Large-scale scanning transmission electron microscopy (nanotomy) of healthy and injured zebrafish brain
Large-scale 2D electron microscopy (EM), or nanotomy, is the tissue-wide application of nanoscale resolution electron microscopy. Others and we previously applied large scale EM to human skin pancreatic islets, tissue culture and whole zebrafish larvae1-7. Here we describe a universally applicable method for tissue-scale scanning EM for unbiased detection of sub-cellular and molecular features. Nanotomy was applied to investigate the healthy and a neurodegenerative zebrafish brain. Our method is based on standardized EM sample preparation protocols: Fixation with glutaraldehyde and osmium, followed by epoxy-resin embedding, ultrathin sectioning and mounting of ultrathin-sections on onehole grids, followed by post staining with uranyl and lead. Large-scale 2D EM mosaic images are acquired using a scanning EM connected to an external large area scan generator using scanning transmission EM (STEM). Large scale EM images are typically ~ 5 - 50 G pixels in size, and best viewed using zoomable HTML files, which can be opened in any web browser, similar to online geographical HTML maps. This method can be applied to (human) tissue, cross sections of whole animals as well as tissue culture1-5. Here, zebrafish brains were analyzed in a noninvasive neuronal ablation model. We visualize within a single dataset tissue, cellular and subcellular changes which can be quantified in various cell types including neurons and microglia, the brain's macrophages. In addition, nanotomy facilitates the correlation of EM with light microscopy (CLEM)8 on the same tissue, as large surface areas previously imaged using fluorescent microscopy, can subsequently be subjected to large area EM, resulting in the nano-anatomy (nanotomy) of tissues. In all, nanotomy allows unbiased detection of features at EM level in a tissue-wide quantifiable manner.
|Keywords||Correlated microscopy, Developmental biology, Issue 111, Large-scale electron microscopy, Nanotomy, Quantitative EM, Scanning EM, Zebrafish|
|Persistent URL||dx.doi.org/10.3791/53635, hdl.handle.net/1765/95949|
|Journal||Journal of Visualized Experiments|
Kuipers, J, Kalicharan, R.D, Wolters, A.H.G. (Anouk H. G.), van Ham, T.J, & Giepmans, B.N.G. (2016). Large-scale scanning transmission electron microscopy (nanotomy) of healthy and injured zebrafish brain. Journal of Visualized Experiments, 2016(111). doi:10.3791/53635