3D high-resolution imaging is essential for understanding the structural organisation and functioning of cells and tissues. Cryo-correlative light and electron microscopy (cryoCLEM) is an approach that combines fluorescence microscopy with cryo-electron tomography (cryoET) to resolve the structure of proteins within their native cellular environment. This approach is mostly used for 2D cell cultures, but there is a growing demand for 3D biological model systems, such as organoids. High-pressure freezing (HPF) can be used to vitrify samples up to 200 μm in thickness, but the featureless ice surface of HPF tissue samples makes it difficult to precisely correlate light and electron images. CryoFIB/SEM volume imaging allows for detailed nanoscale investigation of vitrified samples with multi-micron dimensions, but cryoCLEM volume imaging of tissues is still to be explored. Cryogenic fixation and imaging can overcome some of the drawbacks of room temperature CLEM volume imaging, allowing for the visualisation of cells and tissues in their native state in their natural environment.
Using a Linkam CMS196 stage Beer et al. have demonstrated an innovative, targeted cryoCLEM workflow for tissues, in which cryogenic confocal fluorescence imaging of millimetre-scale volumes is correlated to 3D cryogenic electron imaging directed by a patterned surface generated during high-pressure freezing (HPF). They applied this workflow to study the mineralisation process in scales of zebrafish as a model system for 3D organs. The scales form an interesting model to study bone formation processes, as the elasmoblasts remain active and vital for hours after being removed from the skin. This workflow allowed for uncompromised imaging of tissues in their near-native state over all relevant length scales, from the millimetre down to the nanometre level, opening up future avenues to study structure-function relations of biological materials, in health and disease.
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