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Proceedings Paper

An automated real-time microscopy system for analysis of fluorescence resonance energy transfer
Author(s): André Bernardini; Christoph Wotzlaw; Hans-Gerd Lipinski; Joachim Fandrey
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Paper Abstract

Molecular imaging based on Fluorescence Resonance Energy Transfer (FRET) is widely used in cellular physiology both for protein-protein interaction analysis and detecting conformational changes of single proteins, e.g. during activation of signaling cascades. However, getting reliable results from FRET measurements is still hampered by methodological problems such as spectral bleed through, chromatic aberration, focal plane shifts and false positive FRET. Particularly false positive FRET signals caused by random interaction of the fluorescent dyes can easily lead to misinterpretation of the data. This work introduces a Nipkow Disc based FRET microscopy system, that is easy to operate without expert knowledge of FRET. The system automatically accounts for all relevant sources of errors and provides various result presentations of two, three and four dimensional FRET data. Two examples are given to demonstrate the scope of application. An interaction analysis of the two subunits of the hypoxia-inducible transcription factor 1 demonstrates the use of the system as a tool for protein-protein interaction analysis. As an example for time lapse observations, the conformational change of the fluorophore labeled heat shock protein 33 in the presence of oxidant stress is shown.

Paper Details

Date Published: 5 May 2010
PDF: 8 pages
Proc. SPIE 7723, Optics, Photonics, and Digital Technologies for Multimedia Applications, 772311 (5 May 2010); doi: 10.1117/12.854027
Show Author Affiliations
André Bernardini, Univ. Duisburg-Essen (Germany)
Fachhochschule Dortmund (Germany)
Christoph Wotzlaw, Univ. Duisburg-Essen (Germany)
Hans-Gerd Lipinski, Fachhochschule Dortmund (Germany)
Joachim Fandrey, Univ. Duisburg-Essen (Germany)

Published in SPIE Proceedings Vol. 7723:
Optics, Photonics, and Digital Technologies for Multimedia Applications
Peter Schelkens; Touradj Ebrahimi; Gabriel Cristóbal; Frédéric Truchetet; Pasi Saarikko, Editor(s)

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