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Title: Effects of fluorescence excitation geometry on the accuracy of DNA fragment sizing by flow cytometry

Abstract

We report on various excitation geometries used in ultrasensitive flow cytometry that yield a linear relation between the fluorescence intensity measured from individual strained DNA fragments and the lengths of the fragments (in base pairs). This linearity holds for DNA samples that exhibit a wide range of conformations. The variety of DNA conformations leads to a distribution of dipole moment orientations for the dye molecules intercalated into the DNA. It is consequently important to use an excitation geometry such that all dye molecules are detected with similar efficiency. To estimate the conformation and the extent of elongation of the strained fragments in the flow, fluorescence polarization anisotropy and autocorrelation measurements were performed. Significant extension was observed for DNA fragments under the flow conditions frequently used for DNA fragment sizing. Classical calculations of the fluorescence emission collected over a finite solid angle are in agreement with the experimental measurements and have confirmed the relative insensitivity to DNA conformation of an orthogonal excitation geometry. Furthermore, the calculations suggested a modified excitation geometry that has increased our sizing resolution. (c) 2000 Optical Society of America.

Authors:
 [1];  [1];  [1];  [1];  [1]
  1. Division of Bioscience, Los Alamos National Laboratory, Mail Stop M888, Los Alamos, New Mexico 87545-0001 (United States)
Publication Date:
OSTI Identifier:
20217160
Resource Type:
Journal Article
Journal Name:
Applied Optics
Additional Journal Information:
Journal Volume: 39; Journal Issue: 16; Other Information: PBD: 1 Jun 2000; Journal ID: ISSN 0003-6935
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; FLUORESCENCE SPECTROSCOPY; DNA; CELL FLOW SYSTEMS; SIZE; VISIBLE SPECTRA; LASER SPECTROSCOPY; CORRELATIONS; ANGULAR DISTRIBUTION; EXPERIMENTAL DATA

Citation Formats

Werner, James H., Larson, Erica J., Goodwin, Peter M., Ambrose, W. Patrick, and Keller, Richard A. Effects of fluorescence excitation geometry on the accuracy of DNA fragment sizing by flow cytometry. United States: N. p., 2000. Web. doi:10.1364/AO.39.002831.
Werner, James H., Larson, Erica J., Goodwin, Peter M., Ambrose, W. Patrick, & Keller, Richard A. Effects of fluorescence excitation geometry on the accuracy of DNA fragment sizing by flow cytometry. United States. doi:10.1364/AO.39.002831.
Werner, James H., Larson, Erica J., Goodwin, Peter M., Ambrose, W. Patrick, and Keller, Richard A. Thu . "Effects of fluorescence excitation geometry on the accuracy of DNA fragment sizing by flow cytometry". United States. doi:10.1364/AO.39.002831.
@article{osti_20217160,
title = {Effects of fluorescence excitation geometry on the accuracy of DNA fragment sizing by flow cytometry},
author = {Werner, James H. and Larson, Erica J. and Goodwin, Peter M. and Ambrose, W. Patrick and Keller, Richard A.},
abstractNote = {We report on various excitation geometries used in ultrasensitive flow cytometry that yield a linear relation between the fluorescence intensity measured from individual strained DNA fragments and the lengths of the fragments (in base pairs). This linearity holds for DNA samples that exhibit a wide range of conformations. The variety of DNA conformations leads to a distribution of dipole moment orientations for the dye molecules intercalated into the DNA. It is consequently important to use an excitation geometry such that all dye molecules are detected with similar efficiency. To estimate the conformation and the extent of elongation of the strained fragments in the flow, fluorescence polarization anisotropy and autocorrelation measurements were performed. Significant extension was observed for DNA fragments under the flow conditions frequently used for DNA fragment sizing. Classical calculations of the fluorescence emission collected over a finite solid angle are in agreement with the experimental measurements and have confirmed the relative insensitivity to DNA conformation of an orthogonal excitation geometry. Furthermore, the calculations suggested a modified excitation geometry that has increased our sizing resolution. (c) 2000 Optical Society of America.},
doi = {10.1364/AO.39.002831},
journal = {Applied Optics},
issn = {0003-6935},
number = 16,
volume = 39,
place = {United States},
year = {2000},
month = {6}
}