Signal generation and Raman-resonant imaging by non-degenerate four-wave mixing under tight focusing conditions

The authors demonstrate Raman-resonant imaging based on the simultaneous generation of several nonlinear frequency mixing processes resulting from a 3-color coherent anti-Stokes Raman scattering (CARS) experiment. The interaction of three coincident short-pulsed laser beams simultaneously generates both 2-color (degenerate) CARS and 3-color (non-degenerate) CARS signals, which are collected and characterized spectroscopically - allowing for resonant, doubly-resonant, and non-resonant contrast mechanisms. Images obtained from both 2-color and 3-color CARS signals are compared and found to provide complementary information. The 3-color CARS microscopy scheme provides a versatile multiplexed modality for biological imaging, which may extend the capabilities of label-free non-linear microscopy, e.g. by probing multiple Raman resonances.Doubly-resonant difference CARS image of a section of a C. elegans nematode imaged at the CD resonance (upper half) and the CH2 resonance (lower half).