Super-resolution localization microscopy is based on determining the positions of individual fluorescent markers in a sample. The major challenge in reaching an ever higher localization precision lies in the limited number of collected photons from single emitters. To tackle this issue, it has been shown that one can exploit the increased photostability at low temperatures, reaching localization precisions in the sub-nanometer range. Another crucial ingredient of single-molecule super-resolution imaging is the ability to activate individual emitter within a diffraction-limited spot. Here, we report on photoblinking behavior of organic dyes at low temperature and elaborate on the limitations of this ubiquitous phenomenon for selecting single molecules. We then show that recording the emission polarization not only provides access to the molecular orientation, but it also facilitates the assignment of photons to individual blinking molecules. Furthermore, we employ periodical modulation of the excitation polarization as a robust method to effectively switch fluorophores. We bench mark each approach by resolving two emitters on different DNA origami structures.
Methods, trade-offs and opportunities in high-speed optical microscopy for neural voltage imaging
University of California, Los Angeles（加州大学洛杉矶分校）；Sorbonne Université（索邦大学）；INSERM（法国国家健康与医学研究院）；CNRS（法国国家科学研究中心）；Chan Zuckerberg Biohub（陈-扎克伯格生物枢纽）；Tsinghua University（清华大学）；California NanoSystems Institute（加州纳米系统研究所）
Label-free Imaging of Single-Biomolecule Structure and Interaction by Stimulated Raman Photothermal Encoded Scattering
Boston University（波士顿大学）；Stowers Institute for Medical Research（斯图沃医学研究所）；University of Washington（华盛顿大学）；Northeastern University（东北大学）；Georgia Institute of Technology（佐治亚理工学院）；Emory University（埃默里大学）；University of Virginia School of Medicine（弗吉尼亚大学医学院）；Howard Hughes Medical Institute（霍华德·休斯医学研究所）
Relative roles of multiple scattering and Fresnel diffraction in the imaging of small molecules using electrons, Part II: Differential Holographic Tomography
