Seminar by Prof. Markus Sauer

Julius-Maximilians-Universität Würzburg (Germany)

Molecular Resolution Fluorescence Imaging

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. Three central parameters that determine image resolution in single-molecule localization microscopy experiments are the localization precision (statistical spread of the measured position coordinates), the localization accuracy (systematic deviation between the measured and true position), and the labeling density. In recent years, attention has focused mainly on improving the localization precision as one of the key determinants of image resolution.

For instance, the use of sequential structured illumination in combination with single-molecule detection as used in MINFLUX, SIMPLE and SIMFLUX allowed to improve the localization precision of direct stochastic optical reconstruction microscopy (dSTORM) using the red-absorbing cyanine dyes Alexa Fluor 647 (AF647) and Cy5 in photoswitching buffer to the 1-5 nm range. Such high localization precisions permitted to resolve fluorophores separated by only 6 nm on DNA origami and ~10 nm in nuclear pore complexes (NPCs), respectively. However, the results also sparked a debate about the spatial resolution claimed and the reliability of the metho. In particular, the images revealed a low detection probability of fluorophores when separated by only a few nanometers evidenced by a high number of incomplete DNA origami and missing protein localizations in the biological samples. Here we show that resonance energy transfer between fluorophores separated by less than 10 nm results in accelerated fluorescence blinking and consequently lower localization probabilities impeding sub-10 nm fluorescence imaging. We demonstrate that time-resolved fluorescence detection in combination with photoswitching fingerprint analysis can be used advantageously to determine the number anddistance even of spatially unresolvable fluorophores in the sub-10 nm range. In combination with genetic code expansion (GCE) with unnatural amino acids and bioorthogonal click-labeling with small fluorophores, photoswitching fingerprint analysis enables sub-10 nm resolution fluorescence imaging in cells.