BRAIN STED

Intravital optical super-resolution imaging in the brain

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 Obiettivo del progetto (Objective)

'Synapses are small, highly specialized structures of intercellular contact that play a crucial role in neuronal information processing and memory. While traditional fluorescence microscopy is an extremely powerful tool to study the dynamics of biological processes in vivo with molecular specificity, it has insufficient resolving power to dissect details of synaptic structure and functional organization or the structure of small subcellular components. In contrast, far-field optical super-resolution techniques provide spatial resolution at the nanoscale beyond the limit imposed by the diffraction of light. However, current super-resolution techniques are limited to thin brain preparations or to the surface of thick samples. The central aim of this proposal is to establish optical super-resolution methods for imaging chemical synapses in all layers of the cerebral cortex and in deep lying structures of the brain and to apply these techniques to timely questions in neurobiology. We will develop intravital super-resolution microendoscopy based on the stimulated emission depletion (STED) technique. This will enable intravital microscopy of arbitrary brain regions with diffraction-unlimited resolving power. In addition, we will miniaturize the setup, opening up the investigation of nanoscale structures in the brain of awake, freely moving and behaving animals. With this, we will be able to correlate for the first time synaptic structural or organizational plasticity at the nanoscale with behavioural stimuli in a living animal, including for instance in the hippocampus of an animal exposed to a learning situation. Furthermore, these methods will advance the understanding of the interplay between neurons and glia, in particular at synapses, and will help to shed light on brain structure and function in physiological states as well as in disease.'

Introduzione (Teaser)

EU-funded scientists have developed a super-resolution optical microscope that produces high-quality images of cells deep within living tissues. This opens the door to the study subcellular brain changes in disease processes or learning.

Descrizione progetto (Article)

Fluorescence microscopy is an extremely powerful tool that has enabled scientists to study the dynamics of biological processes in vivo with molecular specificity. However, many important cellular and subcellular structures (such as synapses and spines in the brain) are not resolvable with traditional fluorescence techniques.

As it turns out, the so-called diffraction barrier of far-field (lens-based) optical microscopes can be overcome for nano-scale resolution. However, the usefulness of the methods was limited in the past by insufficient image quality deep inside living tissues. The EU-funded project 'Intravital optical super-resolution imaging in the brain' (BRAIN STED) developed a novel strategy for fluorescence imaging of nano-scale structures and dynamics of living cells and inside tissues, particularly in the brain.

The super-resolution microscope developed by BRAIN STED scientists was tested on living cell culture samples. It demonstrated competitive resolution, high image quality and capability of repeated imaging (to compare tissue changes after experimental manipulations). When tested on neuronal cells in cultured brain tissue and after compensation for optical aberrations, the technique produced high-resolution images of neurons deep within the tissue. Importantly, it enabled decoding the intricate three-dimensional structure of neurons in living tissue samples.

With resolving power not limited by the diffraction barrier the technology paves the way to observation of brain function and nano-scale structures also in living animals. It might in the future be incorporated in a miniaturized imaging device for in-vivo nanoscopy. The technology is likely to shed light on the molecular mechanisms of learning and memory. More generally, it could reveal important links between structure and function in virtually all cells and tissues in the body in health and disease. Further, the super-resolution technology will put the EU at the forefront of an important global market sector.