Open AccessUltra-stable and versatile widefield cryo-fluorescence microscope for single-molecule localization with sub-nanometer accuracy
Author(s) -
Weixing Li,
Simon Christoph Stein,
Ingo Gregor,
Jörg Enderlein
Publication year - 2015
Publication title -
optics express
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.394
H-Index - 271
ISSN - 1094-4087
DOI - 10.1364/oe.23.003770
Subject(s) - cryostat , microscope , optics , materials science , microscopy , fluorescence correlation spectroscopy , numerical aperture , nanometre , optical microscope , fluorescence , fluorescence lifetime imaging microscopy , single molecule experiment , fluorescence microscope , molecule , spectroscopy , photon , optoelectronics , scanning electron microscope , chemistry , physics , wavelength , superconductivity , quantum mechanics , organic chemistry
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.