Targeted 2D histology and ultrastructural bone analysis based on 3D microCT anatomical locations

Abstract

Histological processing of mineralised tissue (e.g. bone) allows examining the anatomy of cells and tissues as well as the material properties of the tissue. However, resin-embedding offers limited control over the specimen position for cutting. Moreover, specific anatomical planes (coronal, sagittal) or defined landmarks are often missed with standard microtome sectioning. Here we describe a method to precisely locate a specific anatomical 2D plane or any anatomical feature of interest (e.g. bone lesions, newly formed bone, etc.) using 3D micro computed tomography (microCT), and to expose it using controlled-angle microtome cutting. The resulting sections and corresponding specimen's block surface offer correlative information of the same anatomical location, which can then be analysed using multiscale imaging. Moreover, this method can be combined with immunohistochemistry (IHC) to further identify any component of the bone microenvironment (cells, extracellular matrix, proteins, etc.) and guide subsequent in-depth analysis. Overall, this method allows to: • Cut your specimens in a consistent position and precise manner using microCT-based controlled-angle microtome sectioning. • Locate and expose a specific anatomical plane (coronal, sagittal plane) or any other anatomical landmarks of interest based on microCT. • Identify any cell or tissue markers based on IHC to guide further in-depth examination of those regions of interest.

Keywords

• Mineralised tissue
• Bone ultrastructure
• Bone extracellular matrix
• Controlled-angle microtome sectioning
• Immunohistochemistry (IHC)
• Second harmonic generation (SHG) imaging