Ultra-stable and versatile widefield cryo-fluorescence microscope for single-molecule localization with sub-nanometer accuracy.

We developed a stand-alone cryostat with optical access to the sample which can be adapted to any epi-fluorescence microscope for single-molecule fluorescence spectroscopy and imaging. The cryostat cools the sample to a cryogenic temperature of 89 K, and allows for imaging single molecules using an air objective with a numerical aperture of 0.7. An important property of this system is its excellent thermal and mechanical stability, enabling long-time observations of samples over several hours with negligible drift. Using this system, we performed photo-bleaching studies of Atto647N dye molecules, and find an improvement of the photostability of these molecules by more than two orders of magnitude. The resulting increased photon numbers of several millions allow for single-molecule localization accuracy of sub-nanometer.