Correlative Light and Electron Microscopy (CLEM): Bringing Together the Best of Both Worlds to Study Neuronal Autophagy

Autophagy is a key protein degradative pathway for primarily long-lived proteins and damaged organelles, contributing to cellular proteostasis. Neurons rely on a particularly effective autophagy machinery, due to their high metabolic activity, ATP demand, and terminally differentiated nature. Autophagy was originally identified with electron microscopy (EM), and it has remained one of the most important tools to study autophagy and its pathway intermediates. However, distinguishing between specific autophagic structures, such as autophagosomes, autolysosomes, and lysosomes, remains challenging, requires substantial expertise, and is often complicated by the large degree of membrane and cargo complexity, underpinned by the heterogeneous electron density within these structures. To accurately identify specific components of the autophagic machinery, correlative light and electron microscopy (CLEM) has emerged as a powerful tool. To discern between autophagic components, specific fluorescent tags can be assigned to macromolecules and structures of interest, and correlated to the ultrastructural detail and subcellular context provided by the electron micrograph. In doing so, a large degree of subjectivity is eliminated, leading to a more accurate characterization and depiction of the total cellular autophagic response and its biological role. This chapter will outline the advantage of CLEM for the study of neuronal autophagy and provide a methodological workflow for both two-dimensional and three-dimensional CLEM approaches.