The custom-built laser-scanning two-photon microscope used for ultrashort pulse microscopy (UPM) has been previously described.19 Briefly, sub-10-fs pulses from a passively mode-locked Ti:Sapphire oscillator with 800 nm center wavelength and 133 nm full-width half maximum (Femtolasers, Vienna, Austria) were pre-compensated (GSM 270, Femtolasers) and coupled by a galvanometer driven X–Y scanner (Cambridge Technology, Cambridge MA) into an upright microscope (Axioskop2 MAT, Carl Ziess). The beam was directed by a 635 nm short pass dichroic mirror (Chroma, Bellows Falls, VT) through the imaging objective to the sample. The two-photon excited GFP signal was then collected through the imaging objective (63X, 1.2 NA and 40X, 0.8 NA from Carl Zeiss) and separated using a 430 nm long-pass dichroic mirror (Chroma), further discriminated with a 525/50 nm bandpass filter (Chroma), and detected with a PMT (Hammamatsu. Bridgewater, NJ). Data acquisition was controlled with custom LabVIEW software (National Instruments, Austin, TX).