Prof. Markus Sauer

Super-resolution imaging based on single-molecule localization

Fluorescence microscopy allows the direct observation of cellular processes with molecular specificity and high temporal resolution in three dimensions. Due to the wave nature of light, however, the spatial resolution had been limited to about half of the wavelength of the light in the imaging plane, significantly larger than most cellular structures. That is, conventional fluorescence microscopes do not provide insight into the structural organization of vital protein assemblies and machineries with a size of a few tens of nanometers. Only recently methods emerged that enable super-resolution imaging with substantially improved optical resolution [1].

I review recent progress in super-resolution fluorescence imaging microscopy using various photoswitchable fluorophores and strategies. Special emphasis will be placed on the design and development of photoswitches and the requirements photoswitches have to fulfill for successful use in super-resolution imaging.

Finally, I introduce a general approach for multicolor super-resolution fluorescence imaging based on reversible photoswitching of standard small organic fluorophores. Photoswitching of organic rhodamine and oxazine fluorophores, i.e. the reversible transition from a fluorescent to a non-fluorescent state in aqueous buffers exploits the formation of long-lived radical anions through reaction with thiol compounds and repopulation of the singlet ground state by reaction with molecular oxygen. We unravel the underlying switching mechanism, investigate the importance of labeling strategies and densities, and demonstrate super-resolution imaging with different commercially available organic fluorophores with high spatial and temporal resolution. Finally, we demonstrate that direct stochastic optical reconstruction microscopy (dSTORM) [2,3] in combination with suited chemical tags can be advantageously used for dynamic live cell imaging at resolutions ~ 20 nm.

[1] M. Heilemann et al., Laser & Photon. Rev. 3 (2009) 180-202.

[2] M. Heilemann et al., Angew. Chem. Int. Ed. 47 (2008) 6172-6176.

[3] M. Heilemann et al., Angew. Chem. Int. Ed. 48 (2009) 6903-6908.