Dipole-spread-function engineering for simultaneously measuring the 3D orientations and 3D positions of fluorescent molecules

• DOI: 10.1364/optica.451899
• Publication Date: 2022-04-05T18:00:13Z
• Authors: Tingting Wu; Jin Lu; Matthew D. Lew
• Keywords: Fluorescent light

Interactions between biomolecules are characterized by where they occur and how organized, e.g., the alignment of lipid molecules to form a membrane. However, spatial angular information mixed within image fluorescent molecule–the microscope’s dipole-spread function (DSF). We demonstrate pixOL algorithm simultaneously optimize all pixels phase mask produce an engineered Green’s tensor–the dipole extension point-spread engineering. The DSF achieves optimal precision measure 3D orientation location single molecule, i.e., 4.1° orientation, 0.44 sr wobble angle, 23.2 nm lateral localization, 19.5 axial localization precisions in simulations over 700 depth range using 2500 detected photons. microscope accurately precisely resolves positions orientations Nile red spherical supported bilayer, resolving both membrane defects differences cholesterol concentration six dimensions.