STED nanoscopy of the living brain

STED nanoscopy of the living brain

STED-Mikroskopie des intakten Gehirns

Sebastian Berning

Doctoral thesis

Date of Examination: 2011-12-13

Date of issue: 2012-05-03

Advisor: Hell, Stefan Prof. Dr.

Referee: Hell, Stefan Prof. Dr.

Referee: Schmidt, Christoph F. Prof. Dr.

Referee: Grubmüller, Helmut Prof. Dr.

Persistent Address: http://hdl.handle.net/11858/00-1735-0000-000D-F0B1-E

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Abstract

English

Over the past decade, a new class of fluorescence microscopes has evolved which thoroughly break the diffraction limit of classical far-field light microscopy and thus provide significantly improved spatial resolution. Especially so-called STED microscopes have shown to provide valuable information otherwise inaccessible when investigating molecular processes in living biological specimens. Naturally, the question arises whether the findings obtained from cultured cells or tissue samples can be directly transferred to the whole living organism. Especially in the case of the brain, where neurons process information only by linking to thousands of neighboring neurons, it is often questionable to deduce the function of the whole organ solely from observations in isolated systems. Imaging directly in the cortex of living higher animals therefore represents the gold standard in the neurosciences. As STED microscopy is becoming increasingly popular for experiments in cultured brain slices, it is important to verify the obtained results accordingly. The scope of this thesis is to establish STED microscopy directly in the living mouse brain. In order to identify potential problems arising from aberrations induced by the refractive index mismatch between the immersion system and the brain tissue, the system is first assessed numerically. Following this, a new microscope in upright configuration is constructed addressing the specific requirements for in-vivo experiments. As a result, it is shown for the first time that super-resolution light microscopy in living higher organisms is indeed possible. Using the newly established protocols for preparation and imaging, < 70nm structural features are routinely observed in transgenic mice expressing the popular fluorescent proteins EGFP and EYFP. Time-lapse recordings of single dendritic spines reveal subtle morphological changes on the nanometer scale as well as the timescales on which they take place. Furthermore, images of glial cells such as astrocytes and oligodendrocytes exhibit small features which could previously only be seen in electron micrographs. Finally, a new recording scheme is demonstrated which enables in-vivo STED microscopy with two super-resolved spectral channels in double-transgenic mice. Initial experiments show that this is a promising method for future studies of interactions at the finest processes of neurons and glial cells.

Keywords: STED; microscopy; microscope; dendrites; mouse; brain