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Spectrally resolved fluorescence lifetime imaging microscopy (SFLIM) and coincidence analysis: new tools to study the organization of biomolecular machines
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Paper Abstract

We present a new technique for high-resolution colocalization of fluorescent dyes. The technique is based on polarization modulated excitation and spectrally-resolved fluorescence lifetime imaging microscopy (SFLIM) as well as on coincidence analysis of the detected photon counts following pulsed laser excitation. The method takes advantage of single fluorescent dyes that can be efficiently excited by a single pulsed diode laser emitting at 635 nm but differ in their emission maxima, and in their fluorescence lifetime. A combined analysis of the fractional intensities and fluorescence lifetimes recorded on two spectrally-separated detectors enables the classification of the portion of each dye per pixel in a point-spread-function (PSF) image with high accuracy, even though only a limited number (generally a few thousand) photons are detected per single dye. From these portions two separate PSF images are calculated and fitted to two-dimensional (2D) Gaussian functions to localize their centers with a precision of a few nanometers. To reveal the number of absorbing and emitting molecules polarization modulated excitation and coincidence analysis of the detected photon counts is used. We demonstrate that by the use of appropriately selected dyes, the presented technique permits (1) the counting of the number of molecules present in the observation volume, and (2) the determination of the distance between two single molecules down to approximately 30 nm with a precision of approximately 10 nm without any chromatic aberrations. The developed techniques are promising for applications in molecular biology, e.g. to determine the number of polymerase molecules active within a transcription factory and/or to measure their distances to nanscent transcripts.

Paper Details

Date Published: 19 June 2003
PDF: 11 pages
Proc. SPIE 4962, Manipulation and Analysis of Biomolecules, Cells, and Tissues, (19 June 2003); doi: 10.1117/12.478917
Show Author Affiliations
Thomas Heinlein, Ruprecht-Karls-Univ. Heidelberg (Germany)
Mike Heilemann, Ruprecht-Karls-Univ. Heidelberg (Germany)
Dirk P. Herten, Ruprecht-Karls-Univ. Heidelberg (Germany)
Christian Muller, Ruprecht-Karls-Univ. Heidelberg (Germany)
Philip Tinnefeld, Ruprecht-Karls-Univ. Heidelberg (Germany)
Kenneth D. Weston, Florida State Univ. (United States)
Markus Sauer, Ruprecht-Karls-Univ. Heidelberg (Germany)

Published in SPIE Proceedings Vol. 4962:
Manipulation and Analysis of Biomolecules, Cells, and Tissues
Dan V. Nicolau; Joerg Enderlein; Robert C. Leif; Daniel L. Farkas, Editor(s)

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