Imaging ONEWORLD - Professor Dr. Markus Sauer 14 February 2022


Scientific Organisers: Stefanie Reichelt, Alex Sossick, Nick Barry, Alessandro Esposito and Kirti Prakash

The meeting will begin at 13:00BST.

As part of the 'Imaging ONEWORLD' series, the focus of these lectures is on microscopy and image analysis methods and how to apply these to your research. Almost all aspects of imaging such as sample preparation, labelling strategies, experimental workflows, ‘how-to’ image and analyse, as well as facilitating collaborations and inspiring new scientific ideas will be covered. Speakers will be available for questions and answers. The organisers, CRUK CI core facility staff, Gurdon Institute, MRC-LMB, MRC Cancer Unit and NPL will be able to continue the discussion and provide advice on your imaging projects.


Speaker

  • Professor Dr. Markus Sauer

    Department of Biotechnology and Biophysics, Biocenter, University of Würzburg

    Markus Sauer studied Chemistry at the University Heidelberg where he received his Diploma in 1991. He finished his PhD 1995 in Physical Chemistry under the supervision of Prof. Dr. Jürgen Wolfrum. 1998 he has been awarded the BioFuture Prize for Detection, Analysis and Handling of Single Molecules, which allowed him to establish his own group for single-molecule fluorescence detection and single-molecule DNA sequencing. Since 2009 he is Professor and Chair of the Department of Biotechnology and Biophysics at the Julius Maximilian University Würzburg. His research interests are single-molecule fluorescence spectroscopy and imaging with a particular focus on super-resolution fluorescence imaging by direct stochastic optical reconstruction microscopy (dSTORM) and its applications in neurobiology and immunology. He has published more than 250 journal papers and coordinates several national super-resolution microscopy projects. 

Abstract

In the last decade, super-resolution microscopy has evolved as a very powerful method for sub-diffraction resolution fluorescence imaging of cells and structural investigations of cellular organelles. Super-resolution microscopy methods can now provide a spatial resolution that is well below the diffraction limit of light microscopy, enabling invaluable insights into the spatial organization of proteins in biological samples. However, current super-resolution measurements become error-prone below 25 nm. An alternative approach to bypass the diffraction limit and enable “super-resolution imaging” on standard fluorescence microscopes, is the physical expansion of the cellular structure of interest. By linking a protein of interest into a dense, cross-linked network of a swellable polyelectrolyte hydrogel, biological specimens can be physically expanded allowing ~70 nm lateral resolution by confocal laser scanning microscopy. Since its first introduction by Boyden and co-workers in 2015, expansion microscopy (ExM) has shown impressive results including the magnified visualization of pre- or post-expansion labeled proteins and RNAs with fluorescent proteins, antibodies, and oligonucleotides, respectively, in cells, tissues, and human clinical specimen. By combining ExM with single-molecule localization microscopy (SMLM) it is potentially possible to approach the resolution of electron microscopy. However, current attempts to combine both methods remained challenging because of protein and fluorophore loss during digestion or denaturation, gelation, and the incompatibility of expanded polyelectrolyte hydrogels with photoswitching buffers. Here we show that re-embedding of expanded hydrogels enables dSTORM imaging of expanded samples and demonstrate that post-labeling ExM resolves the current limitations of super-resolution microscopy. Using reference structures, neurons and brain slices, we demonstrate that post-labeling Ex-SMLM can be used advantageously for super-resolution imaging. It preserves ultrastructural details, improves the labeling efficiency and reduces the positional error arising from linking fluorophores into the gel thus paving the way for super-resolution imaging of immunolabeled endogenous proteins with true molecular resolution.