Selective plane activation for super-resolution imaging inside volumetric samples

We developed structured illumination microscopy (SIM) method for super-resolution imaging of dense objects using selective plane activation of reversibly photoswitchable fluorescent proteins. By integrating sheet illumination for activation with structured illumination for excitation, we achieve a thin, sinusoidal emission pattern in the object plane. This technique, selective-plane-activation structured illumination microscopy (SPA-SIM), improves axial discrimination and eliminates out-of-focus backgrounds, reducing artifacts in the SIM reconstruction process. We observed detailed structures in cell nuclei and spheroids with high spatial resolution and contrast.