Construction of a two-photon microscope and optimisation of illumination pulse duration.

The construction of a two-photon/confocal microscope system is described in detail. For two-photon illumination, a Ti:sapphire modelocked laser generating 62-fs pulses at 715 nm was used. The effect of the optical train on illumination pulse width was examined and the observed increase 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 imaging performance of the two-photon microscope is similar to that of a conventional confocal microscope. With two-photon illumination, the resolution (full width at half-maximum intensity) was 0.42 microM (x-y) and 0.81 microM axially, while with single-photon illumination (at 488 nm in the same instrument with a confocal pinhole detector) the resolution was 0.3 microM (x-y) and 0.75 microM 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 restoration is described.