CONSTRUCTION OF A 2-PHOTON MICROSCOPE AND OPTIMIZATION OF ILLUMINATION PULSE DURATION

The construction of a two-photon/confocal microscope system is describ ed in detail. For two-photon illumination, a Ti:sapphire modelocked la ser generating 62-fs pulses at 715 nm was used. The effect of the opti cal train on illumination pulse width was examined and the observed in crease in pulse duration was almost completely removed by the addition /adjustment of a prism compressor system. The imaging capabilities of the two-photon microscope are demonstrated and it is shown that the im aging performance of the two-photon microscope is similar to that of a conventional confocal microscope. With two-photon illumination, the r esolution (full width at half-maximum intensity) was 0.42 mu m (x-y) a nd 0.81 mu m axially, while with single-photon illumination (at 488 nm in the same instrument with a confocal pinhole detector) the resoluti on was 0.3 mu m (x-y) and 0.75 mu m axially. The results are discussed with regard to the general problem of femtosecond pulse distortion in an optical system and a simple procedure for optimal pulse restoratio n is described.