Expedited large-volume 3-D SEM workflows for comparative microanatomical imaging.

To-date, different electron microscopy (EM) approaches are available (e.g., TEM, SEM, STEM, CLEM, etc.) to collect three-dimensional (3-D) information in tissues and cells from the microscale up to the nanometer scale. However, an abundant amount of possibilities and methodologies exist to reconstruct volumes of biological matter. In this topical paper we outline two specimen manipulation workflows for the generation of 3-D scanning electron microscopy (3-D SEM) data by means of serial-block face/focussed ion beam SEM and array tomography (AT). We applied these commonly used workflows to rodent and zebrafish liver as model experimental systems. In doing so, we outline the specific steps of each procedure and discuss in-depth the strengths vs. limitations for each of the respective 3-D SEM specimen manipulation workflows.

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.