3-D EM exploration of the hepatic microarchitecture - lessons learned from large-volume in situ serial sectioning.

Gerald John Shami,Celien Vreuls,Filip Braet,Eddie Wisse,Minh Huynh,Delfine Cheng

doi:10.1038/srep36744

Abstract

To-date serial block-face scanning electron microscopy (SBF-SEM) dominates as the premier technique for generating three-dimensional (3-D) data of resin-embedded biological samples at an unprecedented depth volume. Given the infancy of the technique, limited literature is currently available regarding the applicability of SBF-SEM for the ultrastructural investigation of tissues. Herein, we provide a comprehensive and rigorous appraisal of five different SBF-SEM sample preparation protocols for the large-volume exploration of the hepatic microarchitecture at an unparalleled X, Y and Z resolution. In so doing, we qualitatively and quantitatively validate the use of a comprehensive SBF-SEM sample preparation protocol, based on the application of heavy metal fixatives, stains and mordanting agents. Employing the best-tested SBF-SEM approach, enabled us to assess large-volume morphometric data on murine parenchymal cells, sinusoids and bile canaliculi. Finally, we integrated the validated SBF-SEM protocol with a correlative light and electron microscopy (CLEM) approach. The combination of confocal scanning laser microscopy and SBF-SEM provided a novel way to picture subcellular detail. We appreciate that this multidimensional approach will aid the subsequent research of liver tissue under relevant experimental and disease conditions.

Highlights

To-date serial block-face scanning electron microscopy (SBF-SEM) dominates as the premier technique for generating three-dimensional (3-D) data of resin-embedded biological samples at an unprecedented depth volume
SsTEM is regarded as a partial effort in the generation of 3-D information, primarily due to the fact that it is a highly arduous and labour-intensive technique that is riddled with several sources of error, such as section compression and the loss of serial sections, which results in an incomplete data set
It should be noted that backscattered electron micrographs are displayed with inverted contrast – an automated function during image acquisition – resembling the appearance of conventional TEM micrographs, thereby assisting with subsequent image analysis and interpretation

Summary

Introduction

To-date serial block-face scanning electron microscopy (SBF-SEM) dominates as the premier technique for generating three-dimensional (3-D) data of resin-embedded biological samples at an unprecedented depth volume. Given the infancy of the technique, limited literature is currently available regarding the applicability of SBF-SEM for the ultrastructural investigation of tissues. The combination of confocal scanning laser microscopy and SBF-SEM provided a novel way to picture subcellular detail. We appreciate that this multidimensional approach will aid the subsequent research of liver tissue under relevant experimental and disease conditions. Throughout the infancy of EM techniques, including both transmission (TEM) and scanning electron microscopy (SEM), the structural information gained was limited to two-dimensional (2-D) space, providing a mere snapshot of innately three-dimensional (3-D) structures. For a review of volume SEM imaging techniques, see {Titze and Genoud15 #60@@author-year}

Methods

Results

Conclusion