Two-photon fluorescence: large area excitation and enhanced sensitivity using waveguide structures

Two-photon fluorescence (TPF) microscopy is characterized by high resolution, three-dimensional imaging capabilities, large penetration depth and reduced photobleaching. It especially qualifies for studies of tissue and living cells. Furthermore, biochemical labeling becomes dispensable in some applications. On the other hand, TPF excitation requires high intensities only available in the focus of ultra-short-pulse lasers. This restriction to very small excitation areas is a major drawback in pharmaceutical screening systems, where a large number of samples have to be analyzed in parallel. Using the evanescent field of planar and structured waveguides, we demonstrate the possibility to induce TPF excitation simultaneously on large areas. Also, the application of diffractive optics for parallel excitation of TPF is investigated and a highly sensitive filtering technique adapted to TPF is presented. These concepts are applied to the excitation of the intrinsic fluorescence of amino acids.