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Proceedings Paper

Fluorescence lifetime imaging of green fluorescent protein in a single living cell
Author(s): Ammasi Periasamy; Kristin K. Sharman; Ramesh C. Ahuja; Masumi Eto; David L. Brautigan
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Paper Abstract

Observing dynamic reorganization and molecular interactions of cellular components on a precise spatial and temporal scale is not possible using existing microscopic techniques. However, fluorescence lifetimes occur on a nanosecond time scale, are independent of local signal intensity and concentration of the fluorophore, and provide sensitive discrimination of the molecular environment. We designed and implemented a fluorescence lifetime imaging microscope (FLIM) using a picosecond-gated multi-channel plate image intensifier, providing two-dimensional time-resolved images of single cell specimen. BHK21 cells were transfected with vectors for green fluorescent protein (GFP) and placed on an infinity-corrected Olympus epi-fluorescence microscope, coupled to a Coherent tunable femtosecond ti-sapphire pulsed laser and a frequency doubler to select an appropriate excitation wave length. After synchronizing the high-speed gated image intensifier to the excitation laser pulses, time-resolved nanosecond images of fluorescent emission were acquired. These images were processed pixel-by-pixel for single exponential decay to obtain an image based on fluorescence lifetime. Although the nucleus appeared brighter than the cytoplasm by fluorescence intensity measurement, FILM showed a uniform lifetime of the GFP fluorescence in both compartments, indicating that the GFP was in similar molecular environments. This technology also has important applications in fluorescence resonance energy transfer (FRET) imaging.

Paper Details

Date Published: 1 June 1999
PDF: 7 pages
Proc. SPIE 3604, Optical Diagnostics of Living Cells II, (1 June 1999); doi: 10.1117/12.349210
Show Author Affiliations
Ammasi Periasamy, Univ. of Virginia (United States)
Kristin K. Sharman, Univ. of Virginia (United States)
Ramesh C. Ahuja, LaVision GmbH (Germany)
Masumi Eto, Health Sciences Ctr./Univ. of Virginia (United States)
David L. Brautigan, Health Sciences Ctr./Univ. of Virginia (United States)

Published in SPIE Proceedings Vol. 3604:
Optical Diagnostics of Living Cells II
Daniel L. Farkas; Robert C. Leif; Bruce J. Tromberg, Editor(s)

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