A WORKFLOW FOR 3D-CLEM investigating liver TISSUE.

Kremer, A; VAN Hamme, E; Bonnardel, J; Borghgraef, P; GuÉrin, C; Guilliams, M; Lippens, S

Correlative light and electron microscopy (CLEM) is a method used to investigate the exact same region in both light and electron microscopy (EM) in order to add ultra-structural information to a light microscopic (usually fluorescent) signal. Workflows combining optical or fluorescent data with electron microscopic images are complex, hence there is a need to communicate detailed protocols and share tips & tricks for successful application of these methods. With the development of volume-EM techniques such as serial blockface scanning electron microscopy (SBF-SEM) and Focussed Ion Beam-SEM, correlation in three dimensions has become more efficient. Volume electron microscopy allows automated acquisition of serial section imaging data that can be reconstructed in three dimensions (3D) to provide a detailed, geometrically accurate view of cellular ultra-structure. In addition, combining volume-EM with high resolution light microscopy (LM) techniques decreases the resolution gap between LM and EM, making retracing of a region of interest and eventual overlays more straightforward. Here, we present a workflow for 3D CLEM on mouse liver, combining high resolution confocal microscopy with SBF-SEM. In this workflow, we have made use of two types of landmarks: (1) near infrared laser branding marks to find back the region imaged in LM in the electron microscope and (2) landmarks present in the tissue but independent of the cell or structure of interest to make overlay images of LM and EM data. Using this approach, we were able to make accurate 3D-CLEM overlays of liver tissue and correlate the fluorescent signal to the ultra-structural detail provided by the electron microscope. This workflow can be adapted for other dense cellular tissues and thus act as a guide for other three-dimensional correlative studies. This article is protected by copyright. All rights reserved.