Oral Presentation 24th Australian Conference on Microscopy and Microanalysis 2016

A comparative appraisal of sample preparation protocols for the application of large-volume reconstruction of the hepatic architecture by means of serial block-face scanning electron microscopy (#18)

Gerald J. Shami 1 , Delfine Cheng 1 , Minh Huynh 2 , Patrick Trimby 2 , Eddie Wisse 3 , Filip Braet 1 2
  1. School of Medical Sciences (Discipline of Anatomy and Histology) — The Bosch Institute, The University of Sydney, Camperdown, NSW, Australia
  2. Australian Centre for Microscopy and Microanalysis , The University of Sydney, Camperdown, NSW, Australia
  3. EMU, University of Maastricht, Maastricht, Netherlands

To-date serial block-face scanning electron microscopy (SBF-SEM) dominates as the premier technique for generating high-resolution, three-dimensional data of resin-embedded biological samples at an unprecedented volume. Given the infancy of SBF-SEM, little information is currently available throughout the literature regarding the suitability of particular specimen preparation protocols for the application of SBF-SEM.

Herein, we provide a comprehensive and comparative appraisal of five different specimen preparation protocols – (1) SCF1, (2) GOT1, (3), TAMOI1, (4), ROUM2, (5) NCMIR3 – which progressively increase in the degree of contrasting, in order to determine their suitability for the large-volume reconstruction of the hepatic architecture, by means of SBF-SEM.

Based on the excellent preservation quality, superior contrasting of subcellular structures and morphometric similarity to tissues prepared under conventional protocols (e.g. SCF & GOT), 3D modelling of hepatocytes the hepatic sinusoids and bile canaliculi was performed on tissues prepared under the NCMIR protocol, in order to visualise the 3D arrangement and structure of these objects within the acquired (1,503,394.77 µm3) volume. Moreover; it was possible to perform large-volume morphometric analysis regarding the volumetric composition of the liver based on segmented data; thus overcoming a major limitation of conventional 2D imaging modalities in failing to encapsulate the entire volume of tissue and cellular features of interest.

In conclusion, we validate the suitability of the NCMIR protocol3 combined with jet-fixation4 for the future investigation of the large-volume reconstruction of the hepatic architecture by means of SBF-SEM.

  1. Shami, G., et al., Assessment of different fixation protocols on the presence of membrane-bound vesicles in Caco-2 cells: A multidimensional view by means of correlative light and 3-D transmission electron microscopy. Micron, 2014. 67: p. 20-29.
  2. Starborg, T., et al., Using transmission electron microscopy and 3View to determine collagen fibril size and three-dimensional organization. Nat. Protocols, 2013. 8(7): p. 1433-1448.
  3. Deerinck, T.J., et al., NCMIR Methods for 3D EM: A new protocol for preparation of biological specimens for serial block face scanning electron microscopy. 2010, National Center for Microscopy and Imaging Research.
  4. Vreuls, C., et al., Jet-Fixation: A Novel Method to Improve Microscopy of Human Liver Needle Biopsies. Hepatology, 2014. 59(2): p. 737-739.