Lateral resolution improvement in two-photon excitation microscopy by aperture engineering

A technique for resolution improvement in two-photon excitation (2PE) fluorescence microscopy based on radially-symmetric annular binary filter (consist of central circular aperture and a concentric peripheral annulus) is proposed. Resolution improvement is achieved by engineering the aperture of the objective lens in a way so as to enhance high spatial frequencies. The structure of the electromagnetic field in the regions of focus and nearby regions are determined. The central lobe of the time-averaged electric energy density is considerably reduced for both linearly- and circularly-polarized illuminated light. An impressive combined comparative percentage improvement of 40% and 53.71% both at low (α = 30) and high (α = 60) aperture angle is obtained for linearly-polarized light. Proposed aperture engineering technique complements conventional, confocal, two-photon fluorescence microscopy, and may facilitate working at low-to-medium magnifications and large free-working distances.