Fiber laser based two-photon FRET measurement of calmodulin and mCherry-E(0)GFP proteins

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. Microsc. Res. Tech. 75:837–843, 2012. © 2011 Wiley Periodicals, Inc.