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 similar to 800 nm. We illustrate the capabilities of our approach for three-dimensional high-resolution microscopy with superresolution images of actin filaments in fixed cells and single-molecule tracking of quantum-dot labeled transmembrane proteins in live HeLa cells. (C) 2012 Optical Society of America