Three-dimensional super-resolution in eukaryotic cells using the double-helix point spread function

Carr, Alexander R., Ponjavic, Aleks, Basu, Srinjan, McColl, James, Santos, Ana Mafalda, Davis, Simon, Laue, Ernest D., Klenerman, David and Lee, Steven F. (2017) Three-dimensional super-resolution in eukaryotic cells using the double-helix point spread function. Biophysical Journal, 112 (7). pp. 1444-1454. ISSN 0006-3495

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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.

Item Type: Article
Faculty \ School: Faculty of Science
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Depositing User: LivePure Connector
Date Deposited: 15 Aug 2022 15:31
Last Modified: 22 Oct 2022 07:53
DOI: 10.1016/j.bpj.2017.02.023


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