Macroautophagy (which we will call autophagy hereafter) is a critical intracellular bulk degradation system that is active at basal rates in eukaryotic cells. This process is embedded in the homeostasis of nutrient availability and cellular metabolic demands, degrading primarily long-lived proteins and specific organelles.. Autophagy is perturbed in many pathologies, and its manipulation to enhance or inhibit this pathway therapeutically has received considerable attention. Although better probes are being developed for a more precise readout of autophagic activity in vitro and increasingly in vivo, many questions remain. These center in particular around the accurate measurement of autophagic flux and its translation from the in vitro to the in vivo environment as well as its clinical application. In this review, we highlight key aspects that appear to contribute to stumbling blocks on the road toward clinical translation and discuss points of departure for reaching some of the desired goals. We discuss techniques that are well aligned with achieving desirable spatiotemporal resolution to gather data on autophagic flux in a multi-scale fashion, to better apply the existing tools that are based on single-cell analysis and to use them in the living organism. We assess how current techniques may be used for the establishment of autophagic flux standards or reference points and consider strategies for a conceptual approach on titrating autophagy inducers based on their effect on autophagic flux . Finally, we discuss potential solutions for inherent controls for autophagy analysis, so as to better discern systemic and tissue-specific autophagic flux in future clinical applications.Abbreviations: GFP: Green fluorescent protein; J: Flux; MAP1LC3/LC3: Microtubule-associated protein 1 light chain 3; nA: Number of autophagosomes; TEM: Transmission electron microscopy; τ: Transition time.
Keywords: Autophagy; autophagy activity; autophagy flux; fluorescence microscopy; in vivo; lightsheet microscopy; steady state; tissue.