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During the last two decades, nonlinear imaging techniques experienced an impressive growth in biological and biomedical imaging applications. New techniques have been developed and applied to several topics in modern biology and biomedical optics. The nonlinear nature of the excitation provides an absorption volume spatially confined to the focal point. The localization of the excitation is maintained even in strongly scattering tissues, allowing deep-tissue high-resolution microscopy. This paper gives a general overview of two-photon fluorescence, second-harmonic generation, and fluorescence lifetime imaging techniques, including a theoretical approach to the physical background, a brief description of the methodologies used, and a list of biological and biomedical applications on tissue imaging.
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