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

作者：Rego E Hesper^*; Shao Lin; Macklin John J; Winoto Lukman; Johansson Goeran A; Kamps Hughes Nicholas; Davidson Michael W; Gustafsson Mats G L

来源：Proceedings of the National Academy of Sciences, 2012, 109(3): E135-E143.

DOI：10.1073/pnas.1107547108

• patterned excitation
• moire
• subdiffraction

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.

• 出版日期2012-1-17