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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.

Original publication

DOI

10.1016/j.bpj.2017.02.023

Type

Journal article

Journal

Biophys J

Publication Date

11/04/2017

Volume

112

Pages

1444 - 1454

Keywords

Algorithms, Animals, Calibration, Cell Nucleus, Diffusion, Eukaryotic Cells, Fluorescence, Humans, Imaging, Three-Dimensional, Jurkat Cells, Mice, Mouse Embryonic Stem Cells, T-Lymphocytes