Nonlinear structured-illumination microscopy with a photoswitchable protein reveals cellular structures at 50-nm resolution

TLDR

Structured‑illumination microscopy can double resolution, but achieving ~50‑nm resolution via saturation requires high light intensities that damage biological samples, limiting its use. The study aims to demonstrate whole‑cell super‑resolution imaging using nonlinear structured‑illumination microscopy enabled by reversible photoswitching of a fluorescent protein at ultralow light intensities. The method employs nonlinear structured‑illumination microscopy with a photoswitchable protein (Dronpa) to generate nonlinearity at ultralow light intensities. The technique achieves ~40‑nm resolution on microtubules and visualizes nuclear pores and actin, establishing it as a biologically compatible super‑resolution method.

Abstract

Using ultralow light intensities that are well suited for investigating biological samples, we demonstrate whole-cell superresolution imaging by nonlinear structured-illumination microscopy. Structured-illumination microscopy can increase the spatial resolution of a wide-field light microscope by a factor of two, with greater resolution extension possible if the emission rate of the sample responds nonlinearly to the illumination intensity. Saturating the fluorophore excited state is one such nonlinear response, and a realization of this idea, saturated structured-illumination microscopy, has achieved approximately 50-nm resolution on dye-filled polystyrene beads. Unfortunately, because saturation requires extremely high light intensities that are likely to accelerate photobleaching and damage even fixed tissue, this implementation is of limited use for studying biological samples. Here, reversible photoswitching of a fluorescent protein provides the required nonlinearity at light intensities six orders of magnitude lower than those needed for saturation. We experimentally demonstrate approximately 40-nm resolution on purified microtubules labeled with the fluorescent photoswitchable protein Dronpa, and we visualize cellular structures by imaging the mammalian nuclear pore and actin cytoskeleton. As a result, nonlinear structured-illumination microscopy is now a biologically compatible superresolution imaging method.