Bleed-through and photobleaching correction in multiphoton FRET microscopy

Masilamani Elangovan; Ammasi Periasamy

doi:10.1117/12.424552

24 April 2001 Bleed-through and photobleaching correction in multiphoton FRET microscopy

Masilamani Elangovan, Ammasi Periasamy

Proceedings Volume 4262, Multiphoton Microscopy in the Biomedical Sciences; (2001) https://doi.org/10.1117/12.424552
Event: BiOS 2001 The International Symposium on Biomedical Optics, 2001, San Jose, CA, United States

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.

Citation Download Citation

Masilamani Elangovan and Ammasi Periasamy "Bleed-through and photobleaching correction in multiphoton FRET microscopy", Proc. SPIE 4262, Multiphoton Microscopy in the Biomedical Sciences, (24 April 2001); https://doi.org/10.1117/12.424552

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