title: Three-Dimensional Super-Resolution in Eukaryotic Cells Using the Double-Helix Point Spread Function.
abstract: Single-molecule localization microscopy, typically based on total internal reflection illumination, has taken our understanding of protein organization and dynamics in cells beyond the diffraction limit. However, biological systems exist in a complicated three-dimensional environment, which has required the development of new techniques, including the double-helix point spread function (DHPSF), to accurately visualize biological processes. The application of the DHPSF approach has so far been limited to the study of relatively small prokaryotic cells. By matching the refractive index of the objective lens immersion liquid to that of the sample media, we demonstrate DHPSF imaging of up to 15-μm-thick whole eukaryotic cell volumes in three to five imaging planes. We illustrate the capabilities of the DHPSF by exploring large-scale membrane reorganization in human T cells after receptor triggering, and by using single-particle tracking to image several mammalian proteins, including membrane, cytoplasmic, and nuclear proteins in T cells and embryonic stem cells.
authors.affiliation: Department of Chemistry, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Alexander R
authors.initials: AR
authors.lastname: Carr
authors.affiliation: Department of Chemistry, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Aleks
authors.initials: A
authors.lastname: Ponjavic
authors.affiliation: Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Srinjan
authors.initials: S
authors.lastname: Basu
authors.affiliation: Department of Chemistry, University of Cambridge, Cambridge, United Kingdom.
authors.forename: James
authors.initials: J
authors.lastname: McColl
authors.affiliation: Radcliffe Department of Clinical Medicine and Medical Research Council Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom.
authors.forename: Ana Mafalda
authors.initials: AM
authors.lastname: Santos
authors.affiliation: Radcliffe Department of Clinical Medicine and Medical Research Council Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom.
authors.forename: Simon
authors.initials: S
authors.lastname: Davis
authors.affiliation: Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Ernest D
authors.initials: ED
authors.lastname: Laue
authors.affiliation: Department of Chemistry, University of Cambridge, Cambridge, United Kingdom.
authors.forename: David
authors.initials: D
authors.lastname: Klenerman
authors.affiliation: Department of Chemistry, University of Cambridge, Cambridge, United Kingdom. Electronic address: sl591@cam.ac.uk.
authors.forename: Steven F
authors.initials: SF
authors.lastname: Lee
mesh_terms: D000465:Algorithms; D000818:Animals; D002138:Calibration; D002467:Cell Nucleus / Q000378:metabolism; D004058:Diffusion; D005057:Eukaryotic Cells / Q000378:metabolism*; D005453:Fluorescence; D006801:Humans; D021621:Imaging, Three-Dimensional; D019169:Jurkat Cells; D051379:Mice; D000066450:Mouse Embryonic Stem Cells / Q000166:cytology; D013601:T-Lymphocytes / Q000378:metabolism