Primary spherical aberration in two-color (two-photon) excitation fluorescence microscopy with two confocal excitation beams.

We study the effects of primary spherical aberration on the three-dimensional point spread function (PSF) of the two-color (two-photon) excitation (2CE) (2PE) fluorescence microscope with two confocal excitation beams that are separated by an angle theta. The two excitation wavelengths lambda1 and lambda2 are related to the single-photon excitation wavelength lambda(e) by: 1/lambda(e) = 1/lambda1 + 1/lambda2. The general case is considered where both focused beams independently suffer from spherical aberration. For theta = 0, pi/2, and pi, the resulting deterioration of the PSF structure is evaluated for different values of the spherical aberration coefficients via the Linfoot's criteria of fidelity, structural content, and correlation quality. The corresponding degradation of the peak 2CE fluorescence intensity is also determined. Our findings are compared with that of the 2PE fluorescence (lambda1 = lambda2) under the same aberration conditions. We found that the 2CE microscope is more robust against spherical aberration than its 2PE counterpart, with the pi/2 configuration providing the clearest advantage. The prospect of aberration correction in the two-beam 2CE microscope is also discussed.