Simultaneous orientation and 3D localization microscopy with a Vortex point spread function.

ABSTRACT: Estimating the orientation and 3D position of rotationally constrained emitters with localization microscopy typically requires polarization splitting or a large engineered Point Spread Function (PSF). Here we utilize a compact modified PSF for single molecule emitter imaging to estimate simultaneously the 3D position, dipole orientation, and degree of rotational constraint from a single 2D image. We use an affordable and commonly available phase plate, normally used for STED microscopy in the excitation light path, to alter the PSF in the emission light path. This resulting Vortex PSF does not require polarization splitting and has a compact PSF size, making it easy to implement and combine with localization microscopy techniques. In addition to a vectorial PSF fitting routine we calibrat

SUBMITTER: Hulleman CN 

PROVIDER: S-EPMC8505439 | biostudies-literature | 2021 Oct

REPOSITORIES: biostudies-literature