A binary annular phase mask to regulate spherical aberration and allow super-localization in single-particle tracking over extended depth-of-focus

Abstract

Important applications of single-particle tracking (SPT) aim at deciphering the diffusion properties of single fluorescent nanoparticles immersed in heterogeneous environments, such as multi-cellular biological tissues. To maximize the particle localization precision in such complex environments, high numerical aperture objectives are often required, which intrinsically restrict depth-of-focus (DOF) to less than a micrometer and impedes recording long trajectories when particles escape the plane of focus. In this work, we show that a simple binary phase mask can work with the spherical aberration inevitably induced by thick sample inhomogeneities, to extend the DOF of a single-molecule fluorescence microscope over more than 4 μm. The effect of point-spread-function (PSF) engineering over spherical aberration regularizes inhomogeneities of the PSF along the optical axis by restricting it to a narrow distribution. This allows the use of a single fitting function (i.e. Gaussian function) to localize single emitters over the whole extended DOF. Application of this simple approach on diffusing nanoparticles demonstrate that SPT trajectories can be recorded on significantly longer times.