Combining two-photon excitation with fluorescence lifetime imaging

Abstract

Two-photon excitation (TPE) imaging with a microscope objective that is properly matched to the sample results in a two- to four-times larger penetration depth than with confocal laser scanning microscopy. The pulsed light source present in a TPE microscope makes time-gated fluorescence lifetime imaging an attractive alternative for quantitative imaging of ion concentrations. Using TPE fluorescence lifetime imaging, in-depth imaging of pH in biofilm can be accomplished.