Nonlinear chemical imaging microscopy: Near-field third harmonic generation imaging of human red blood cells

Third harmonic generation (THG) imaging using a near-field scanning optical microscope (NSOM) is demonstrated for the first time. A femtosecond, tunable near-infrared laser was used to generate both nonresonant and resonantly enhanced third harmonic radiation in human red blood cells. We show that resonantly enhanced THG is a chemically specific bulk probe in NSOM imaging by tuning the excitation source onto and off of resonance with the Soret transition of oxyhemoglobin. Additionally, we provide evidence that tightly focused, nonresonant, far-field THG imaging experiments do not produce contrast that is truly surface specific.