True molecular resolution imaging in cells: Fluorescence imaging with a spatial resolution in the one-digit nanometer range

by Prof. Markus Sauer (Univ Würzburg)

Thursday 13 Jun 2024, 15:15 → 16:00 Europe/Stockholm

FB53 (AlbaNova Main Building)

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 with a spatial resolution of 20-30 nm. Recently, refined single-molecule localization microscopy methods such as MINFLUX and DNA-PAINT achieved superior localization precisions enabling the separation of two fluorophores separated by only 1 nm. However, translation of such high localization precisions into sub-10 nm spatial resolution in biological samples remains challenging. I will discuss different possibilities to bypass these limitations. One is based on physical expansion of the cellular structure by linking a protein of interest into a dense, cross-linked network of a swellable polyelectrolyte hydrogel. By combining Expansion Microscopy (ExM) with super-resolution Structured Illumination Microscopy it is possible to realize four-color fluorescence imaging in cells with ~20 nm spatial resolution. I will show how the spatial resolution can be further pushed to the one-digit nanometer range in cells by combining ExM with dSTORM. Another approach uses resonance energy transfer between fluorophores separated by less than 10 nm. Using time-resolved fluorescence detection in combination with photoswitching fingerprint analysis interfluorophore distances of only a few nanometers can resolved. I will show how the method can be used advantageously to determine the number and distance even of spatially unresolvable fluorophores in the sub-10 nm range. I will demonstrate the performance of the different one-digit nanometer imaging methods using biological reference structures and protein-based PicoRulers generated by genetic code expansion (GCE) with unnatural amino acids and bioorthogonal click-labeling with small fluorophores. Finally, I will demonstrate that time-resolved photoswitching fingerprint analysis can pave the way for molecular-resolution fluorescence imaging even in living cells.