Sub-60-nm 3D super-resolution imaging via saturated I5S

To investigate fine structure in biological samples, over recent decades, super-resolution methods in light microscopy have been honed. Here, we report a technique integrating nonlinearity arising from fluorophore saturation with a new version of I5S to produce quasi-isotropic three-dimensional (3D) wide-field (WF) super-resolution imaging. This extended technique—saturated I5S (SI5S)—promises lateral and axial resolutions below 60 nm when the illuminating light intensity approaches the saturation threshold in simulation. A linear processing algorithm is used to acquire SI5S image reconstructions, and comparisons with 3D structured illumination microscopy (3D-SIM) and I5S show that a substantial improvement in resolution can be realized using SI5S. The notable improvement in 3D resolution means that SI5S could facilitate volumetric visualization of intracellular fine structure within biological samples.