Monitoring Autophagy by Optical Microscopy.

Thanks to the advances in optical microscope technology and our knowledge of autophagic biomarkers, single-molecule events of autophagy are now accessible to human eyes. Different proteins are involved hierarchically in the biogenesis and maturation of autophagosomes. Detecting these autophagy-related proteins either by immunostaining or fluorescent protein labelling makes the dynamic autophagic process visible. However, low antibody specificity and weak endogenous expression of autophagy-related proteins in certain tissues limit the applicability of immunostaining in autophagy detection. To cope with this, live-cell imaging combined with various fluorescent probes has been developed and employed in monitoring autophagy. As the most widely used autophagic biomarker, LC3 can be used to visualize autophagosomes, and fluorescent probes targeting LC3, i.e., RFP/mCherry-GFP-LC3, and GFP-LC3-RFP-LC3ΔG, can examine autophagy flux dynamically and quantitatively. In addition, the application of novel fluorophores such as Keima helps to detect the temporal and spatial characteristics of autophagy. Furthermore, selective autophagy can be clarified by labelling corresponding substrates and autophagosomes or lysosomes simultaneously. With the help of two-photon microscopy, the process of autophagy in live animals has been uncovered. Here, we summarize the methods for observing autophagy by optical microscopy and the selection of fluorescent markers.