title: Single molecule localization deep within thick cells; a novel super-resolution microscope.
abstract: A novel 3D imaging system based on single-molecule localization microscopy is presented to allow high-accuracy drift-free (<0.7 nm lateral; 2.5 nm axial) imaging many microns deep into a cell. When imaging deep within the cell, distortions of the point-spread function result in an inaccurate and very compressed Z distribution. For the system to accurately represent the position of each blink, a series of depth-dependent calibrations are required. The system and its allied methodology are applied to image the ryanodine receptor in the cardiac myocyte. Using the depth-dependent calibration, the receptors deep within the cell are spread over a Z range that is many hundreds of nanometers greater than implied by conventional analysis. We implemented a time domain filter to detect overlapping blinks that were not filtered by a stringent goodness of fit criterion. This filter enabled us to resolve the structure of the individual (30 nm square) receptors giving a result similar to that obtained with electron tomography.
authors.affiliation: Department of Chemistry, University of British Columbia, Vancouver, British Columbia, V6T 1Z1, Canada.
authors.forename: Reza
authors.initials: R
authors.lastname: Tafteh
authors.affiliation: Department of Cellular and Physiological Sciences, Life Sciences Institute, University of British Columbia, Vancouver, BC, V6T 1Z3, Canada.
authors.forename: David R L
authors.initials: DR
authors.lastname: Scriven
authors.affiliation: Department of Cellular and Physiological Sciences, Life Sciences Institute, University of British Columbia, Vancouver, BC, V6T 1Z3, Canada.
authors.forename: Edwin D W
authors.initials: ED
authors.lastname: Moore
authors.affiliation: Department of Chemistry, University of British Columbia, Vancouver, British Columbia, V6T 1Z1, Canada. kcchou@chem.ubc.ca.
authors.forename: Keng C
authors.initials: KC
authors.lastname: Chou
mesh_terms: D000818:Animals; D002138:Calibration; D005456:Fluorescent Dyes / Q000378:metabolism; D021621:Imaging, Three-Dimensional / Q000379:methods*; D008853:Microscopy / Q000379:methods*; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism*; D051381:Rats; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D019837:Ryanodine Receptor Calcium Release Channel
keywords: cardiomyocyte; drift correction; high-density localization; ryanodine receptor; single-molecule localization