Challenges of microtome-based serial block-face scanning electron microscopy in neuroscience

J Microsc. 2015 Aug;259(2):137-142. doi: 10.1111/jmi.12244. Epub 2015 Apr 23.

Abstract

Serial block-face scanning electron microscopy (SBEM) is becoming increasingly popular for a wide range of applications in many disciplines from biology to material sciences. This review focuses on applications for circuit reconstruction in neuroscience, which is one of the major driving forces advancing SBEM. Neuronal circuit reconstruction poses exceptional challenges to volume EM in terms of resolution, field of view, acquisition time and sample preparation. Mapping the connections between neurons in the brain is crucial for understanding information flow and information processing in the brain. However, information on the connectivity between hundreds or even thousands of neurons densely packed in neuronal microcircuits is still largely missing. Volume EM techniques such as serial section TEM, automated tape-collecting ultramicrotome, focused ion-beam scanning electron microscopy and SBEM (microtome serial block-face scanning electron microscopy) are the techniques that provide sufficient resolution to resolve ultrastructural details such as synapses and provides sufficient field of view for dense reconstruction of neuronal circuits. While volume EM techniques are advancing, they are generating large data sets on the terabyte scale that require new image processing workflows and analysis tools. In this review, we present the recent advances in SBEM for circuit reconstruction in neuroscience and an overview of existing image processing and analysis pipelines.

During the last decades, numerous technologies have been developed in order to visualize the brain at high resolution with Volume Electron Microscopy. The visualization in three dimensions and the analysis of an entire brain from an animal model where all cells are visible and can be followed and where all synapses can be examined and counted is considering as a milestone by numerous neuroscientists. One of the technique that could be used to acquire images of an entire brain at nanometre scale is the microtome‐based block‐face scanning electron microscopy developed by W. Denk and his collaborators. In this approach, a microtome integrated within the specimen chamber of a scanning electron microscope cuts away nanometres thin slice of the specimen, thus allowing the imaging of the next ‘layer’. These individual 2D images are then assembled to a 3D image of all neurons and their projections. The recent improvements of the technique due to better microscopes and better sample preparation are pushing the limits of data quality and acquisition speed. It is creating data sets requiring important storage space. At the level of analysis, new algorithms are developed to reconstruct manually or with semiautomated interfaces all the neurons present in a volume.

Keywords: Microtome; SBFSEM; registration; segmentation; serial block-face scanning electron microscopy (SBEM); three-dimensional reconstruction; tiling.

Publication types

  • Review

MeSH terms

  • Animals
  • Brain / ultrastructure
  • Connectome
  • Histocytological Preparation Techniques
  • Imaging, Three-Dimensional / methods
  • Microscopy, Electron, Scanning / instrumentation
  • Microscopy, Electron, Scanning / methods*
  • Microtomy*
  • Neural Pathways / ultrastructure*
  • Neurons / ultrastructure
  • Neurosciences / methods*
  • Synapses / ultrastructure