Calculated two-photon fluorescence correction factors for reflective scan engines

Excitation laser spatial and temporal characteristics at the objective focal point are critical to the performance of two-photon scanning microscopes. Optical aberrations in scanning systems increase the microscope objective focal spot area and introduce pulse time broadening in the deflected beam, resulting in degradation of two-photon-induced fluorescence across the scan field. The geometrical pulse broadening is investigated for what is believed to be the first time and then combined with a focused spot area to provide a normalized two-photon fluorescence intensity correction factor. This factor, calculated using OSLO optical software, is compared for four reflective scan engines and allows compensation of the detected signal with position across the scan field. This new metric highlights that a parabolic mirror afocal relay exhibits superior performance as a reflective scan engine for two-photon scanning microscopy.