Effect of the detector size and the fluorescence wavelength on the resolution of three- and two-photon confocal microscopy

Image formation in three- and two-photon confocal fluorescence scanning microscopy is analysed under the paraxial approximation. The analysis incorporates the effect of a finite-sized detector and the effect of the fluorescence wavelength. The resolution in the transverse and axial directions is examined for edge and layer objects, respectively. For a given excitation energy gap, three-photon confocal and non-confocal fluorescence microscopes feature a resolution close to that of their two-photon counterparts. However, the resolution of the three-photon microscopes is improved by up to 40-50% when the same excitation wavelength is used. This may prove advantageous in practical three-photon fluorescence microscopy.