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.
Highlights
Single-molecule imaging (SMI) methods, including superresolution techniques such as photoactivated localization microscopy (PALM) [1] and stochastic optical reconstruction microscopy [(d)STORM] [2,3,4], are widely used to study the dynamics and organization of proteins within cells at a resolution below the diffraction limit [5]
The majority of SMI has been carried out using total internal reflection fluorescence microscopy (TIRFM) [6], which is restricted to probing interfaces
We demonstrate that the technique can be applied to multiple cell types, including T cells and embryonic stem (ES) cells, enabling new biophysical studies on the 3D organization and dynamics of membrane, cytoplasmic, and nuclear proteins
Summary
Single-molecule imaging (SMI) methods, including superresolution techniques such as photoactivated localization microscopy (PALM) [1] and (direct) stochastic optical reconstruction microscopy [(d)STORM] [2,3,4], are widely used to study the dynamics and organization of proteins within cells at a resolution below the diffraction limit [5]. Other excitation confinement techniques, such as light-sheet microscopy, are becoming widely used owing to their ability to image above the coverslip with high contrast [7], enabling intracellular imaging and minimizing surface-induced perturbations of protein dynamics [8]. Like TIRFM, these methods typically employ two-dimensional (2D) imaging, which complicates the study of curved and irregular structures above the coverslip. This limitation has motivated the development of a number of three-dimensional (3D) SMI techniques, which allow for complete sampling of the protein distribution with high precision in all dimensions.
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