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Three-Dimensional Super-Resolution in Eukaryotic Cells Using the Double-Helix Point Spread Function

Published version
Peer-reviewed

Type

Article

Change log

Authors

Carr, AR 
Ponjavic, A 
McColl, J 
Santos, AM 

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.

Description

Keywords

algorithms, animals, calibration, cell nucleus, diffusion, eukaryotic cells, fluorescence, humans, imaging, three-dimensional, Jurkat cells, mice, mouse embryonic stem cells, T-lymphocytes

Journal Title

Biophysical Journal

Conference Name

Journal ISSN

0006-3495
1542-0086

Volume Title

112

Publisher

Elsevier (Cell Press)
Sponsorship
Engineering and Physical Sciences Research Council (EP/M003663/1)
Wellcome Trust (206291/Z/17/Z)
Medical Research Council (MR/P019471/1)
Engineering and Physical Sciences Research Council (EP/L027631/1)
Medical Research Council (MR/M010082/1)
Wellcome Trust (via University of Oxford) (207547/Z/17/Z)
We thank the Royal Society for the University Research Fellowship of S.F.L. (UF120277). This work was kindly funded by the Engineering and Physical Sciences Research Council (EP/M003663/1) and by the Wellcome Trust.