Examples, Molecules, and Methods for Super-Resolution Imaging in Cells with Single Molecules

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• Professor W. E. Moerner, Department of Chemistry, Stanford University, Stanford, California
• Wednesday 29 June 2011, 17:00-18:00
• Pfizer Lecture Theatre, Department of Chemistry, Lensfield Rd, CB2 1EW.

If you have a question about this talk, please contact David Klenerman.

Since the first optical detection and spectroscopy of a single molecule in a condensed phase material1, much has been learned about the ability of single molecules to probe local nanoenvironments and individual behavior in biological and nonbiological materials in the absence of ensemble averaging that can obscure heterogeneity. Because each single fluorophore acts a light source roughly 1 nm in size, microscopic imaging of individual fluorophores leads naturally to superlocalization, or determination of the position of the molecule with precision beyond the optical diffraction limit, simply by digitization of the point-spread function from the single emitter. For example, the shape of single filaments in a living cell can be extracted simply by allowing a single molecule to move through the filament2. The addition of photoinduced control of single-molecule emission allows imaging beyond the diffraction limit (super-resolution) and a new array of acronyms (PALM, STORM , F-PALM etc.) and advances have appeared, but a mechanism-independent term for these methods is Single-Molecule Active Control Microscopy (SMACM). In terms of applications of this method, we have used the native blinking and switching of a common yellow-emitting variant of green fluorescent protein (EYFP) reported more than a decade ago3 to achieve sub-40 nm super-resolution imaging of several protein structures in the bacterium Caulobacter crescentus: the quasi-helix of the actin-like protein MreB4, the cellular distribution of the DNA binding protein HU5 , and the recently discovered division spindle composed of ParA filaments6. Even with these advances, better emitters would provide more photons and improved resolution, and a new photoactivatable small-molecule emitter has recently been synthesized and targeted to specific structures in living cells to provide super-resolution images7. Finally, a new optical method for extracting three-dimensional position information based on a double-helix point spread function enables quantitative tracking of single mRNA particles in living yeast cells with 15 ms time resolution and 25-50 nm spatial precision8. These examples illustrate the power of single-molecule super-resolution optical imaging in extracting new structural and functional information in living cells.

[1] W. E. Moerner and L. Kador, , “Optical Detection and Spectroscopy of Single Molecules in Solids,” Phys. Rev. Lett. 62, 2535 (1989). [2] S. Y. Kim, Z. Gitai, A. Kinkhabwala, L. Shapiro, and W. E. Moerner, “Single Molecules of the Bacterial Actin MreB Undergo Directed Treadmilling Motion in Caulobacter crescentus,” Proc. Nat. Acad. Sci. (USA) 103, 10929-10934 (2006). [3] R. M. Dickson, A. B. Cubitt, R. Y. Tsien, and W. E. Moerner, “On/Off Blinking and Switching Behavior of Single Green Fluorescent Protein Molecules,” Nature 388, 355 (1997). [4] J. S. Biteen, M. A. Thompson, N. Tselentis, G. R. Bowman, L. Shapiro, and W. E. Moerner, “Superresolution Imaging in Live Caulobacter Crescentus Cells Using Photoswitchable EYFP ,” Nature Meth. 5, 947-949 (2008). [5] Steven F. Lee, Michael A. Thompson, Monica Schwartz, Lucy Shapiro, and W. E. Moerner, “Super-Resolution Imaging of the Nucleoid-Associated Protein HU in Caulobacter crescentus,” Biophys. J. Lett. (appearing April 2011). [6] Jerod L. Ptacin, Steven F. Lee, Ethan C. Garner, Esteban Toro, Michael Eckart, Luis R. Comolli, W.E. Moerner, and Lucy Shapiro, “A spindle-like apparatus guides bacterial chromosome segregation,” Nature Cell Biology 12, 791-798 (2010). [7] Hsiao-lu D. Lee, Samuel J. Lord, Shigeki Iwanaga, Ke Zhan, Hexin Xie, Jarrod C. Williams, Hui Wang, Grant R. Bowman, Erin D. Goley, Lucy Shapiro, Robert J. Twieg, Jianghong Rao, and W. E. Moerner, “Superresolution Imaging of Targeted Proteins in Fixed and Living Cells Using Photoactivatable Organic Fluorophores,” J. Am. Chem. Soc. 132, 15099-15101 (2010). [8] Michael A. Thompson, Jason M. Casolari, Majid Badieirostami, Patrick O. Brown, and W.E. Moerner, “Three-dimensional tracking of single mRNA particles in S. cerevisiae using a Double-Helix Point Spread Function,” Proc. Nat. Acad. Sci. (USA) 107, 17864-17871 (2010).

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