All-solid-state ultrafast lasers facilitate multiphoton excitation fluorescence imaging

Improvements in ultrafast laser technology have enabled a new excitation mode for optical sectioning fluorescence microscopy: multiphoton excitation fluorescence imaging. The primary advantages of this technique over laser scanning confocal imaging derive from the localized excitation volume; additional advantages accrue from the longer wavelength of the excitation source. Recent advances in all-solid-state, ultrafast (subpicosecond) laser technology should allow the technique to gain widespread use as a commercial instrument. In this paper, we review: optical sectioning fluorescence microscopy, multiphoton excitation fluorescence laser scanning microscopy, developments in laser physics which have enabled all-solid-state lasers to be used as excitation sources for multiphoton excitation fluorescence imaging, and provide current data for all-solid-state ultrafast lasers. A direct comparison between confocal (488 nm) imaging and two-photon excitation (1047 nm) imaging of a mouse brain slice stained with the lipophilic dye FM4-64 shows two-photon imaging can provide usable images more than twice as deep as confocal imaging. Multi-mode images (both two- and three-photon excitation) are presented for fixed and living cells as examples of multiphoton excitation fluorescence imaging applied to developmental biology. Also, a comparison of the axial resolution of our system is presented for confocal imaging (488 nm) and two-photon imaging (1047 nm) with and without a confocal pinhole aperture.