Correlative 3D imaging: CLSM and FIB-SEM tomography using high-pressure frozen, freeze-substituted biological samples.

Correlative light and electron microscopy aims at combining data from different imaging modalities, ideally from the same area of the one sample, in order to achieve a more holistic view of the hierarchical structural organization of cells and tissues. Modern 3D imaging techniques opened up new possibilities to expand morphological studies into the third dimension at the nanometer scale. Here we present an approach to correlate 3D light microscopy data with volume data from focused ion beam-scanning electron microscopy. An adapted sample preparation method based on high-pressure freezing for structure preservation, followed by freeze-substitution for multimodal en bloc imaging, is described. It is based on including fluorescent labeling during freeze-substitution, which enables histological context description of the structure of interest by confocal laser scanning microscopy prior to high-resolution electron microscopy. This information can be employed to relocate the respective structure in the electron microscope. This approach is most suitable for targeted small 3D volume correlation and has the potential to extract statistically relevant data of structural details for systems biology.