Dependence of Two-Photon eGFP Bleaching on Femtosecond Pulse Spectral Amplitude and Phase

Photobleaching is a key limitation in two-photon imaging of fluorescent proteins with femtosecond pulsed excitation. We present measurements of the dependence of eGFP photobleaching on the spectral amplitude and phase of the pulses used. A strong dependence on the excitation wavelength was confirmed and measured over a 800-950 nm range. A fiber continuum light source and pulse shaping techniques were used to investigate photobleaching with broadband, 15 fs transform limited, pulses with differing spectral amplitude and phase. Narrow band pulses, >150 fs transform limited, typical of femtosecond laser sources used in two-photon imaging applications, were also investigated for their photobleaching dependence on pulse dispersion and bandwidth. The bleach rate for broadband pulses was found to be primarily determined by the second harmonic spectrum of the excitation light. On the other hand, for narrow band excitation pulses with similar center wavelengths improvement in bleach rate was found to be mostly dependent on reducing the pulse length. A simple model to predict the relative bleach rates for broadband pulses is presented and compared to the experimental data.