Two-photon microscopy using an Yb(3+)-doped fiber laser with variable pulse widths.

Most of the two-photon fluorescence microscopes are based on femtosecond Ti:Sapphire laser sources near the 800 nm wavelength. Here, we introduce a new confocal two-photon microscope system using a mode-locked Yb(3+)-doped fiber laser. The mode-locked fiber laser produces 13 ps pulses with large positive chirping at a repetition rate of 36 MHz with an average power of 80 mW. By using an external grating pair pulse compressor, the pulse width and the frequency chirping of the laser output are controlled for optimum two-photon excitation. For a given objective lens, the optimum condition was obtained by monitoring the two-photon-induced-photocurrent in a GaAsP photodiode at the sample position. The performance of this pulse width optimized two-photon microscope system was demonstrated by imaging Vybrant DiI-stained dorsal root ganglion cells in 2 and 3 dimensions.