Three dimensional point spread function of a fluorescence microscope with two-color confocal-theta excitation

Abstract

Two-photon fluorescence microscopy has proven to be an effective biological imaging tool primarily because of the inherent quadratic dependence of the fluorescence point spread function (PSF) on the excitation intensity. This effectively confines the fluorescence volume to a very tight region resulting to an improvement in the axial resolution − the very property that spurred its use in near-field imaging of single molecules and optical microfabrication.
Recently, it was discovered that by modifying the spatial distribution of the illumination and detection PSF, further enhancement of the effective resolution is achievable. Such an improvement can be realized by a simple change in optical geometry manifested in Confocal-theta microscopy, where the detection and illumination axes are at an angular distance with respect to each other. This decreases the overlap of the intensity distribution, leading to increased resolution in the axial direction.
In this paper, we present a modification of the confocal-theta arrangement by employing dual excitation sources with wavelengths λ₁ and λ₂ to induce two-photon fluorescence at the sample. Specifically, the three-dimensional fluorescence PSF is computed for coherent (λ₁ = λ₂) and incoherent (λ₁ ≠ λ₂) excitation schemes. Fluorescence detection is carried out along one of the optical axes.