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Journal Article
Research Support, Non-U.S. Gov't
Combining confocal and single molecule localisation microscopy: A correlative approach to multi-scale tissue imaging.
Methods : a Companion to Methods in Enzymology 2015 October 16
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.
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