Effects of primary spherical aberration in two-color excitation microscopy with two confocal beams

Abstract

We evaluate the effects of primary spherical aberrations in the point spread function (PSF) of the two-color excitation (2CE) microscope when the excitation beams are separated by an angle θ. The single-photon excitation wavelength λ_e is related to the 2CE wavelengths λ₁ and λ₂ by 1/λ_e = 1/λ₁ + 1/λ₂. Spatio-temporal simultaneity, which is affected by misalignments and aberrations, is a basic requirement of multi-photon excitation.
Previously, we have shown the advantage of 2CE against two-photon excitation (2PE) fluorescence in aberration-free misalignment. In this study, the effect of primary spherical aberrations is considered and the degradation of the peak 2CE fluorescence intensity (F_2c^p) is observed with varying spherical aberration coefficients for θ = 0, π/2 and π. The deterioration of the fluorescence PSF is also evaluated using the Linfoot's criteria of fidelity, structural content and correlation quality. We compare the 2CE PSF behavior with that of the 2PE fluorescence (λ₁ = λ₂) under the same aberration conditions.
At θ = π/2, the 2CE microscope is more robust against primary spherical aberration than its 2PE counterpart, yielding the same PSF albeit with a reduced peak power. Our calculations reveal that 2CE imaging is more resilient to primary spherical aberration than two-beam 2PE.