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

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.

[1]  Heng Li,et al.  Improved localization accuracy in double-helix point spread function super-resolution fluorescence microscopy using selective-plane illumination , 2014, Other Conferences.

[2]  Partho Ghosh,et al.  Structure of the complex between human T-cell receptor, viral peptide and HLA-A2 , 1996, Nature.

[3]  W. E. Moerner,et al.  Localizing and tracking single nanoscale emitters in three dimensions with high spatiotemporal resolution using a double-helix point spread function. , 2010, Nano letters.

[4]  Lucien E. Weiss,et al.  Precise Three-Dimensional Scan-Free Multiple-Particle Tracking over Large Axial Ranges with Tetrapod Point Spread Functions , 2015, Nano letters.

[5]  J. J. Macklin,et al.  A general method to improve fluorophores for live-cell and single-molecule microscopy , 2014, Nature Methods.

[6]  D. Klenerman,et al.  The changing point-spread function: single-molecule-based super-resolution imaging , 2014, Histochemistry and Cell Biology.

[7]  E. Betzig,et al.  Imaging live-cell dynamics and structure at the single-molecule level. , 2015, Molecular cell.

[8]  Ying S Hu,et al.  Single-molecule super-resolution light-sheet microscopy. , 2014, Chemphyschem : a European journal of chemical physics and physical chemistry.

[9]  Ying S Hu,et al.  Superresolution imaging reveals nanometer- and micrometer-scale spatial distributions of T-cell receptors in lymph nodes , 2016, Proceedings of the National Academy of Sciences.

[10]  Matthew D Lew,et al.  Corkscrew point spread function for far-field three-dimensional nanoscale localization of pointlike objects. , 2011, Optics letters.

[11]  J. Biteen,et al.  Nanoscopic Cellular Imaging: Confinement Broadens Understanding. , 2016, ACS nano.

[12]  W E Moerner,et al.  Single-molecule spectroscopy and imaging of biomolecules in living cells. , 2010, Analytical chemistry.

[13]  Matthew D. Lew,et al.  Easy-DHPSF open-source software for three-dimensional localization of single molecules with precision beyond the optical diffraction limit. , 2013, Protocol exchange.

[14]  Adam S. Backer,et al.  Optimal point spread function design for 3D imaging. , 2014, Physical review letters.

[15]  Mark Bates,et al.  Three-Dimensional Super-Resolution Imaging by Stochastic Optical Reconstruction Microscopy , 2008, Science.

[16]  Ji Won Yoon,et al.  DySCo: Quantitating Associations of Membrane Proteins Using Two-Color Single-Molecule Tracking , 2009, Biophysical journal.

[17]  M. Brameshuber,et al.  Two-color single molecule tracking combined with photobleaching for the detection of rare molecular interactions in fluid biomembranes , 2010 .

[18]  L. Zakharova.  Evolution of adaptive immunity , 2009, Biology Bulletin.

[19]  S. Bromley,et al.  The immunological synapse: a molecular machine controlling T cell activation. , 1999, Science.

[20]  M. Garcia-Parajo,et al.  Chromatin Fibers Are Formed by Heterogeneous Groups of Nucleosomes In Vivo , 2015, Cell.

[21]  Matthew D. Lew,et al.  Three-dimensional localization precision of the double-helix point spread function versus astigmatism and biplane. , 2010, Applied physics letters.

[22]  Wesley R. Legant,et al.  High density three-dimensional localization microscopy across large volumes , 2016, Nature Methods.

[23]  M. Davidson,et al.  Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics , 2015, Science.

[24]  Akihiro Kusumi,et al.  Tracking single molecules at work in living cells. , 2014, Nature chemical biology.

[25]  Alexander Y Katsov,et al.  fast multicolor 3 d imaging using aberration-corrected multifocus microscopy , 2012 .

[26]  M. Saxton.  Single-particle tracking: the distribution of diffusion coefficients. , 1997, Biophysical journal.

[27]  Michael M. Kazhdan,et al.  Poisson surface reconstruction , 2006, SGP '06.

[28]  Michael Loran Dustin,et al.  Analysis of two-dimensional dissociation constant of laterally mobile cell adhesion molecules. , 2007, Biophysical journal.

[29]  M. Heilemann,et al.  Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes. , 2008, Angewandte Chemie.

[30]  Wei Zhang,et al.  The Nucleosome Remodeling and Deacetylase Complex NuRD Is Built from Preformed Catalytically Active Sub-modules , 2016, Journal of molecular biology.

[31]  Michael J Rust,et al.  Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM) , 2006, Nature Methods.

[32]  J. Lippincott-Schwartz,et al.  Imaging Intracellular Fluorescent Proteins at Nanometer Resolution , 2006, Science.

[33]  X. Zhuang,et al.  Isotropic 3D Super-resolution Imaging with a Self-bending Point Spread Function , 2014, Nature photonics.

[34]  B. Hendrich,et al.  CHD4 in the DNA-damage response and cell cycle progression: not so NuRDy now , 2013, Biochemical Society transactions.

[35]  W E Moerner,et al.  Quantitative multicolor subdiffraction imaging of bacterial protein ultrastructures in three dimensions. , 2013, Nano letters.

