Quantitative fibre analysis of single-molecule localization microscopy data

Single molecule localization microscopy (SMLM) methods produce data in the form of a spatial point pattern (SPP) of all localized emitters. Whilst numerous tools exist to quantify molecular clustering in SPP data, the analysis of fibrous structures has remained understudied. Taking the SMLM localization coordinates as input, we present an algorithm capable of tracing fibrous structures in data generated by SMLM. Based upon a density parameter tracing routine, the algorithm outputs several fibre descriptors, such as number of fibres, length of fibres, area of enclosed regions and locations and angles of fibre branch points. The method is validated in a variety of simulated conditions and experimental data acquired using the image reconstruction by integrating exchangeable single-molecule localization (IRIS) technique. For this, the nanoscale architecture of F-actin at the T cell immunological synapse in both untreated and pharmacologically treated cells, designed to perturb actin structure, was analysed.

[1]  Akihiro Kusumi,et al.  Paradigm shift of the plasma membrane concept from the two-dimensional continuum fluid to the partitioned fluid: high-speed single-molecule tracking of membrane molecules. , 2005, Annual review of biophysics and biomolecular structure.

[2]  E. Betzig,et al.  Formin-generated actomyosin arcs propel T cell receptor microcluster movement at the immune synapse , 2016, The Journal of cell biology.

[3]  David J. Williamson,et al.  Live-Cell Super-resolution Reveals F-Actin and Plasma Membrane Dynamics at the T Cell Synapse. , 2017, Biophysical journal.

[4]  Alexander Babich,et al.  F-actin polymerization and retrograde flow drive sustained PLCγ1 signaling during T cell activation , 2012, The Journal of cell biology.

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

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

[7]  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.

[8]  W. Webb,et al.  Precise nanometer localization analysis for individual fluorescent probes. , 2002, Biophysical journal.

[9]  David J. Williamson,et al.  Bayesian cluster identification in single-molecule localization microscopy data , 2015, Nature Methods.

[10]  Daniel Choquet,et al.  SR-Tesseler: a method to segment and quantify localization-based super-resolution microscopy data , 2015, Nature Methods.

[11]  M. Rao,et al.  Active Remodeling of Cortical Actin Regulates Spatiotemporal Organization of Cell Surface Molecules , 2012, Cell.

[12]  Suliana Manley,et al.  Functional nanoscale organization of signaling molecules downstream of the T cell antigen receptor. , 2011, Immunity.

[13]  Mark M Davis,et al.  TCR and Lat are expressed on separate protein islands on T cell membranes and concatenate during activation , 2010, Nature Immunology.

[14]  M. Rao,et al.  Nanoclusters of GPI-Anchored Proteins Are Formed by Cortical Actin-Driven Activity , 2008, Cell.

[15]  Nanoscopic compartmentalization of membrane protein motion at the axon initial segment , 2016, bioRxiv.

[16]  Juliette Griffié,et al.  Quantification of fibrous spatial point patterns from single‐molecule localization microscopy (SMLM) data , 2017, Bioinform..

[17]  T. Svitkina,et al.  Natural Killer Cell Lytic Granule Secretion Occurs through a Pervasive Actin Network at the Immune Synapse , 2011, PLoS biology.

[18]  Prabuddha Sengupta,et al.  Probing protein heterogeneity in the plasma membrane using PALM and pair correlation analysis , 2011, Nature Methods.

[19]  M. Maruoka,et al.  Multitarget super-resolution microscopy with high-density labeling by exchangeable probes , 2015, Nature Methods.

[20]  Igor Orlov,et al.  ClusterViSu, a method for clustering of protein complexes by Voronoi tessellation in super-resolution microscopy , 2016, Scientific Reports.

[21]  David Baddeley,et al.  Visualization of Localization Microscopy Data , 2010, Microscopy and Microanalysis.

[22]  Christian Eggeling,et al.  Cytoskeletal actin dynamics shape a ramifying actin network underpinning immunological synapse formation , 2017, Science Advances.

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

[24]  Michael W. Davidson,et al.  Actin Depletion Initiates Events Leading to Granule Secretion at the Immunological Synapse , 2015, Immunity.

[25]  Sjoerd Stallinga,et al.  Co-Orientation: Quantifying Simultaneous Co-Localization and Orientational Alignment of Filaments in Light Microscopy , 2015, PloS one.

[26]  T. Takenawa,et al.  Spatial and temporal regulation of actin polymerization for cytoskeleton formation through Arp2/3 complex and WASP/WAVE proteins. , 2002, Cell motility and the cytoskeleton.

[27]  P. Verveer,et al.  Coordinate-based colocalization analysis of single-molecule localization microscopy data , 2011, Histochemistry and Cell Biology.

[28]  Astrid Magenau,et al.  Pre-existing clusters of the adaptor Lat do not participate in early T cell signaling events , 2011, Nature Immunology.

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

[30]  Astrid Magenau,et al.  Quantitative analysis of three-dimensional fluorescence localization microscopy data. , 2013, Biophysical journal.

[31]  Astrid Magenau,et al.  PALM imaging and cluster analysis of protein heterogeneity at the cell surface , 2010, Journal of biophotonics.

[32]  B. Ripley Modelling Spatial Patterns , 1977 .

[33]  Katharina Gaus,et al.  Method for co-cluster analysis in multichannel single-molecule localisation data , 2014, Histochemistry and Cell Biology.

[34]  Akihiro Kusumi,et al.  Rapid hop diffusion of a G-protein-coupled receptor in the plasma membrane as revealed by single-molecule techniques. , 2005, Biophysical journal.

[35]  Pakorn Kanchanawong,et al.  Extracting microtubule networks from superresolution single-molecule localization microscopy data , 2017, Molecular biology of the cell.

[36]  Juliette Griffié,et al.  Topographic prominence as a method for cluster identification in single‐molecule localisation data , 2015, Journal of biophotonics.

[37]  Akihiro Kusumi,et al.  Three-dimensional reconstruction of the membrane skeleton at the plasma membrane interface by electron tomography , 2006, The Journal of cell biology.

[38]  Guy M. Hagen,et al.  ThunderSTORM: a comprehensive ImageJ plug-in for PALM and STORM data analysis and super-resolution imaging , 2014, Bioinform..

[39]  F. Simmel,et al.  Single-molecule kinetics and super-resolution microscopy by fluorescence imaging of transient binding on DNA origami. , 2010, Nano letters.

[40]  Patrick Rubin-Delanchy,et al.  3D Bayesian cluster analysis of super-resolution data reveals LAT recruitment to the T cell synapse , 2017, Scientific Reports.

[41]  Michael D. Mason,et al.  Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. , 2006, Biophysical journal.