Dark-field third-harmonic imaging

Abstract

Coherent cancellation of third-harmonic generation (THG) in a tightly focused laser beam is shown to enable a label-free imaging of individual neurons in representative brain tissues. The intrinsic coherence of third-harmonic buildup and cancellation combined with the nonlinear nature of the process enhances the locality of the dark signal in THG, translating into a remarkable sharpness of dark-field THG images. Unique advantages of this technique for high-contrast subcellular-resolution neuroimaging are demonstrated by comparing THG images of hippocampus and somatosensory cortex in a mouse brain with images visualizing fluorescent protein biomarkers.