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Fiber Laser Based Two-Photon FRET Measurement of Calmodulin and mCherry-E0GFP Proteins
Adany, Peter Johnson, Carey K. Hui, Rongqing
Adany, Peter
Johnson, Carey K.
Hui, Rongqing
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Abstract
The speed and accuracy of Förster Resonance Energy Transfer (FRET) measurements can be improved by rapidly alternating excitation wavelengths between the donor and acceptor fluorophore. We demonstrate FRET efficiency measurements based on a fiber laser and photonic crystal fiber as the source for two-photon excitation (TPE). This system offers the potential for rapid wavelength switching with the benefits of axial optical sectioning and improved penetration depth provided by TPE. Correction of FRET signals for cross excitation and cross emission was achieved by switching the excitation wavelength with an electrically controlled modulator. Measurement speed was primarily limited by integration times required to measure fluorescence. Using this system, we measured the FRET efficiency of calmodulin labeled with Alexa Fluor 488 and Texas Red dyes. In addition, we measured two-photon induced FRET in an E0GFP-mCherry protein construct. Results from one-photon and two-photon excitation are compared to validate the rapid wavelength switched two-photon measurements.
Description
This is the peer reviewed version of the following article: Adany, P., Johnson, C. K. and Hui, R. (2012), Fiber laser based two-photon fret measurement of calmodulin and mcherry-E0GFP proteins. Microsc. Res. Tech., 75: 837–843. doi:10.1002/jemt.22002, which has been published in final form at http://doi.org/10.1002/jemt.22002. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.
Date
2012-06
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Wiley
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Keywords
Wavelength tuning, Photonic crystal fiber, Alexa Fluor 488, Texas Red, Green fluorescent protein
Citation
Adany, P., Johnson, C. K. and Hui, R. (2012), Fiber laser based two-photon fret measurement of calmodulin and mcherry-E0GFP proteins. Microsc. Res. Tech., 75: 837–843. doi:10.1002/jemt.22002