Focal adhesions (FAs) are the main cellular structures to link the intracellular cytoskeleton to the extracellular matrix. FAs mediate cell adhesion, are important for cell migration and are involved in many (patho)-physiological processes. Here we examined FAs and their associated actin fibres using correlative fluorescence and scanning electron microscopy (SEM). We used fluorescence images of cells expressing paxillin-GFP to define the boundaries of FA complexes in SEM images, without using SEM contrast enhancing stains. We observed that SEM contrast was increased around the actin fibre entry site in 98% of FAs, indicating increases in protein density and possibly also phosphorylation levels in this area. In nearly three quarters of the FAs, these nanostructures had a fork shape, with the actin forming the stem and the high-contrast FA areas the fork. In conclusion, the combination of fluorescent and electron microscopy allowed accurate localisation of a highly abundant, novel fork structure at the FA-actin interface.

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Correlation between light, mostly fluorescence, and elec-tron microscopy (EM) is needed to identify biological molecules within their ultrastructural context and/or to relate the ultrastructure to preceding dynamics of biolog-ical molecules. Recent development of labels, sample preparation techniques, and microscopy tools allow researchers to bridge the gap between these two modalities, while dedicated, integrated microscopes merge the two techniques into one. This not only allows broader possi-bilities for implementation of CLEM (correlative light and electron microscopy) in analytical sciences but also enables novel applications crossing boundaries between the traditional microscopes. We provide an overview of the different CLEM approaches, including common labels and sample preparation techniques, and focus attention specifically on the advanced instrumentation and the novel opportunities and challenges these bring for the chemical and biological sciences.@en