Protein Diffusion Analysis and Aberration Correction in Single Molecule Localization Microscopy

Single molecule localization microscopy (SMLM) is a powerful tool to measure the spatial localization of molecules with tens of nanometer precision and tens of millisecond time—resolution. When applied to living cells, it can provide the spatial and temporal information of molecular localization and diffusive behaviors of individual proteins in vivo. To fully characterize the distribution of molecular behavior, analysis of a large number of individual measurements is required. The work presented here focuses on the accurate extraction of protein diffusive states from experimental measurements using aberration corrected SMLM. The computational analysis framework fits well-sampled experimental distributions. The robustness of this approach is demonstrated using single-molecule trajectories acquired at different exposure times. The diffusive states are resolved. The results indicate that the fluorescent protein mEos3.2 undergoes confined Brownian diffusing in live Y. enterocolitica cells. To further the quality and quantity of single-molecule localizations, a phase-retrieved vectorial PSF model is introduced to account for aberrations in ultra-wide fields-of-view imaging. The spatially-variant aberrations in two color channels of a 3D single-molecule microscope are quantified. By computationally correcting spatially-variant aberrations during data post-processing, emitters can be localized with improved precision throughout the ultra-wide field-of-view.