Three-dimensional diffusion coefficient measurement by a large depth-of-field rotating point spread function

A prominent challenge in single-molecule localization microscopy is the real-time, fast, and accurate localization of nano-objects moving in three-dimensional (3D) samples. A well-established method for 3D single-molecule localization is the double-helix pointspread-function (DH-PSF) engineering, which uses additional optical elements to make the PSF exhibit different rotation angles with different nanoparticle depths. However, the compact main lobe size, effective detection depth, and precise conversion between rotation angle and depth are necessary, posing challenges to the DH-PSF generation method. Here we generate a more compact DH-PSF using Fresnel-zone-based spiral phases, and the pure phase mask achieves high transmission efficiency. The final generated DH-PSFs have a linear rotation rate at each axial position, showing a more accurate rotation angle and depth conversion. The Cramer–Rao lower limit calculation results show that the axial depth of DH-PSF extends to ∼ 11 µ m with an axial localization precision of ∼ 45 n m at 3000 photons and average background noise of 15. We measured the diffusion coefficient of nanospheres in different concentrations of glycerol using the generated DH-PSF. The measured results are within 6% error from the theoretical values, indicating the superior performance of the DH-PSF for nanoparticle diffusion coefficient measurements.