An accelerated procedure for approaching and imaging of optically branded region of interest in tissue.

Abstract

Many areas of biology have benefited from advances in light microscopy (LM). However, one limitation of the LM approach is that numerous critically important aspects of subcellular machineries are well beyond the resolution of conventional LM. For studying these, electron microscopy (EM) remains the technique of choice to visualize and identify macromolecules at the ultrastructural level. The most powerful approach is combining both techniques, LM and EM (i.e., to apply correlative light/electron microscopy, CLEM) to image exactly the same region of interest. This combination allows, for example, to immuno-localize proteins by LM and then to visualize the ultrastructural context of the same region of the sample. However, the identification and correlation of the regions of interest (ROIs) at the levels of LM and EM remains a major challenge, mostly due to the difficulties with correlation along the Z-axis for both modalities. In this chapter, we address this difficulty and describe an approach for performing CLEM in tissue samples using marks from near-infrared branding as indicators of a ROI, and then using serial block face-scanning electron microscopy (SBF-SEM) to identify and approach this ROI. Once a ROI has been approached, serial sections are collected on grids for high-resolution imaging by transmission EM, and subsequent correlation with LM images showing labeled proteins.