Nanoscale three-dimensional single particle tracking by light-sheet-based double-helix point spread function microscopy.

The double-helix point spread function (DH-PSF) microscopy has become an essential tool for nanoscale three-dimensional (3D) localization and tracking of single molecules in living cells. However, its localization precision is limited by fluorescent contrast in thick samples because the signal-to-noise ratio of the system is low due to the inherent low transfer function efficiency and background fluorescence. Here we combine DH-PSF microscopy with light-sheet illumination to eliminate out-of-focus background fluorescence for high-precision 3D single particle tracking. To demonstrate the capability of the method, we obtain the single fluorescent bead image with light-sheet illumination, with three-dimensional localization accuracy better than that of epi-illumination. We also show that the single fluorescent beads in agarose solution can be tracked, which demonstrates the possibility of our method for the study of dynamic processes in complex biological specimens.