Volume electron microscopy in axon regeneration research: insights into mitochondria, endoplasmic reticulum, and...

Analysing organelle structures in tissue samples that preserve the in vivo environment, rather than in isolated cells, can provide valuable information on the mechanisms underlying nerve recovery and repair. However, determining organelle ultrastructure within tissue samples remains challenging with conventional microscopic techniques. To address this limitation, volume electron microscopy, particularly focused ion beam/scanning electron microscopy (FIB/SEM), has provided novel insights into organelle morphology, distribution, and membrane contacts, in three-dimensions and even within intact tissues. This review highlights the application of FIB/SEM for exploring the three-dimensional organization of mitochondria in motor neuron cell bodies and along the axon initial segments (AIS), where simultaneous investigation of intracellular and extracellular environments is difficult using other approaches. These analyses have revealed novel findings regarding mitochondrial distribution under healthy conditions and its dramatic alteration following injury, as well as microglial attachment around the AIS. Furthermore, FIB/SEM has enabled detailed characterization of the complex endoplasmic reticulum (ER) architecture within motor neuron cell bodies. Three-dimensional reconstructions have demonstrated a distinct uneven distribution of the ER in healthy neurons and revealed disruption of this organization following injury. In addition, ER-plasma membrane (ER-PM) contacts have been characterized as sheet-like structures, and quantitative analyses have shown significant increases in ER-PM contacts after injury. The novel findings obtained through FIB/SEM provide new perspectives on the cellular mechanisms underlying neuroregeneration and highlight the value of volume electron microscopy in advancing our understanding of nerve repair processes.