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

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.

Unobstructed Multiscale Imaging of Tissue Sections for Ultrastructural Pathology Analysis by Backscattered Electron Scanning Microscopy

“…Many popular protocols for processing tissues and cells for backscattered electron scanning electron microscopy (BSE-SEM) imaging, especially for “three-dimensional” volume scanning electron microscopy (SEM) imaging, rely on mordanting agents such as tannic acid or thiocarbohydrazide to increase the deposition of heavy metals to improve membrane contrast and the signal-to-noise ratio and to avoid sample charging. 14–16 These protocols have drawbacks when working with tissues other than lipid-rich nervous tissue (e.g., muscle, kidney, lung) and can result in unequal staining and infiltration, overstaining (obscuring fine details by excessive heavy metal deposition), and poor reproducibility due to diverse tissue composition and sample sizes 15–18 (and our own observations). Furthermore, there is a lack of reference publications and validation for these protocols for applications in diagnostic and investigative pathology where phenotypes of healthy and diseased tissues need to be compared with published reference “gold standard” EM images in pathology journals, tissue atlases, and ultrastructural pathology text books.…”

“…This was important to evaluate as the most recent work with BSE-SEM has explored lipid-rich nervous tissue, 10,11,13 and much less is known about the processing and imaging quality for alternative tissues with BSE-SEM. 12,18 For this purpose, a diverse set of mouse and rat tissues were evaluated, including brain, kidney, skin, cardiac muscle, and intestine (Fig. 3A–E).…”

Field-Emission Scanning Electron Microscope as a Tool for Large-Area and Large-Volume Ultrastructural Studies

“…Since BSE provide information about material composition, they are used as a signal source for imaging of the cut surface of tissue blocks or the tissue sections mounted to the solid substrates in the majority of methods developed to study subcellular and cellular structures of animal tissue, organs, and even entire organisms in 2D and 3D modes [ 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 ]. The effective, high-resolution ultrastructural imaging of biological samples using BSE signals is dependent on three aspects.…”

“…Efficient, high-quality imaging using BSE requires a more intensive infiltration of tissue with heavy metals than for STEM and TEM imaging. Many protocols for sample fixation and en bloc contrasting for BSE imaging [ 24 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , 53 ] have been proposed depending on the tissue properties and sample size ( Table 1 ). The sections of tissues fixed with routine TEM methods and contrasted after cutting can also be imaged using BSE detection; however, they frequently require longer acquisition times; therefore, they are less useful for the digitalization of very large sample areas.…”

“…This limitation can be overcome by using a modern field-emission scanning electron microscope (FE-SEM) with high-sensitivity detection, which enables the creation of TEM-like images from the surface of resin-embedded biological specimens. Several FE-SEM-based techniques for two-dimensional (2D) and three-dimensional (3D) ultrastructural studies of cells, tissues, organs, and organisms have been developed during the 21st century [ 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 ]. These techniques have created a new era in structural biology and have changed the role of the scanning electron microscope (SEM) in biological and medical laboratories.…”