaging of organelle transport in adult Drosophila in vivo, which exploits the accessibility of the wing neurons for microscopic observation and does not require surgical procedures . In this chapter, we describe methods to image mitochondrial transport in different neuronal population of the wing using anaesthetized adult animals. We have previously showed the suitability of this tissue for structured illumination microscopy (SIM) of neuronal membranes and nuclear markers . With the aim of improving the resolution for in vivo imaging of mitochondrial trafficking, here we describe the application of Super-Resolution Radial Fluctuation (SRRF) microscopy [36, 37] to live imaging in the wing nerve of adult Drosophila. Finally, we report the generation of new transgenic flies encoding the photosensitisers KillerRed and SuperNova targeted to the mitochondria and describe photostimulation protocols to induce mitochondrial damage rapidly, coupled to the imaging of the redox state and motility of the organelles. We believe this will expand the utility of the wing nerve system and provide additional tools for the study of mitochondrial function and transport in vivo.