title: Combining confocal and single molecule localisation microscopy: A correlative approach to multi-scale tissue imaging.
abstract: Many biological questions require information at different spatial scales that include molecular, organelle, cell and tissue scales. Here we detail a method of multi-scale imaging of human cardiac tissue by correlatively combining nano-scale data of direct stochastic optical reconstruction microscopy (dSTORM) with cellular and tissue level data provided by confocal microscopy. By utilising conventional fluorescence dyes the same cellular structures can be imaged with both modalities. Human cardiac tissue was first imaged at the nanoscale to identify macro-molecular membrane complexes containing the cardiac muscle proteins junctophilin (JPH) and the ryanodine receptor (RyR). The distribution of these proteins and an additional cell membrane marker (wheat germ agglutinin, WGA) were subsequently imaged by confocal microscopy. By segmenting dSTORM data into membrane and non-membrane components we demonstrate increased colocalization of RyR with JPH at the plasma-membrane as compared to intracellular compartments. Strategies for antibody labelling, quality control, locating and aligning structures between modalities, and analysis of combined multi-scaled data sets are described.
authors: Department of Physiology, University of Auckland, Auckland, New Zealand.
authors: David J
authors: DJ
authors: Crossman
authors: Department of Physiology, University of Auckland, Auckland, New Zealand.
authors: Yufeng
authors: Y
authors: Hou
authors: Biomedical Physics, University of Exeter, UK.
authors: Izzy
authors: I
authors: Jayasinghe
authors: Department of Physiology, University of Auckland, Auckland, New Zealand; Department of Cell Biology, Yale University, New Haven, USA.
authors: David
authors: D
authors: Baddeley
authors: Department of Physiology, University of Auckland, Auckland, New Zealand; Biomedical Physics, University of Exeter, UK. Electronic address: c.soeller@exeter.ac.uk.
authors: Christian
authors: C
authors: Soeller
mesh_terms: D006801:Humans; D008565:Membrane Proteins; D018613:Microscopy, Confocal / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D057054:Molecular Imaging / Q000379:methods*; D009206:Myocardium / Q000166:cytology* / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel
chemical_list: D008565:Membrane Proteins; D019837:Ryanodine Receptor Calcium Release Channel; C412507:junctophilin
keywords: Correlative microscopy; Human heart tissue; Single molecule localisation microscopy; Super-resolution; dSTORM