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Nonlinear Microscopy without Fluorescence: Seeing the Needle in the Haystack with Femtosecond Pulse Shaping

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We use rapidly updatable, femtosecond pulse shaping and multidimensional spectroscopy to make new targets accessible by nonlinear optical imaging. For example, we observe two-photon absorption (TPA), sum frequency absorption (SFA) and self phase modulation (SPM)). Detection of TPA and related effects, such as the local quantum yield (fluorescence/absorption) permits direct observation of important endogenous molecular markers which are invisible in multiphoton fluorescence microscopy; it also permits excitation in the long-wavelength water windows which have significantly reduced scattering, but little endogenous two-photon fluorescence. The fundamental problem is that at the powers one might reasonably apply to tissue (e.g. 5 mW from a modelocked laser) typically 10−6- of the light is removed by TPA, with the rest lost to scattering and linear absorption; and SPM does not broaden the spectrum in the dramatic way associated with (for example) continuum generation.

© 2009 Optical Society of America

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