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

Alexander R. Carr, Aleks Ponjavic, Srinjan Basu, James McColl, Ana Mafalda Santos, Simon Davis, Ernest D. Laue, David Klenerman, Steven F. Lee

Research output: Contribution to journalArticlepeer-review

27 Citations (SciVal)

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.

Original languageEnglish
Pages (from-to)1444-1454
Number of pages11
JournalBiophysical Journal
Volume112
Issue number7
DOIs
Publication statusPublished - 11 Apr 2017

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