Fiber Laser Based Two-Photon FRET Measurement of Calmodulin and mCherry-E0GFP Proteins
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Issue Date
2012-06Author
Adany, Peter
Johnson, Carey K.
Hui, Rongqing
Publisher
Wiley
Type
Article
Article Version
Scholarly/refereed, author accepted manuscript
Metadata
Show full item recordAbstract
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.
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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
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