We show experiments proving the feasibility of scanning fluorescence microscopy by three-photon excitation. Three-photon excitation fluorescence axial images are shown of polystyrene beads stained with the fluorophore 2,5-bis(4-biphenyl)oxazole (BBO). Three-photon excitation is performed at an excitation wavelength of 900 nm and with pulses of 130 fs duration provided by a mode-locked titanium-sapphire laser. Fluorescence is collected between 350 and 450 nm. The fluorescence image signal features a third-order dependence on the excitation power, also providing intrinsic 3-D imaging. The resolution of a three-photon excitation microscope is increased over that of a comparable two-photon excitation microscope.

SummaryWe report three-dimensional (3D) microscopy with nearly isotropic resolution in the l/5 ¹ l/10 range. Our approach combines 4Pi-confocal two-photon fluorescence microscopy with image restoration. The 3D resolution is demonstrated with densely clustered beads as well as with F-actin fibers in mouse fibroblast cells. A comparison with unrestored twophoton confocal images reveals a total reduction of the uncertainty volume up to a factor of 15.

We examined the fluorescence spectral properties of the DNA stains DAPI (4',6-diamidino-2-phenylindole, hydrochloride) and Hoechst 33342 (bis-benzimide, or 2,5'-bi'1H-benzimidazole2'-(4-ethoxyphenyl)-5-(4-methyl-1-piperazi nyl)) with two-photon (2h nu) and three-photon (3h nu) excitation using femtosecond pulses from a Ti:sapphire laser from 830 to 885 nm. The mode of excitation of DAPI bound to DNA changed from two-photon at 830 nm to three-photon at 885 nm. In contrast, Hoechst 33342 displayed only two-photon excitation from 830 to 885 nm. DAPI-DNA displayed the same emission spectra and decay times for 2h nu and 3h nu excitation. Hoechst 33342-DNA displayed the same intensity decay for excitation at 830 and 885 nm. Both probes displayed higher anisotropies for multiphoton excitation as compared to one-photon excitation with ultraviolet wavelengths, and DAPI-DNA displays a higher anisotropy for 3h nu at 885 nm than for 2h nu at 830 nm. We used 970-nm excitation of DAPI-stained chromosomes to obtain the first three-dimensional images with three-photon excitation. Three-photon excitation of DAPI-stained chromosomes at 970 nm was demonstrated by the power dependence in the fluorescence microscope.

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The combination of two-photon excitation 4Pi-confocal fluorescence microscopy with image restoration leads to a fundamental improvement of three-dimensional resolution in the imaging of transparent, fluorescent specimens. The improvement is exemplified by randomly dispersed fluorescent beads and with actin filaments in a mouse fibroblast cell. For an illumination wavelength of 810 nm, we obtained lateral and axial full-width at half-maxima of point-like objects of 120-140 nm, and 70-100 nm, respectively. Fluorescent beads that are 150 nm apart are imaged with an intensity dip of ϳ25%. This amounts to a ϳsixfold improvement of the axial resolution over standard two-photon confocal microscopy. In the cell, the 3D-images reveal details otherwise not resolvable with focused light.