A video-rate scanning two-photon excitation microscope (TPEM) has been successfully constructed and tested. The TPEM, based on a Nikon RCM-8000, incorporates a femtosecond pulsed laser, a pre-chirper, and a non-confocal detection box for ratio imaging. Fig. 1 shows the schematic layout of the main components of the instrument, each of which is briefly discussed below.Laser System: A Tsunami Ti: Sapphire laser (from Spectra-Physics) is optically pumped by a 5 W green laser (Millennia from Spectra-Physics) and is capable of generating 100 fs pulses at a repetition rate of 82 MHz and an average power of 0.8 W. The output wavelength is tunable from 690 to 1050 nm with three optical sets, each covering part of the spectrum with some overlapping.Pre-chirper: After leaving the Tsunami, the laser beam enters an optic unit known as a pre-chirper which pre-chirps laser pulses to compensate for the group velocity dispersion which will result when the laser beam goes through the microscope optics.