[36]  Chrysanthe Preza,et al.  Characterization of a three-dimensional double-helix point-spread function for fluorescence microscopy in the presence of spherical aberration , 2013, Journal of biomedical optics.

[37]  Michael M. Kazhdan,et al.  Screened poisson surface reconstruction , 2013, TOGS.

[38]  S. Hess,et al.  Three-dimensional sub–100 nm resolution fluorescence microscopy of thick samples , 2008, Nature Methods.

[39]  Stefan W. Hell,et al.  Effect of the specimen refractive index on the imaging of a confocal fluorescence microscope employing high aperture oil immersion lenses , 1995 .

[40]  J. Mege,et al.  Quantification of cell surface roughness; a method for studying cell mechanical and adhesive properties. , 1986, Journal of theoretical biology.

[41]  M. Neil,et al.  Remodelling of Cortical Actin Where Lytic Granules Dock at Natural Killer Cell Immune Synapses Revealed by Super-Resolution Microscopy , 2011, PLoS biology.

[42]  Kishan Dholakia,et al.  A Compact Airy Beam Light Sheet Microscope with a Tilted Cylindrical Lens References and Links , 2022 .

[43]  Matthew D. Lew,et al.  Three-dimensional superresolution colocalization of intracellular protein superstructures and the cell surface in live Caulobacter crescentus , 2011, Proceedings of the National Academy of Sciences.

[44]  Paul D. Dunne,et al.  Remarkably low affinity of CD4/peptide-major histocompatibility complex class II protein interactions , 2016, Proceedings of the National Academy of Sciences.

[45]  B. Pernis.  Internalization of lymphocyte membrane components. , 1985, Immunology today.

[46]  M. Dahan,et al.  ViSP: representing single-particle localizations in three dimensions , 2013, Nature Methods.

[47]  M. Moser.  Faculty Opinions recommendation of Polarized release of T-cell-receptor-enriched microvesicles at the immunological synapse. , 2014 .

[48]  R. Alon,et al.  Three-dimensional localization of T-cell receptors in relation to microvilli using a combination of superresolution microscopies , 2016, Proceedings of the National Academy of Sciences.

[49]  D. Braun,et al.  Single-Molecule Imaging in Living Drosophila Embryos with Reflected Light-Sheet Microscopy. , 2016, Biophysical journal.

[50]  James McColl,et al.  The T Cell Receptor Triggering Apparatus Is Composed of Monovalent or Monomeric Proteins , 2011, The Journal of Biological Chemistry.

[51]  Jordan R. Myers,et al.  Ultra-High Resolution 3D Imaging of Whole Cells , 2016, Cell.

[52]  William J. Fitzgerald,et al.  Single-molecule level analysis of the subunit composition of the T cell receptor on live T cells , 2007, Proceedings of the National Academy of Sciences.

[53]  Omer Dushek,et al.  Mechanisms for T cell receptor triggering , 2011, Nature Reviews Immunology.

[54]  Mark Bates,et al.  Multicolor Super-Resolution Imaging with Photo-Switchable Fluorescent Probes , 2007, Science.

[55]  Clare M. Waterman,et al.  Advances in light-based imaging of three-dimensional cellular ultrastructure. , 2012, Current opinion in cell biology.

[56]  Ulrich Kubitscheck,et al.  Light Sheet Microscopy for Single Molecule Tracking in Living Tissue , 2010, PloS one.

[57]  M. Dahan,et al.  Whole-cell, multicolor superresolution imaging using volumetric multifocus microscopy , 2014, Proceedings of the National Academy of Sciences.

[58]  D. Klenerman,et al.  A Quantitative Comparison of Single-Dye Tracking Analysis Tools Using Monte Carlo Simulations , 2013, PloS one.

[59]  M. Tokunaga,et al.  Highly inclined thin illumination enables clear single-molecule imaging in cells , 2008, Nature Methods.

[60]  S. Hell,et al.  Aberrations in confocal fluorescence microscopy induced by mismatches in refractive index , 1993 .

[61]  Y. Korchev,et al.  Plasma membrane topography and interpretation of single-particle tracks , 2010, Nature Methods.

[62]  C. Carman,et al.  Antigen Recognition Is Facilitated by Invadosome-like Protrusions Formed by Memory/Effector T Cells , 2012, The Journal of Immunology.

[63]  S. Simon,et al.  Imaging with total internal reflection fluorescence microscopy for the cell biologist , 2010, Journal of Cell Science.

[64]  Wesley R. Legant,et al.  3D imaging of Sox2 enhancer clusters in embryonic stem cells , 2014, eLife.

[65]  Wesley R. Legant,et al.  Single-Molecule Dynamics of Enhanceosome Assembly in Embryonic Stem Cells , 2014, Cell.

[66]  R. Hochstrasser,et al.  Wide-field subdiffraction imaging by accumulated binding of diffusing probes , 2006, Proceedings of the National Academy of Sciences.

[67]  Colin R. F. Monks,et al.  Three-dimensional segregation of supramolecular activation clusters in T cells , 1998, Nature.

[68]  C. Baldari,et al.  Regulation of Vesicular Traffic at the T Cell Immune Synapse: Lessons from the Primary Cilium , 2015, Traffic.

[69]  H. Qian,et al.  Single particle tracking. Analysis of diffusion and flow in two-dimensional systems. , 1991, Biophysical journal.