High-Speed Nonlinear Interferometric Vibrational Imaging of Biological Tissue With Comparison to Raman Microscopy

Vibrational contrast imaging of the distribution of complex biological molecules requires the use of techniques that provide broadband spectra with sufficient resolution. Coherent anti-Stokes Raman scattering (CARS) microscopy is currently limited in meeting these requirements due to the presence of a nonresonant background and its inability to target multiple resonances simultaneously. We present nonlinear interferometric vibrational imaging (NIVI), a technique based on CARS that uses femtosecond pump and Stokes pulses to retrieve broadband vibrational spectra over 200 cm(-1) (full-width at half maximum). By chirping the pump and performing spectral interferometric detection, the anti-Stokes pulses are resolved in time. This phase-sensitive detection allows suppression of not only the nonresonant background, but also of the real part of the nonlinear susceptibility chi((3)), improving the spectral resolution and features to make them comparable to those acquired with spontaneous Raman microscopy, as shown for a material sample and mammary tissue.