DOI: 10.1073/pnas.0409487102. Single molecule high-resolution colocalization of Cy3 and Cy5 attached to macromolecules measures intramolecular distances through time

Single molecule high-resolution colocalization of Cy3 and Cy5 attached to macromolecules measures intramolecular distances through time

10.1073/pnas.0409487102

Crossref journal-article

Proceedings of the National Academy of Sciences

Proceedings of the National Academy of Sciences (341)

Abstract

Here we present a technique called single-molecule high-resolution colocalization (SHREC) of fluorescent dyes that allows the measurement of interfluorophore distances in macromolecules and macromolecular complexes with better than 10-nm resolution. By using two chromatically differing fluorescent molecules as probes, we are able to circumvent the Rayleigh criterion and measure distances much smaller than 250 nm. The probes are imaged separately and localized individually with high precision. The registration between the two imaging channels is measured by using fiduciary markers, and the centers of the two probes are mapped onto the same space. Multiple measurements can be made before the fluorophores photobleach, allowing intramolecular and intermolecular distances to be tracked through time. This technique's lower resolution limit lies at the upper resolution limit of single molecule FRET (smFRET) microscopy. The instrumentation and fluorophores used for SHREC can also be used for smFRET, allowing the two types of measurements to be made interchangeably, covering a wide range of interfluorophore distances. A dual-labeled duplex DNA molecule (30 bp) was used as a 10-nm molecular ruler to confirm the validity of the method. We also used SHREC to study the motion of myosin V. We directly observed myosin V's alternating heads while it walked hand-over-hand along an actin filament.

Bibliography

Churchman, L. S., Ãkten, Z., Rock, R. S., Dawson, J. F., & Spudich, J. A. (2005). Single molecule high-resolution colocalization of Cy3 and Cy5 attached to macromolecules measures intramolecular distances through time. Proceedings of the National Academy of Sciences, 102(5), 1419â1423.

Authors 5

L. Stirling Churchman (first)
Zeynep Ökten (additional)
Ronald S. Rock (additional)
John F. Dawson (additional)
James A. Spudich (additional)

References 23 Referenced 301

Dates

Type	When
Created	20 years, 7 months ago (Jan. 24, 2005, 8:26 p.m.)
Deposited	3 years, 4 months ago (April 12, 2022, 2:05 p.m.)
Indexed	1 week, 1 day ago (Aug. 22, 2025, 12:46 a.m.)
Issued	20 years, 7 months ago (Jan. 24, 2005)
Published	20 years, 7 months ago (Jan. 24, 2005)
Published Online	20 years, 7 months ago (Jan. 24, 2005)
Published Print	20 years, 6 months ago (Feb. 1, 2005)

Funders 0

None

BibTeX

@article{Churchman_2005, title={Single molecule high-resolution colocalization of Cy3 and Cy5 attached to macromolecules measures intramolecular distances through time}, volume={102}, ISSN={1091-6490}, url={http://dx.doi.org/10.1073/pnas.0409487102}, DOI={10.1073/pnas.0409487102}, number={5}, journal={Proceedings of the National Academy of Sciences}, publisher={Proceedings of the National Academy of Sciences}, author={Churchman, L. Stirling and Ökten, Zeynep and Rock, Ronald S. and Dawson, John F. and Spudich, James A.}, year={2005}, month=jan, pages={1419–1423} }

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