Development of a Novel Super Resolution Microscopy Technique using an Electron Beam for High Resolution Imaging

The requirement for spatial resolution that surpasses the limitations of classical light microscopy has proceeded along two paths; super-resolution light microscopy and electron microscopy. Optical microscopy offers multicolour flexibility for use in live cell imaging. Conversely, electron microscopes require a vacuum, and are essentially monochromatic; biological materials require dehydration and laborious treatments. Correlative light and electron microscopy endeavours to combine the two imaging approaches to maximise spatial resolution and correlate these images with live cell dynamics. The JEOL ClairScope atmospheric scanning electron microscope (ASEM) enables wide-field fluorescence microscopy on living cells, and can then obtain electron microscope images on the same sample in a hydrated environment. In many cases this would use conventional heavy metal stains; this project aimed to use the instrument for investigations of specific biological interactions, using immunogold. The labelling protocols were verified using the model system of actin in cells. The achievable resolution of the ClairScope was determined to be about 20 nm. Immunogold labelling was used to investigate the recycling dynamics of vesicles in neuroblastoma cells. The use of fluid phase markers such as FM dyes were used in attempts to correlate fluorescence and ASEM, although it was discovered that in an aqueous environment these markers were not suitable. The immunogold labelling system was further employed to investigate of the previously reported association of the protein kinase A (PKA) subunit RIα. Puncta of this protein were observed to form during differentiation of neuroblastoma cells following treatment with cAMP. Various techniques in fluorescence microscopy were employed in the study of RIα localisation. Photobleaching revealed that RIα puncta freely exchanged with cytoplasmic molecules. Further work is required to shed light on the signalling mechanism of neuroblastoma differentiation