Studying synapses in human brain with array tomography and electron microscopy

Abstract

Postmortem studies of synapses in human brain are problematic due to the axial resolution limit of light microscopy and the difficulty preserving and analyzing ultrastructure with electron microscopy. Array tomography overcomes these problems by embedding autopsy tissue in resin and cutting ribbons of ultrathin serial sections. Ribbons are imaged with immunofluorescence, allowing high-throughput imaging of tens of thousands of synapses to assess synapse density and protein composition. The protocol takes approximately 3 days per case, excluding image analysis, which is done at the end of the study. Parallel processing for transmission electron microscopy (TEM) using a protocol modified to preserve structure in human samples allows complimentary ultrastructural studies. Incorporation of array tomography and TEM into brain banking is a potent way of phenotyping synapses in well-characterized clinical cohorts to develop clinico-pathological correlations at the synapse level. This will be important for research in neurodegenerative disease, developmental diseases, and psychiatric illness.

Quantitative immunocytochemistry at the ultrastructural level: a stereology-based approach to molecular nanomorphomics

“…However, physical ultrathin sectioning underlies various forms of array tomography: for example, ribbons of sections can be deposited on suitable substrates and viewed by SEM (Micheva and Smith 2007;Wacker and Schroeder 2013) or uncoated block-faces of TEM sections can be viewed by using SEM in backscattering mode (Denk and Horstmann 2004). Both SEM and TEM can be used in conjunction with array tomography and ICC (Micheva et al 2010;Kay et al 2013).…”

The Intersection of Amyloid Beta and Tau at Synapses in Alzheimer’s Disease

“…In these mice, the accumulation of oligomeric Aβ around plaques negatively correlated with the linear synapse loss approaching the plaque edge, and synapses containing Aβ were significantly smaller than neighboring postsynaptic densities, supporting the idea that oligomeric Aβ contributes to synapse shrinkage and collapse (Koffie et al, 2009). We then extended this technique to human autopsy tissue (Kay et al, 2013), and examined whether Aβ was present at synapses around plaques in postmortem AD brain tissue (Figure 4). We confirmed using array tomography that oligomeric Aβ is present in both pre and postsynaptic puncta, and furthermore we found an association of increased Aβ at synapses that also contain apolipoprotein Eε4 (apoE4) (Koffie et al, 2012).…”

Morphomics: An integral part of systems biology of the human placenta

“…streptavidin, IgG) can be used. Molecular characterization can be achieved also by combining immunocytochemistry with array tomography and SEM or TEM [86,101].…”

“…Alternatively, serial sections prepared for TEM can be examined using SEM in backscattering mode [85]. Software is available for 3D reconstruction and for quantification [86].…”