PSF shaping using adaptive optics for three-dimensional single-molecule super-resolution imaging and tracking

We present a novel approach for three-dimensional localization of single molecules using adaptive optics. A 52-actuator deformable mirror is used to both correct aberrations and induce two-dimensional astigmatism in the point-spread-function. The dependence of the z-localization precision on the degree of astigmatism is discussed. We achieve a z-localization precision of 40 nm for fluorescent proteins and 20 nm for fluorescent dyes, over an axial depth of ~800 nm. We illustrate the capabilities of our approach for three-dimensional high-resolution microscopy with super-resolution images of actin filaments in fixed cells and single-molecule tracking of quantum-dot labeled transmembrane proteins in live HeLa cells.