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 resolution and depth volume. Given the relative infancy of the technique, limited literature is currently available regarding the applicability of SBF-SEM for the ultrastructural investigation of tissues that are inherently low in contrast, including hepatic tissue. Such tissues, relative to neural tissue – which has been extensively investigated by means of SBF-SEM – require dedicated SBF-SEM specimen preparation protocols combined with optimised imaging conditions in order to collect large-volume 3-D image data, at appropriate resolution which is devoid of imaging artefacts that are inherent to SEM investigations of uncoated samples.
Therefore, in this study, we provide a comprehensive and rigorous appraisal of different tissue preparation protocols for the large-volume exploration of hepatic architecture at an unparalleled XY and Z resolution. In so doing, we qualitatively and quantitatively validate the use of a novel and time-saving SBF-SEM tissue preparation protocol, which involves the concomitant application of heavy metal fixatives, stains and mordanting agents for the subsequent imaging of contrast-rich SBF-SEM data, 3-D reconstruction and modelling of the hepatic architecture. We further extrapolate large-volume morphometric data relating to key tissue features of the murine liver, including hepatocytes, the hepatic sinusoids and bile canaliculi network (Fig. 1).
Furthermore, we combine our validated SBF-SEM specimen preparation protocol with a correlative light and electron microscopy approach, to combine the respective advantages of confocal scanning laser microscopy and SBF-SEM in order to selectively picture particular subcellular hepatic details in a novel manner. We propose that this correlated multidimensional light and electron imaging approach will allow the study of different liver diseases under relevant experimental tissue models.
To cite this abstract:Gerald Shami, Delfine Cheng, Minh Huynh, Celien Vreuls, Eddie Wisse, Filip Braet; 3-D EM exploration of the hepatic microarchitecture – lessons learned for large-volume in situ serial sectioning. The 16th European Microscopy Congress, Lyon, France. http://emc-proceedings.com/abstract/3-d-em-exploration-of-the-hepatic-microarchitecture-lessons-learned-for-large-volume-in-situ-serial-sectioning/. Accessed: May 27, 2017
EMC Abstracts - http://emc-proceedings.com/abstract/3-d-em-exploration-of-the-hepatic-microarchitecture-lessons-learned-for-large-volume-in-situ-serial-sectioning/