Three-photon-excitation microscopy: Theory, experiment and applications

We derive the three-dimensional resolution of three-photon excitation fluorescence microscopy and compare it with its single and two-photon excitation counterpart. We perform three-photon excitation microscopy in the 900-970 nm range and compare the experimentally determined axial resolution with that of two-photon excitation microscopy at the same excitation wavelength. At an excitation wavelength of 900 nm, we measure a resolution increase of 35% of three-photon over two-photon excitation microscopy. This is in good agreement with the theoretically predicted resolution increase. At 900 nm and an aperture of 1.4 oil immersion we obtain an axial resolution of 587 ± 27 nm. Three-photon images are displayed of DAPI-labelled metaphase chromosomes. The images are recorded by using a train of subpicosecond pulses of a mode-locked Titanium-Sapphire laser emitting at a wavelength of 970 nm. We also discuss the role of the pulse length and that of annular apertures in three-photon microscopy. Further, we point out the implications of three-photon excitation for other far-field fluorescence microscopies. Our findings also reveal the potential of superresolution for increasing the information density in three-photon induced three-dimensional data storage.