Examination of the spatial distribution of dyes and polymers in thin films by two-photon microscopy

Two-photon absorption induced fluorescence microscopy was used as a tool for the examination of the spatial distribution of a thin dye film. The two-photon absorption induced fluorescence signal is essentially the same as that produced by excitation with a single photon of equivalent energy. When femtosecond pulses are focused into a sample there is an intrinsic spatial selectivity of the two-photon emission signal, since it is dependent upon the square of the light intensity. This has tremendous implications in fluorescence microscopy. Since two-photon absorption is confined in a small region at the focal waist of an objective lens, photodamage and photobleaching of the sample are significantly reduced. In addition, the two-photon signal has inherent z-axis spatial resolution, which facilitates the construction of 3-D images. In the present work an application of this technique to a thin him of a dye is presented. The method can generally be applied to thin films made from photonic polymers.