Bridging Gaps in Imaging by Applying EM Tomography and Serial Block Face SEM, Including a New Genetically Encoded Tag for Correlated Light and 3D Electron Microscopy of Intact Cells, Tissues and Organisms: Integrating the Resulting Correlated Image Data Using the Whole Brain Catalog

Abstract

A grand goal in neuroscience research is to understand how the interplay of structural, chemical and electrical signals in and between cells of nervous tissue gives rise to behavior. We are rapidly approaching this horizon as neuroscientists make use of an increasingly powerful arsenal of tools and technologies for obtaining data, from the level of molecules to nervous systems, and engage in the arduous and challenging process of adapting and assembling neuroscience data at all scales of resolution and across disciplines into computerized databases. This talk will highlight projects where development and application of new contrasting methods and imaging tools have allowed us to see otherwise hidden relationships between cellular, subcellular and molecular constituents of nervous systems. New chemistries for carrying out correlated light and electron microscopy will be described, as well as recent advances in large-scale high-resolution 3D reconstruction with TEM and SEM based methods. The Whole Brain Catalog (WBC), a Google Earth-like open-source virtual model of the mouse brain, will also be described. The WBC is as an example of an informatics framework and web-based tool whose purpose is partly to facilitate integration of 3D image data from multiple microscopy methods and to enable the linking of information derived from other analytical approaches to imaging data shared in the publically accessible catalog.

Differential effects of early or late exposure to prenatal maternal immune activation on mouse embryonic neurodevelopment

“…Postfixed brains were sectioned to 50-μm sagittal slices by using a VT1200S vibratome (Leica Biosystem) and stored at −20 °C in cryoprotectant (30% glycerol and 30% ethylene glycol in PBS [50 mM] [pH 7.4]). Three brain sections in which the dorsal hippocampus was present [coronal section 12-15 ( 75 ), roughly equivalent to lateral 2.0 to 2.8 mm ( 76 )], were processed for EM by using osmium-thiocarbohydrazide-osmium postfixation ( 77 ) ( SI Appendix , section 1.4.1 ). Samples were sectioned into ∼70- to 75-nm ultrathin sections by using an Ultracut UC7 ultramicrotome (Leica Biosystems).…”

Differential effects of early or late exposure to prenatal maternal immune activation on mouse embryonic neurodevelopment

“…Post-fixed brains were sectioned to 50 μm sagittal slices using a VT1200S vibratome (Leica Biosystem), and stored at −20 °C in cryoprotectant (30% glycerol, 30% ethylene glycol in PBS [50mM] (pH 7.4)). Three brain sections in which the dorsal hippocampus was present (Coronal section 12-15,(36)), roughly equivalent to lateral 2.0-2.8 mm(37)), were processed for electron microscopy using osmium-thiocarbohydrazide-osmium post-fixation(38) (see Supplement 1.4 ). Samples were sectioned into ~70-75 nm ultrathin sections using an Ultracut UC7 ultramicrotome (Leica Biosystems).…”

Serial block face‐scanning electron microscopy: A tool for studying embryonic development at the cell–matrix interface

“…Other groups are developing smaller genetically encoded tags that can be tracked in real time with a fluorescence microscope, but which generate an identifiable dark stain during preparation for block face imaging. A singlet oxygen generator, which is half the size of green fluorescent protein (GFP), has been successfully used to follow synaptic cell adhesion molecule transport in intact mouse brain (Shu et al, ) and a small ascorbate peroxidase has been used to track a mitochondrial protein in vitro (Martell et al, ). The development of site‐specific tags will allow GFP‐like discoveries on an EM scale.…”