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Proceedings Paper

Bleed-through and photobleaching correction in multiphoton FRET microscopy
Author(s): Masilamani Elangovan; Ammasi Periasamy
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Paper Abstract

Fluorescence resonance energy transfer (FRET) microscopy provides a tool to visualize the protein with high spatial and temporal resolution. In multi-photon FRET microscopy one experiences considerably less photobleaching compared to one-photon excitation since the illumination is the diffraction limited spot and the excitation is infrared-pulsed laser light. Because of the spectral overlap involved in the selection of the fluorophore pair for FRET imaging, the spectral bleed-through signal in the FRET channel is unavoidable. We describe in this paper the development of dedicated software to correct the bleed-through signal due to donor and acceptor fluorophore molecules. We used living cells expressed with BFP-RFP (DsRed)-C/EBP(alpha) proteins in the nucleus. We acquired images of different combinations like donor alone, acceptor alone, and both acceptor and donor under similar conditions. We statistically evaluated the percentage of bleed-through signal from one channel to the other based on the overlap areas of the spectra. We then reconstructed the images after applying the correction. Characterization of multi-photon FRET imaging system taking into account the intensity, dwell time, concentration of fluorophore pairs, objective lens and the excitation wavelength are described in this paper.

Paper Details

Date Published: 24 April 2001
PDF: 9 pages
Proc. SPIE 4262, Multiphoton Microscopy in the Biomedical Sciences, (24 April 2001); doi: 10.1117/12.424552
Show Author Affiliations
Masilamani Elangovan, Univ. of Virginia (United States)
Ammasi Periasamy, Univ. of Virginia (United States)

Published in SPIE Proceedings Vol. 4262:
Multiphoton Microscopy in the Biomedical Sciences
Ammasi Periasamy; Peter T. C. So, Editor(s)

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