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Proceedings Paper

Fluorescence intensity decay shape analysis microscopy (FIDSAM) for quantitative and sensitive live-cell imaging
Author(s): Sébastien Peter; Kirstin Elgass; Marcus Sackrow; Katharina Caesar; Anne-Kathrin Born; Katharina Maniura; Klaus Harter; Alfred J. Meixner; Frank Schleifenbaum
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Paper Abstract

Fluorescence microscopy became an invaluable tool in cell biology in the past 20 years. However, the information that lies in these studies is often corrupted by a cellular fluorescence background known as autofluorescence. Since the unspecific background often overlaps with most commonly used labels in terms of fluorescence spectra and fluorescence lifetime, the use of spectral filters in the emission beampath or timegating in fluorescence lifetime imaging (FLIM) is often no appropriate means for distinction between signal and background. Despite the prevalence of fluorescence techniques only little progress has been reported in techniques that specifically suppress autofluorescence or that clearly discriminate autofluorescence from label fluorescence. Fluorescence intensity decay shape analysis microscopy (FIDSAM) is a novel technique which is based on the image acquisition protocol of FLIM. Whereas FLIM spatially resolved maps the average fluorescence lifetime distribution in a heterogeneous sample such as a cell, FIDSAM enhances the dynamic image contrast by determination of the autofluorescence contribution by comparing the fluorescence decay shape to a reference function. The technique therefore makes use of the key difference between label and autofluorescence, i.e. that for label fluorescence only one emitting species contributes to fluorescence intensity decay curves whereas many different species of minor intensity contribute to autofluorescence. That way, we were able to suppress autofluorescence contributions from chloroplasts in Arabidopsis stoma cells and from cell walls in Arabidopsis hypocotyl cells to background level. Furthermore, we could extend the method to more challenging labels such as the cyan fluorescent protein CFP in human fibroblasts.

Paper Details

Date Published: 24 February 2010
PDF: 9 pages
Proc. SPIE 7568, Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues VIII, 75681N (24 February 2010); doi: 10.1117/12.840644
Show Author Affiliations
Sébastien Peter, Eberhard Karls Univ. Tübingen (Germany)
Kirstin Elgass, Eberhard Karls Univ. Tübingen (Germany)
Marcus Sackrow, Eberhard Karls Univ. Tübingen (Germany)
Katharina Caesar, Eberhard Karls Univ. Tübingen (Germany)
Anne-Kathrin Born, Swiss Federal Labs. for Materials Testing and Research (Switzerland)
Katharina Maniura, Swiss Federal Labs. for Materials Testing and Research (Switzerland)
Klaus Harter, Eberhard Karls Univ. Tübingen (Germany)
Alfred J. Meixner, Eberhard Karls Univ. Tübingen (Germany)
Frank Schleifenbaum, Eberhard Karls Univ. Tübingen (Germany)

Published in SPIE Proceedings Vol. 7568:
Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues VIII
Daniel L. Farkas; Dan V. Nicolau; Robert C. Leif, Editor(s)

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