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Proceedings Paper

Two-channel fluorescence lifetime microscope with two colour laser excitation, single-molecule sensitivity, and submicrometer resolution
Author(s): Felix Koberling; Michael Wahl; Matthias Patting; Hans-Juergen Rahn; Peter Kapusta; Rainer Erdmann
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Paper Abstract

We present results from a two channel confocal microscope set-up allowing one to efficiently record two-colour as well as polarization resolved time-correlated single molecule fluorescence data. In addition to their spectral characteristics, single molecules can be distinguished by their fluorescence lifetime and polarization. This provides independent distinctive information and results in enhanced detection sensitivity. The set-up we present uses two picosecond diode lasers (440nm and 635 nm) for fluorescence excitation and a piezo scanner for sample movement. A learning scan algorithm permits very fast piezo scanner movement and offers a superior positioning accuracy on single molecules. The time-correlated photon counting system uses Time-Tagged Time-Resolved (TTTR) data aquisition, in which each photon is recorded individually. This method allows for the reconstruction not only fluorescence decay constants of each pixel for the purpose of Fluorescence Lifetime Imaging (FLIM) but also to analyze the fluorescence fluctuation correlation function on a single spot of interest. Cross-correlation between two channels can be used to eliminate detector artifacts. Finally, fluorescence antibunching can also be analyzed. We show results obtained with immobilized and diffusing red and blue excited fluorescently labelled latex microspheres, as well as from single fluorophore molecules.

Paper Details

Date Published: 14 October 2003
PDF: 12 pages
Proc. SPIE 5143, Novel Optical Instrumentation for Biomedical Applications, (14 October 2003); doi: 10.1117/12.500610
Show Author Affiliations
Felix Koberling, PicoQuant GmbH (Germany)
Michael Wahl, PicoQuant GmbH (Germany)
Matthias Patting, PicoQuant GmbH (Germany)
Hans-Juergen Rahn, PicoQuant GmbH (Germany)
Peter Kapusta, PicoQuant GmbH (Germany)
Rainer Erdmann, PicoQuant GmbH (Germany)

Published in SPIE Proceedings Vol. 5143:
Novel Optical Instrumentation for Biomedical Applications
Albert-Claude Boccara, Editor(s)

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