Abstract

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 θ. The two excitation wavelengths λ₁ and λ₂ are related to the single-photon excitation wavelength λ_e by: 1/λ_e = 1/λ₁ + 1/λ₂. The general case is considered where both focused beams independently suffer from spherical aberration. For θ = 0, π/2, and π, 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 (λ₁ = λ₂) under the same aberration conditions. We found that the 2CE microscope is more robust against spherical aberration than its 2PE counterpart, with the π/2 configuration providing the clearest advantage. The prospect of aberration correction in the two-beam 2CE microscope is also discussed.

© 2003 Optical Society of America

Two-color excitation fluorescence microscopy through highly scattering media

Carlo Mar Blanca and Caesar Saloma
Appl. Opt. 40(16) 2722-2729 (2001)

Reduced effects of spherical aberration on penetration depth under two-photon excitation

Djenan Ganic, Xiaosong Gan, and Min Gu
Appl. Opt. 39(22) 3945-3947 (2000)

Behavior of the point-spread function in photon-limited confocal microscopy

Giovanni Tapang and Caesar Saloma
Appl. Opt. 41(8) 1534-1540 (2002)

Effects of defocus and primary spherical aberration on images of a straight edge in confocal microscopy

Min Gu and Colin J. R. Sheppard
Appl. Opt. 33(4) 625-630 (1994)

Two-color two-photon 4Pi fluorescence microscopy

Jianfang Chen and Katsumi Midorikawa
Opt. Lett. 29(12) 1354-1356 (2004)