3D Reconstruction and Analysis of Thin Subcellular Neuronal Structures using Focused-Ion Beam Scanning Electron Microscopy Data

Recent advances in scanning electron microscope technologies now permit the rapid three-dimensional (3D) analysis of ultrathin subcellular processes. Here, a methodological pipeline is presented to identify, visualize, and analyze thin neuronal processes, such as those that project into the presynaptic boutons of other neurons (termed 'spinules'). Using freely available software packages, this protocol demonstrates how to use a decision tree to identify common neuronal subcellular structures using morphological criteria within focused ion beam scanning electron microscopy (FIB-SEM) image volumes, with particular attention on identifying a diversity of spinules projecting into presynaptic boutons. In particular, this protocol describes how to trace spinules within neuronal synapses to produce 3D reconstructions of these thin subcellular projections, their parent neurites, and postsynaptic partners. Additionally, the protocol includes a list of freely available opensource software programs for analyzing FIB-SEM data and offers tips (e.g., smoothing, lighting) toward improving 3D reconstructions for visualization and publication. This adaptable protocol offers an entry point into the rapid nanoscale analysis of subcellular structures within FIB-SEM image volumes.

Automated summary

This article presents a methodological pipeline for the rapid 3D analysis of subcellular structures, focusing on identifying and tracing structures within thin neuronal processes. It also includes a list of freely available software programs for data analysis and offers tips for improving 3D reconstructions.