COLLOQUIUM: Counting Molecules One at a time with Super-resolution Localization Microscopy

Speaker:  Dr. Sanghyuk Lee, Berkeley

Topic:  “Counting Molecules One at a time with Super-resolution Localization Microscopy – Peeking into sub-10nm Intracelluar Structures – ”

Time: 11:00 am, Monday, March 24, 2014

Place:  P-148 (refreshments will be served at 10:45 in P-145A)



Although fluorescence microscopy has played a crucial role in biology for a long time, it has suffered from its low spatial resolution. Super-resolution localization microscopy circumvents this limitation by stochastically separating two photoactivatable fluorescent molecules in time axis and localizing their individual position with high accuracy. However, even the super-resolution localization microscopy is not good enough to resolve a macromolecular structure less than 10 nm. At this small length scale of biological world, typically a few number of proteins oligomerize to form a functional unit such as channel, pore or motor. In this case, the accurate information about the number of molecules can greatly compensate for the lack of true molecular resolution in the current generation super-resolution microscopy. Although the molecular counting is almost naturally built in the super-resolution localization microscopy, the phenomena of fluorescence blinking can introduce a significant error. In the first half of my talk, I will explain how we can minimize the error and thus achieve accurate counting of molecules with super-resolution localization microscopy through better understanding of the kinetics of fluorescence blinking. Then, I will demonstrate how this powerful combination of the sub-diffraction-limit spatial resolution and the accurate single-molecule counting could resolve the in vivo macromolecular structure of SpoIIIE DNA-translocase that transports a DNA into a correct daughter cell through division septum during sporulation of Bacillus subtilis.


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