Resolving single-molecule assembled patterns with superresolution blink-microscopy

In this paper we experimentally combine a recently developed AFM-based molecule-by-molecule assembly (singlemolecule cut-and-paste, SMCP) with subdiffraction resolution fluorescence imaging. Using "Blink- Microscopy", which exploits the fluctuating emission of single molecules for the reconstruction of superresolution images, we resolved SMCP assembled structures with features below the diffraction limit. Artificial line patterns then served as calibration structures to characterize parameters, such as the labeling density, that can influence resolution of Blink-Microscopy besides the localization precision of a single molecule. Finally, we experimentally utilized the adjustability of blink parameters to demonstrate the general connection of photophysical parameters with spatial resolution and acquisition time in superresolution microscopy.