title: Polarization sensitive, three-dimensional, single-molecule imaging of cells with a double-helix system.
abstract: Double-helix point spread function photoactivation-localization microscopy allows three-dimensional (3D) superresolution imaging of objects smaller than the optical diffraction-limit. We demonstrate polarization sensitive detection with 3D super-localization of single-molecules and unveil 3D polarization specific characteristics of single-molecules within the intracellular structure of PtK1 cells expressing photoactivatable green fluorescent protein. The system modulates orthogonal polarization components of single-molecule emissions with a single spatial light modulator and detects them separately with a single detector. Information obtained from the two polarization channels demonstrates polarization based contrast in 3D superresolution imaging. Further, we show that the 3D information from the two channels can be optimally combined to yield up to 30% improvement in localization precision relative to a single polarization channel system.
authors.affiliation: Department of Electrical and Computer Engineering, University of Colorado, Boulder, CO 80309, USA. pavani@colorado.edu
authors.forename: Sri Rama Prasanna
authors.initials: SR
authors.lastname: Pavani
authors.forename: Jennifer G
authors.initials: JG
authors.lastname: DeLuca
authors.forename: Rafael
authors.initials: R
authors.lastname: Piestun
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D017076:Computer-Aided Design; D004247:DNA / Q000648:ultrastructure; D004847:Epithelial Cells / Q000648:ultrastructure*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D007089:Image Enhancement / Q000295:instrumentation*; D021621:Imaging, Three-Dimensional / Q000295:instrumentation*; D008856:Microscopy, Fluorescence / Q000295:instrumentation*; D008859:Microscopy, Polarization / Q000295:instrumentation*; D048328:Potoroidae; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014404:Tubulin / Q000648:ultrastructure*
chemical_list: D014404:Tubulin; D004247:DNA