Advanced multi-colour super-resolution imaging

这个跨学科的博士项目旨在开发用于超分辨率显微镜的新型多色光电开关，这可以导致生物学理解并支持未来医疗保健的改进。荧光显微镜是以相对非侵入性方式研究细胞的首选方法。随着超分辨率方法的发展（获得2014年诺贝尔化学奖），经典的~200 nm衍射极限可以被克服。超分辨率显微镜的分辨率小于20 nm，在生物学的不同领域有着广泛的应用。单分子定位显微镜（SMLM）是超分辨技术中的一个重要分支，它不仅提供了最高的空间分辨率，还提供了定量信息。一种非常常见的SMLM技术称为直接随机光学重建显微镜（dSTORM），它是基于有机染料，通过使用化学缓冲液使其光开关。这些染料体积小，可化学修饰，可用于几乎任何蛋白质的化学计量标记，甚至在活细胞中。dSTORM能够深入了解细胞的结构组织，但需要非常可靠的分子光开关，并且通常仅限于单一颜色。该项目的目的是根据远红/近红外光谱范围内染料物理特性的变化开发多色光电开关，其中多色信息由定制开发的软件解码。该方法的主要优点是在一个波长下单独照射，而不需要在较短波长下激活，单次采集，减少自发荧光和较少的光毒性，利用完美的缓冲液条件，并且不需要通过图像配准校正色差。光开关的特点是使用荧光和吸收光谱在合奏水平，以及固定的DNA在单分子水平。该方法将通过不同细胞参考结构（如细胞骨架）的多色SMLM成像进行基准测试。最后，所提出的方法将非常适合于研究蛋白质在密集的亚细胞器中的分布，其中配准误差可能会影响蛋白质位置的映射。

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.