Intravital STED microscopy of synaptic proteins in the mouse cortex
Far-field light microscopy is a powerful technique for imaging structures inside living cells, tissue or living animals. However, fine details or substructures of the cell cannot be visualized with conventional light microscopy because of the diffraction-limited resolution (~ 200 – 350 nm). This barrier had been overcome by a whole family of super-resolution microscopy or nanoscopy concepts such as STED, RESOLFT, PALM, STORM etc. Amongst these techniques, STED microscopy stands out for its fast recording speed; its inherent 3D sectioning capability; the potential to image deep within tissue; and the possibility of exploiting a vast array of commercially available fluorescent dyes including standard fluorescent proteins, such as GFP, YFP or red-emitting fluorescent proteins. Moreover, the recorded image is a linear response of the number of fluorophores without any mathematical processing being involved. Therefore, STED microscopy is ideally suited for imaging within tissue and as such for imaging and quantifying morphological changes in the living mouse brain.
I will present applications of STED microscopy to image neuronal structures in the brain of living mice. We image the cerebral cortex of a living mouse through a glass window, so that we can observe the dynamics of dendritic spines in the molecular layer of the visual cortex. We had superresolved actin and its morphological changes in the cortex of an anaesthetized mouse, which was the first STED microscopy of a dendritic sub-structure in a living mouse. Recently, we have pioneered in vivo superresolution of the postsynaptic scaffolding molecule PSD95, one of the key components in the organization of synapses that is thought to control synaptic strength by anchoring postsynaptic receptors. We have shown for the first time the dynamic organization of PSD95 over several hours in the visual cortex of a living mouse.
These results show that STED nanoscopy is a highly suitable tool for research in neuroscience which can play a substantial role in the study of learning and memory.