Biophysical Society Thematic Meeting| Lima 2019

Revisiting the Central Dogma of Molecular Biology at the Single-Molecule Level

Thursday Speaker Abstracts

FAR-FIELD FLUORESCENCE NANOSCOPY WITH SUB-10 NM RESOLUTION Fernando D. Stefani 1,2 ; 1 Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Centro de Investigaciones en Bionanociencias (CIBION), Buenos Aires, Argentina 2 Universidad de Buenos Aires, Departamento de Física, Facultad de Ciencias Exactas y Naturales, Buenos Aires , Argentina Far-field fluorescence nanoscopy is a family of methods that has revolutionized biological imaging by providing sub-diffraction spatial resolution while keeping the low invasiveness of visible light interrogation. Making use of on-off switching of molecular emission, these methods break any fundamental limitation to the achievable spatial resolution. In practice, however, the resolution is limited by the total number of excitation-emission or on-off cycles that a molecule can perform or withstand. Under biological conditions, the lateral resolution is typically limited to about 20 – 50 nm. Axial resolution is typically worse, in the range of 60 – 120 nm. Imaging with this level of detail has constituted a significant advance in the field, enabling the discovery and characterization of sub-cellular structures and pathways in their natural environment. Still, resolving supramolecular protein structures, as well as protein-protein interactions in full detail requires another push to the resolution to get into sub-10 nm regime, which is the typical size of structural proteins and complexes. Here, three recent advances from our lab that aim to achieve biological imaging with sub-10 nm resolution will be presented. First, a new and simpler implementation of MINFLUX will be described. Second, a successful combination of STED- FRET will be shown, which is able to super-resolve biomolecular direct interactions. Finally, a TIRF nanoscopy based on DNA-PAINT that can deliver sub-10 nm in three dimensions, and that can be implemented on any wide-field single molecule fluorescence microscope, will be presented.

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