Combining two-photon excitation with fluorescence lifetime imaging

The in-depth imaging properties of two-photon excitation (TPE) imaging are discussed. In particular, a comparison is made between the in-depth imaging properties of confocal laser scanning microscopy (CLSM) and TPE microscopy, using dental biofilm as the test specimen. Two measurements are carried out, either at one excitation and two emission wavelengths or at two excitation and one emission wavelength. Results from these measurements reveal that 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 CLSM.