The objective of the proposed work is to push the boundaries of biological imaging, so that it will be routinely possible to image specific cellular molecules with exceptional spatial resolution (2-3 nm) and high sensitivity (i.e., single molecules can be detected). If we are successful, our technologies will allow any cell biologist to study any biological process, inside living cells, with unprecedented accuracy. Current technology allows detection of single molecules in vitro but not routinely inside living cells, and sub- diffraction far-field optical imaging can currently be performed at 20-30 nm resolution on fixed, but not living cells. In our view, the major challenge to improving single-molecule and super-resolution imaging is the lack of suitable fluorescent probes. We plan to solve this problem in three parts. First, we will design and synthesize new fluorescent probes which are extremely bright and photostable, but can also be delivered into the cytoplasm of living cells and remain well-solubilized. Second, we will develop novel methods for conjugating these fluorophores to specific cellular proteins of interest. Third, we will develop physical methods for enhancing the fluorescence emission of existing fluorophores, while minimizing background from cellular autofluorescence. All these new molecules and techniques will be collectively applied to perform ultra-sensitive, high resolution optical imaging on fixed and living cells.

Public Health Relevance

If successful, our research will produce imaging technologies that will benefit the entire community of cell biologists, by allowing single-molecule and super-resolution optical imaging of biological processes in living cells with unprecedented detail.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM086214-04
Application #
8217278
Study Section
Special Emphasis Panel (ZRG1-BST-Q (51))
Program Officer
Deatherage, James F
Project Start
2009-04-01
Project End
2014-01-31
Budget Start
2012-02-01
Budget End
2014-01-31
Support Year
4
Fiscal Year
2012
Total Cost
$497,592
Indirect Cost
$62,482
Name
Massachusetts Institute of Technology
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
001425594
City
Cambridge
State
MA
Country
United States
Zip Code
02139
Yao, Jennifer Z; Uttamapinant, Chayasith; Poloukhtine, Andrei et al. (2012) Fluorophore targeting to cellular proteins via enzyme-mediated azide ligation and strain-promoted cycloaddition. J Am Chem Soc 134:3720-8
Cohen, Justin D; Zou, Peng; Ting, Alice Y (2012) Site-specific protein modification using lipoic acid ligase and bis-aryl hydrazone formation. Chembiochem 13:888-94
Zhang, Ruobing; Rothenberg, Eli; Fruhwirth, Gilbert et al. (2011) Two-photon 3D FIONA of individual quantum dots in an aqueous environment. Nano Lett 11:4074-8
Simonson, Paul D; Rothenberg, Eli; Selvin, Paul R (2011) Single-molecule-based super-resolution images in the presence of multiple fluorophores. Nano Lett 11:5090-6
Simonson, Paul D; Deberg, Hannah A; Ge, Pinghua et al. (2010) Counting bungarotoxin binding sites of nicotinic acetylcholine receptors in mammalian cells with high signal/noise ratios. Biophys J 99:L81-3
Dempsey, Graham T; Bates, Mark; Kowtoniuk, Walter E et al. (2009) Photoswitching mechanism of cyanine dyes. J Am Chem Soc 131:18192-3