Tuning axial and lateral localization precision in 3D super-resolution microscopy with variable astigmatism

Astigmatism imaging is a three-dimensional (3D) single molecule fluorescence microscopy approach that yields super-resolved spatial information on a rapid time scale from a single image. It is ideally suited for resolving structures on a sub-micrometer scale and temporal behavior in the millisecond regime. While traditional astigmatism imaging utilizes a cylindrical lens, adaptive optics enables the astigmatism to be tuned for the experiment. We demonstrate here how the precisions in x, y, and z are inter-linked and vary with the astigmatism, z-position, and photon level. This experimentally driven and verified approach provides a guide for astigmatism selection in biological imaging strategies.