Optical far-field microscopy of single molecules with 3.4 nm lateral resolution

Optical far‐field imaging of single molecules in a frozen solution at 1.2 K with a lateral resolution of 3.4 nm is reported. The mechanical stability of the fluorescence microscope, especially of the low‐temperature insert, allows for the localization of fluorescing molecules with a reproducibility of better than 5 nm within observation times up to 10 min. For observation times of 9 h the reproducibility of the lateral position is limited to about 20 nm due to mechanical drift. Lateral position and orientation of 314 single molecules, present within the confocal detection volume of ~10 µm³, are obtained. The possibility to correct for mechanical drift by monitoring the position of a spatial reference in the sample is demonstrated.