3-D EM exploration of the hepatic microarchitecture - lessons learned from large-volume in situ serial sectioning.
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.
School of Medical Sciences (Discipline of Anatomy and Histology) - The Bosch Institute, The University of Sydney, NSW 2006, Australia.
Gerald John
GJ
Shami
School of Medical Sciences (Discipline of Anatomy and Histology) - The Bosch Institute, The University of Sydney, NSW 2006, Australia.
Delfine
D
Cheng
Australian Centre for Microscopy and Microanalysis (ACMM), The University of Sydney, NSW 2006, Australia.
Minh
M
Huynh
Department of Pathology, Amphia Hospital, Breda, The Netherlands.
Celien
C
Vreuls
Australian Centre for Microscopy and Microanalysis (ACMM), The University of Sydney, NSW 2006, Australia.|Maastricht Multimodal Molecular Imaging Institute, Division of Nanoscopy, University of Maastricht, 6200 MD Maastricht, The Netherlands.|Department of Internal Medicine, University of Maastricht, 6200, MD Maastricht, The Netherlands.
Eddie
E
Wisse
School of Medical Sciences (Discipline of Anatomy and Histology) - The Bosch Institute, The University of Sydney, NSW 2006, Australia.|Australian Centre for Microscopy and Microanalysis (ACMM), The University of Sydney, NSW 2006, Australia.|Cellular Imaging Facility, Charles Perkins Centre, The University of Sydney, NSW 2006, Australia.
Filip
F
Braet
D000818:Animals; D005260:Female; D021621:Imaging, Three-Dimensional; D008099:Liver / Q000648:ultrastructure*; D008855:Microscopy, Electron, Scanning; D000072356:Parenchymal Tissue / Q000648:ultrastructure; D017208:Rats, Wistar; D059629:Signal-To-Noise Ratio