ID-1-IN-6081 Placing molecules in a cellular context using light, electron and X-ray microscopy

Fluorescence microscopy is a powerful tool for localising proteins within biological samples. However, information is limited to the distribution of the tagged protein, telling us little about the ultrastructure of the surrounding cells and tissues, which may be intimately involved in the biological process under study. Electron microscopy overcomes the resolution limitation inherent in light microscopy and can reveal the ultrastructure of cells and tissues. However, protein localisation tends to be complex and is often dependent on the availability of ‘EM-friendly’ antibodies. Correlative light and electron microscopy (CLEM) combines the benefits of fluorescence and electron imaging, revealing protein localisation against the backdrop of cellular architecture.

In this talk, I will introduce several ways in which we are extending CLEM. We developed ‘correlative light and volume EM’ to enable visualisation of rare events in cells, tissues and whole model organisms, by combining correlative workflows with microscopes that automatically collect large stacks of high resolution images (Focused Ion Beam SEM and Serial Block Face SEM). We applied this technique to study disrupted nuclear envelopes in lipid-depleted mammalian cells and developing blood vessels in zebrafish. We are further developing this technique, to make CLVEM faster and more accurate, using an in-resin fluorescence (IRF) protocol for mammalian cells and tissues. GFP and mCherry are preserved through processing into resin, so that we can directly detect fluorophores and cellular structure in the same ultrathin resin section. We are now developing the next generation of integrated light and electron microscopes to image structural and functional information simultaneously. Finally we are combining cryo-fluorescence microscopy with cryo soft X-ray microscopy to study cellular events in whole mammalian cells, preserved as close to native state as possible within a vacuum.