Continuous wave excitation two-photon fluorescence microscopy exemplified with the 647-nm ArKr laser line

We report on efficient two-photon fluorescence imaging in beam scanning microscopy by exciting UV dyes at the 647-nm line of a continuous wave ArKr mixed gas laser. For a numerical aperture of 1.4 (oil), we used an illumination power of up to 210 mW at the sample. High-resolution images were obtained for DAPI-labelled cell nuclei within 4-60 s. Our method is a simple two-photon alternative to UV confocal imaging with the potential of becoming a very useful feature of laser scanning microscopy.