Enhancing Two-Color Absorption, Self-Phase Modulation and RamanMicroscopy Signatures in Tissue with Femtosecond Laser PulseShaping

摘要

Nonlinear microscopies (most commonly, two-photon fluorescence, second harmonic generation, and coherent anti-Stokes Raman scattering (CARS)) have had notable successes in imaging a variety of endogenous and exogenous targetsin recent years. These methods generate light at a color different from any of the exciting laser pulses, which makes thesignal relatively easy to detect. Our work has focused on developing microscopy techniques using a wider range ofnonlinear signatures (two-photon absorption of nonfluorescent species, self phase modulation) which have some specificadvantages – for example, in recent papers we have shown that we can differentiate between different types of melaninin pigmented lesions, image hemoglobin and its oxygenation, and measure neuronal activity. In general, these signaturesdo not generate light at a different color and we rely on the advantages of femtosecond laser pulse shaping methods toamplify the signals and make them visible (for example, using heterodyne detection of the induced signal with one of theco-propagating laser pulses). Here we extend this work to stimulated Raman and CARS geometries. In the simplestexperiments, both colors arise from filtering a single fs laser pulse, then modulating afterwards; in other cases, wedemonstrate that spectral reshaping can retain high frequency resolution in Raman and CARS geometries withfemtosecond laser pulses.