Near-Degenerate Four-Wave-Mixing Microscopy

Fluorescence microscopy has been widely used to explore the nanoscale world because of its superb sensitivity, but it is limited to fluorescent samples. Hence, various spectroscopic contrasts have been explored for imaging nonfluorescent species. Here we report a multiphoton microscopy based on single-beam near-degenerate four wave mixing (ND-FWM), by detecting a coherent signal generated by the sample at frequencies close to the “edge” of the spectrally “truncated” incident femtosecond pulses. ND-FWM microscopy allows label-free biomedical imaging with high sensitivity and spatial resolution. In particular, by achieving a nearly perfect phase matching condition, ND-FWM generates almost the highest nonlinear coherent signal in a bulk medium and provides a contrast mechanism different from other nonlinear imaging techniques. More importantly, we developed an electronic resonant version of ND-FWM for absorbing but nonfluorescent molecules. Ultrasensitive chromophore detection (∼50 molecules) and hemoglobin imaging are demonstrated, by harnessing a fully (triply) resonant enhancement of the nonlinear polarization and using optical heterodyne detection.