ALL-SOLID-STATE ULTRAFAST LASERS FACILITATE MULTIPHOTON EXCITATION FLUORESCENCE IMAGING

Improvements in ultrafast laser technology have enabled a new excitati on mode for optical sectioning fluorescence microscopy: multiphoton ex citation fluorescence imaging, The primary advantages of this techniqu e over laser scanning confocal imaging derive from the localized excit ation volume; additional advantages accrue from the longer wavelength of the elicitation source. Recent advances in all-solid-state, ultrafa st (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 flu orescence laser scanning microscopy, developments in laser physics whi ch have enabled all-solid-state lasers to be used as excitation source s for multiphoton excitation fluorescence imaging, and provide current data for all-solid-state ultrafast lasers, A direct comparison betwee n confocal (488 nm) imaging and two-photon excitation (1047 nm) imagin g 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 a s confocal imaging, Multi-mode images (both two- and three-photon exci tation) are presented for fixed and living cells as examples of multip hoton excitation fluorescence imaging applied to developmental biology , Also, a comparison of the axial resolution of our system is presente d for confocal imaging (488 mn) and two-photon imaging (1047 nm) with and without a confocal pinhole aperture.