Quantification of fibrous spatial point patterns from single‐molecule localization microscopy (SMLM) data

Motivation: Unlike conventional microscopy which produces pixelated images, SMLM produces data in the form of a list of localization coordinates—a spatial point pattern (SPP). Often, such SPPs are analyzed using cluster analysis algorithms to quantify molecular clustering within, for example, the plasma membrane. While SMLM cluster analysis is now well developed, techniques for analyzing fibrous structures remain poorly explored. Results: Here, we demonstrate a statistical methodology, based on Ripley's K‐function to quantitatively assess fibrous structures in 2D SMLM datasets. Using simulated data, we present the underlying theory to describe fiber spatial arrangements and show how these descriptions can be quantitatively derived from pointillist datasets. We also demonstrate the techniques on experimental data acquired using the image reconstruction by integrating exchangeable single‐molecule localization (IRIS) approach to SMLM, in the context of the fibrous actin meshwork at the T cell immunological synapse, whose structure has been shown to be important for T cell activation. Availability and Implementation: Freely available on the web at https://github.com/RubyPeters/Angular‐Ripleys‐K. Implemented in MatLab. Contact: dylan.owen@kcl.ac.uk Supplementary information: Supplementary data are available at Bioinformatics online.

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

[2]  N. Bertaux,et al.  Quantitative nanoscale imaging of orientational order in biological filaments by polarized superresolution microscopy , 2016, Proceedings of the National Academy of Sciences.

[3]  P. Annibale,et al.  Identification of clustering artifacts in photoactivated localization microscopy , 2011, Nature Methods.

[4]  Mark Bates,et al.  Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging , 2011, Nature Methods.

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

[6]  P. Annibale,et al.  Quantitative Photo Activated Localization Microscopy: Unraveling the Effects of Photoblinking , 2011, PloS one.

[7]  Suliana Manley,et al.  Quantitative evaluation of software packages for single-molecule localization microscopy , 2015, Nature Methods.

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

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

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

[11]  P. Annibale,et al.  Photoactivatable Fluorescent Protein mEos2 Displays Repeated Photoactivation after a Long-Lived Dark State in the Red Photoconverted Form , 2010 .

[12]  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.

[13]  Erwin Frey,et al.  Actin-binding proteins sensitively mediate F-actin bundle stiffness. , 2006, Nature materials.

[14]  Akihiro Kusumi,et al.  Ultrafine membrane compartments for molecular diffusion as revealed by single molecule techniques. , 2004, Biophysical journal.

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

[16]  Matthew F Krummel,et al.  Mediation of T-cell activation by actin meshworks. , 2010, Cold Spring Harbor perspectives in biology.

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

[18]  T. Pollard,et al.  Annual review of biophysics and biomolecular structure , 1992 .

[19]  Robert G. Parton,et al.  Direct visualization of Ras proteins in spatially distinct cell surface microdomains , 2003, The Journal of cell biology.

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

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

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

[23]  Mike Heilemann,et al.  The effect of photoswitching kinetics and labeling densities on super-resolution fluorescence imaging. , 2010, Journal of biotechnology.

[24]  J. Burkhardt,et al.  The actin cytoskeleton in T cell activation. , 2008, Annual review of immunology.

[25]  Gerhard J Schütz,et al.  Varying label density allows artifact-free analysis of membrane-protein nanoclusters , 2016, Nature Methods.

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

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

[28]  Michael Loran Dustin,et al.  The immunological synapse , 2002, Arthritis research.

[29]  Erwin Frey,et al.  Cytoskeletal bundle mechanics. , 2007, Biophysical journal.

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

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