In-Depth Imaging Using Two-Photon Excitation Microscopy

Abstract Two-Photon Excitation (TPE) microscopy is a powerful tool for the imaging of biological specimen. The most important advantages compared with conventional confocal microscopy are: intrinsic optical sectioning, reduced phototoxcicity and photobleaching, and an increased penetration into the specimen. The increased penetration arises from the use of the longer wavelength excitation light, in the red or near infra-red. Here, a quantitative in-depth TPE imaging study is presented on dental biofilm. The biofilm contains a mixed culture of a large number of oral bacteria with a typical size of ≈ 1 μm diameter. They exhibit a complex microstructure and heterogeneous physiological and biochemical properties. The high density of the biofilms limits the depth range over which confocal images can be acquired to 25-40 μm. Using a two-photon excitation microscope fluorescence intensity images could be recorded over the whole thickness (100 μm) of the biofilm sample.