Near-field second harmonic imaging of granular membrane structures in natural killer cells

Second Harmonic Generation (SHG) has been used in combination with a near-field scanning optical microscope (NSOM) to image biological samples for the first time. Near-field SHG images of azure B stained mouse natural killer (NK) cells were recorded at four different wavelengths using a tunable near-infrared femtosecond laser. The surface specificity of SHG allows membrane structures to be selectively imaged with subdiffraction spatial resolution under ambient conditions. Resonance enhancement of SHG is shown to provide a chemically specific contrast mechanism in NSOM imaging, revealing submicron features associated with the cytolytic granular membrane.