Background: Histological variations in the microanatomical organisation of the functional liver units or hepatic lobules adversely affect general body function. Typically, assessment of liver pathology is based on the examination of paraffin-embedded histological slides counterstained with dyes using bright-field light microscopy (BFLM). In some instances, further clinical electron microscopy is recommended to assess the extent of ultrastructural damage to the microvasculature, to determine the nature of hepatic inclusions, or to stage fibrosis, hepatitis, and malignancy in more detail.

          Methods: Unfortunately, the above histopathology practice lacks the ability to cross-correlate observations on the same tissue sample and within a large tissue volume. With the recent advent of smart tissue preparation methodologies and three-dimensional (3-D) imaging practices, this can be circumvented in a relatively swift manner, allowing the same tissue to be examined across different microscopy platforms. Herein, we outline the combined application of X-ray micro-computed tomography (Micro-CT), BFLM and 3-D backscattered electron microscopy (BSEM) on liver tissue as an alternative good practice multimodal imaging approach.

          Results: A workflow is presented facilitating the collection of combined structure-function 3-D data (i.e., X, Y & Z) on liver architecture from the micron down to the nanometre scale using the same tissue preparation protocol. We illustrated the strength of this combined microscopy methodology to characterise various aspects of the hepatic vasculature, ranging from such large vessels as branches of the hepatic portal vein and hepatic artery, down to the smallest sinusoidal capillaries. Moreover, we were able to further characterise the subcellular features of a range of hepatic sinusoidal cells including, liver sinusoidal endothelial cells, liver-associated natural liver cells and Kupffer cells. Above all, we demonstrate the capabilities of this specimen manipulation and microscopy workflow to generate quality microscopic detail and subsequently extract relevant quantitative 3-D information.

          Conclusions: This contribution illustrates the capability that advanced histology imaging can bring to the gastroenterologist and/or pathologist in the fine structure-function assessment of the liver unit, its associated microvasculature, and the individual make-up of parenchymal and non-parenchymal tissue cells. This good practice further allows the instant generation of combined quantitative data about size, shape and volume changes of key microanatomical structures across multiple length scales. 

Figures:

(A) 2-D back projected Micro-CT image showing a vessel (white arrowhead), surrounded by parenchymal cells (Hep) and the hepatic sinusoids (S). (B) 3-D rendering of the hepatic microvascular network corresponding to (A). (C) Low power BFLM micrograph revealing the histological arrangement of toluidine blue stained liver. The inset indicates an area of interest that was investigated by means of BSEM (E-J). (D) 3-D model of the hepatic microvascular corresponding to (C). (E) BSEM image corresponding to (C). (F) Intermediate magnification BSEM image corresponding to (E-bounding box). (G-J) Corresponding 3-D models of various hepatic sinusoidal cells.

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EMC Abstracts - https://emc-proceedings.com/abstract/combined-multidimensional-microscopy-as-a-histopathology-imaging-tool-for-the-holistic-evaluation-of-the-hepatic-microvascular-and-associated-tissue-cells/