Pulsed laser fluorophore deposition: a method for measuring the axial resolution in two-photon fluorescence microscopy

Abstract

We report the generation of thin (< 50 nm) fluorescence layers on a glass substrate in the focal region of a high-numerical-aperture lens by employing subpicosecond pulses of 0.75-TW/cm(2) peak intensity. The conditions for generating the fluorescence layers are described. We find that the fluorescence molecules at the glass interface are less affected by bleaching than those in the surrounding area. The fluorescence layers are suited for measuring and monitoring the axial resolution of two-photon fluorescence microscopes.