Increasing the resolution of single pair fluorescence resonance energy transfer measurements in solution via molecular cytometry.

Increasing the resolution of single pair fluorescence resonance energy transfer measurements in solution via molecular cytometry. Research Abstract Details 

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Increasing the resolution of single pair fluorescence resonance energy transfer measurements in solution via molecular cytometry. Abstract Text:

James H Werner,Evan R McCarney,Richard A Keller,Kevin W Plaxco,Peter M Goodwin,

We report a method to increase the resolution of single pair fluorescence resonance energy transfer (spFRET) measurements in aqueous solutions. Solution-based spFRET measurements of fluorescently labeled biological molecules (proteins, RNA, DNA) are often used to obtain histograms of molecular conformation without resorting to sample immobilization. However, for solution-phase spFRET studies, the number of photons detected from a single molecule as it diffuses through an open confocal volume element are quite limited. An "average" transit may yield on the order of 40 photons. Shot noise on the number of detected photons substantially limits the resolution of the measurement. The method reported here uses a hydrodynamically focused sample stream to ensure molecules traverse the full width of an excitation laser beam. This substantially increases the average number of photons detected per molecular transit (approximately 85 photons/molecule), which increases measurement precision. In addition, this method minimizes another source of heterogeneity present in diffusive measures of spFRET: the distribution of paths taken through the excitation laser beam. We demonstrate here using a FRET labeled protein sample (a FynSH3 domain) that superior resolution (a factor of approximately 2) can be obtained via molecular cytometry compared to spFRET measurements based upon diffusion through an open confocal volume element.

Increasing the resolution of single pair fluorescence resonance energy transfer measurements in solution via molecular cytometry. Publishing Authors By Initials

JH Werner,ER McCarney,RA Keller,KW Plaxco,PM Goodwin,

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Increasing the resolution of single pair fluorescence resonance energy transfer measurements in solution via molecular cytometry. Journal Published:

PUBLICATION TYPE: Research Support, U.S. Gov't,

Journal: Analytical chemistry

VOLUME: 79

Page Numbers: 3509-13

Journal Abbreviation:

ISSN: 0003-2700

DAY: 27

MONTH: 03

YEAR: 2007

Increasing the resolution of single pair fluorescence resonance energy transfer measurements in solution via molecular cytometry. Information

Number of References:

LANGUAGE: eng

NlmUniqueID: 370536

Increasing the resolution of single pair fluorescence resonance energy transfer measurements in solution via molecular cytometry. Keywords Mesh Terms:

KEYWORDS: Water

MESH TERMS: chemistry

Chemical & Substance for Abstract: Increasing the resolution of single pair fluorescence resonance energy transfer measurements in solution via molecular cytometry. Information

Substance Name: Water

Registry Number: 7732-18-5

Grant and Affiliation Information for Increasing the resolution of single pair fluorescence resonance energy transfer measurements in solution via molecular cytometry.

AFFILIATION: Center for Integrated Nanotechnologies, and Bioscience Division, Los Alamos National Laboratory, Los Alamos, New Mexico 87545, USA. jwerner@lanl.gov

Country: United States

AGENCY: United States NIGMS

GRANT: R01GM62868-01A2

ACRONYM: GM

MEDLINETA: Anal Chem

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