Giepmans BNG, Adams SR, Ellisman MH, Tsien RY (2006) The fluorescent toolbox for assessing protein location and function. Xu C, Webb WW (1996) Measurement of two-photon excitation cross sections of molecular fluorophores with data from 690 to 1050 nm. Kobat D, Horton NG, Xu C (2011) In vivo two-photon microscopy to 1.6-mm depth in mouse cortex. Helmchen F, Denk W (2005) Deep tissue two-photon microscopy. A simple model to predict the relative bleach rates for broadband pulses is presented and compared to the experimental data.ĭenk W, Strickler JH, Webb WW (1990) Two-photon laser scanning fluorescence microscopy. 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. The bleach rate for broadband pulses was found to be primarily determined by the second harmonic spectrum of the excitation light. 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. 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. A strong dependence on the excitation wavelength was confirmed and measured over a 800–950 nm range. We present measurements of the dependence of eGFP photobleaching on the spectral amplitude and phase of the pulses used. Photobleaching is a key limitation in two-photon imaging of fluorescent proteins with femtosecond pulsed excitation.
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