The versatile electron microscope: an ultrastructural overview of autophagy

Abstract

Both light microscopy (LM) and electron microscopy (EM) are able to reveal important information about the formation and function of various autophagic compartments. In this article we will outline the various techniques that are emerging in EM, focusing on analyzing three-dimensional morphology, collectively known as volume electron microscopy (volume EM), as well as on methods that can be used to localize proteins and antigenic epitopes. Large cell volumes can now be visualized at the EM level by using one of the two complementary imaging techniques, namely Serial Block-face Scanning Electron Microscopy (SB-SEM) or Focused Ion Beam Scanning Electron Microscopy (FIB-SEM). These two block-face imaging methods reveal ultrastructural information from all membrane-bound organelles such as autophagic compartments to be visualized in a three-dimensional space, in association with their surrounding organelles. Another method which falls into the volume EM category is dual-axis electron tomography (ET). This method is more suited to reconstructing smaller volumes from areas of interest that require nano-structural detail to be confirmed such as membrane contact sites (MCSs) between autophagic compartments and various organelles. Further to this, to complement the morphological identification of autophagic compartments, immunolabeling can be carried out at the EM level to confirm the nature of various autophagic compartments depending on the localization of various antigens at a sub-cellular level. To determine this, various immunolabeling techniques can be carried out, namely the pre-embedding or the post-embedding immunolabeling methods. Examples of both of these methods will be described in this chapter. Correlative light-electron microscopy (CLEM) can be used to visualize the same autophagic organelles under the LM, followed by high-resolution imaging under the EM. Finally, cryofixation has revolutionized the EM field by allowing rapid immobilization of cells and tissue in the near native state, so samples are no longer prone to artefacts induced by chemical fixation. Collectively, this chapter will discuss the aforementioned capabilities of the EM in more detail, with a particular focus on autophagy, namely the impact of EM in the study of the morphology and biogenesis of the phagophore/isolation membrane (referred to as the phagophore hereafter).

Keywords: Autophagy; Correlative light-electron microscopy; Cryofixation; Electron tomography; ImmunoEM; Volume EM.