Advanced multi-colour super-resolution imaging

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This interdisciplinary PhD project aims to develop novel multicolour photoswitches for use in super-resolution microscopy, which can lead to biological understanding and underpin future improvements in healthcare.Fluorescence microscopy is the method of choice to study cells in a comparatively non-invasive way. With the development of super-resolution methods (awarded the 2014 Nobel Prize in Chemistry) the classical ~200 nm diffraction limit can be overcome. Achieving resolutions <20 nm, super-resolution microscopy has found wide-spread applications in different fields of biology. Within the family of super-resolution techniques single-molecule localization microscopy (SMLM) stands out as it provides, besides highest spatial resolution, also quantitative information.A very common SMLM technique termed direct stochastic optical reconstruction microscopy (dSTORM) is based on organic dyes that are made photoswitchable by using chemical buffers. These dyes are small, chemically modifiable, and can be used for stoichiometric labelling of almost any protein even in living cells. dSTORM enables fascinating insights into the structural organization of a cell, but requires extremely reliable molecular photoswitches and is often limited to a single colour. The aim of this project is to develop multicolour photoswitches based on changes in the photophysical properties of dyes in the far-red / near-infrared spectral range, where multicolour information is decoded by custom-developed software. Key advantages of the method are sole irradiation at one wavelength without the need of activation at shorter wavelengths, single acquisition, reduced autofluorescence and less phototoxicity, utilizing perfect buffer conditions, and no demand for correcting chromatic aberration through image registration. Photoswitches will be characterized using fluorescence and absorption spectroscopy at the ensemble level as well as on immobilized DNA at the single-molecule level. The method will be benchmarked by multicolour SMLM imaging of different cellular reference structures such as the cytoskeleton. Finally, the proposed method will be ideally suited to study the distribution of proteins in densely packed subcellular organelles, where registration errors might affect the mapping of protein positions.