Clear, extended field-of-view single-molecule imaging by highly inclined swept illumination

Highly inclined and laminated optical sheet (HILO) illumination facilitates high-contrast single-molecule imaging inside cells with a single objective lens. However, the beam thickness is strongly coupled to the illumination area, limiting its usage. Here, we present highly inclined swept tile illumination microscopy (HIST). By sweeping a thin HILO beam with confocal slit detection, HIST provides a 2-fold thinner illumination and >40-fold larger imaging area than conventional HILO microscopy, enabling 3D single-molecule imaging with high signal-to-background ratio. We demonstrate single-molecule mRNA imaging with a few probes or a single probe in cultured cells and mouse brain tissues.