Counting molecules one at a time by Pointillism optical nanoscopy

Super-resolution fluorescence localization microscopy, represented by PALM and STORM, has rapidly emerged as a revolutionary tool for revealing intercellular processes with unprecedented resolution in vivo. Although the methods are based on single-molecule detection of Photoactivatable Fluorescence Proteins (PA-FPs), the fluorescence markers specialized for PALM, the intrinsic fluorescence blinking of PA-FPs has been a big practical obstacle to the application of PALM for molecular counting. To solve this problem, I first have fully characterized, through in vitro single-molecule study, the blinking properties of two popular PA-FPs, mEos2 and Dendra2. Secondly, based on the measured single-molecule properties, I have developed a set of experimental and analytical methods to minimize the error in counting multiple of the PA-FPs. Finally, I have applied the super-resolution microscopy and the newly developed molecular counting method to the study of organization and function of SpoIIIE, the DNA-pumping motor protein during sporulation of B. subtilis bacterial cells.

Relevant publication:
1. Counting single photoactivatable fluorescent molecules by photoactivated localization microscopy (PALM), S. Lee, J. Y. Shin, A. Lee and C. Bustamante, Proc. Natl. Acad. Sci. USA, 109 17436-17441 (2012)

2. Dynamic SpoIIIE assembly mediates septal membrane fission during Bacillus subtilis sporulation, T. C. Flemming, J. Y. Shin, S. Lee, E. Becker, K. C. Huang, C. Bustamante and K. Pogliano, Genes & Development 24 1160-1172 (2010)