Li Weixing, Stein Simon C, Gregor Ingo, Enderlein Jörg
Opt Express. 2015 Feb 9;23(3):3770-83. doi: 10.1364/OE.23.003770.
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.
我们开发了一种独立的低温恒温器，其具有对样品的光学通道，可适配于任何用于单分子荧光光谱和成像的落射荧光显微镜。该低温恒温器将样品冷却至89 K的低温，并允许使用数值孔径为0.7的空气物镜对单分子进行成像。该系统的一个重要特性是其出色的热稳定性和机械稳定性，能够在数小时内对样品进行长时间观察，且漂移可忽略不计。利用该系统，我们对Atto647N染料分子进行了光漂白研究，发现这些分子的光稳定性提高了两个多数量级。由此产生的数百万个增加的光子数使得单分子定位精度达到亚纳米级。
