help button home button Biophys. J.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH
Biophys. J. BioFAST: First Published May 12, 2006. doi:10.1529/biophysj.106.084004
© 2006 by the Biophysical Society.

A more recent version of this article appeared on August 1, 2006.
This Article
Right arrow Full Text (Rapid PDF)
Right arrow Supplement
Right arrow All Versions of this Article:
biophysj.106.084004v1
91/3/1023    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Author home page(s):
Jeff Gelles
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Friedman, L. J.
Right arrow Articles by Gelles, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Friedman, L. J.
Right arrow Articles by Gelles, J.

SPECTROSCOPY, IMAGING, OTHER TECHNIQUES

Viewing dynamic assembly of molecular complexes by multi-wavelength single-molecule fluorescence

Larry J. Friedman 1, Johnson Chung 1 and Jeff Gelles 1*

1 Brandeis Univ.

* To whom correspondence should be addressed. E-mail: gelles{at}brandeis.edu.

Submitted on February 24, 2006
Revised on March 27, 2006
Accepted on 14 April 2006


  Abstract
Complexes of macromolecules that transiently self-assemble, perform a particular function, and then dissociate are a recurring theme in biology. Such systems often have a large number of possible assembly/disassembly intermediates and complex, highly branched reaction pathways. Measuring the single-step kinetic parameters in these reactions would help to identify the functionally significant pathways. We have therefore constructed a novel single-molecule fluorescence microscope capable of efficiently detecting the co-localization of multiple components in a macromolecular complex when each component is labeled using a different color fluorescent dye. In this through-objective excitation, total internal reflection instrument, the dichroic mirror conventionally used to spectrally segregate the excitation and emission pathways was replaced with small broadband mirrors. This design spatially segregates the excitation and emission pathways and thereby permits efficient collection of the spectral range of emitted fluorescence when three or more dyes are used. In a test experiment with surface-immobilized single-stranded DNA molecules, we directly monitored the time course of a hybridization reaction with three different oligonucleotides, each labeled with a different color dye. The experiment reveals which of the possible reaction intermediates were traversed by each immobilized molecule, measures the hybridization rate constants for each oligonucleotide, and characterizes kinetic interdependences of the reaction steps.

Key Words: TIRF, hybridization, kinetics, microscope




This article has been cited by other articles:


Home page
 RNAHome page
D. J. Crawford, A. A. Hoskins, L. J. Friedman, J. Gelles, and M. J. Moore
Visualizing the splicing of single pre-mRNA molecules in whole cell extract
RNA, January 1, 2008; 14(1): 170 - 179.
[Abstract] [Full Text] [PDF]

Home page
 Biophys. JHome page
N. K. Lee, A. N. Kapanidis, H. R. Koh, Y. Korlann, S. O. Ho, Y. Kim, N. Gassman, S. K. Kim, and S. Weiss
Three-Color Alternating-Laser Excitation of Single Molecules: Monitoring Multiple Interactions and Distances
Biophys. J., January 1, 2007; 92(1): 303 - 312.
[Abstract] [Full Text] [PDF]


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH
Copyright © 2006 by the Biophysical Society.