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Beisker, W. ; Eisert, W.G.

Double-beam autocompensation for fluorescence polarization measurements in flow cytometry.

Biophys. J. 47, 607-612 (1985)
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The degree of depolarization of fluorescent light emitted from an organic dye, which is used as molecular probe, is a powerful tool in probing the microenvironment. By fluorescence depolarization the macromolecular structure can be investigated as well as the mobility of the marker molecule itself or of the complex found by the probe. Additional information such as energy transfer rates, donor-acceptor distances, and orientations are also measurable. These data are of particular interest if they can be measured from whole cells. Using flow cytometry, we can analyze a large number of cells with high statistical significance in a short period of time. We describe a newly developed double-beam epi-illumination arrangement for fluorescence polarization measurements that uses an autocompensation technique. This new technique permits the various depolarizing effects within the optical as well as the electronic components of the system to be continually compensated for on a cell by cell basis. Simultaneous measurements of other cell parameters for cell cycle analysis by total fluorescence intensity remains possible. The sensitivity of the system to measure polarization was determined as ± 0.006 p (0≤p≤0.5 in isotropic media), which amounts to ± 1.2% of the maximum p value. Polarization data for latex microspheres plotted in the histogram mode were measured with a standard deviation of 0.006, which proved the high resolution and the high performance of the system.
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Publication type Article: Journal article
Document type Scientific Article
Corresponding Author
ISSN (print) / ISBN 0006-3495
e-ISSN 1542-0086
Journal Biophysical Journal
Quellenangaben Volume: 47, Issue: 5, Pages: 607-612 Article Number: , Supplement: ,
Publisher Elsevier
Non-patent literature Publications
Reviewing status Peer reviewed