Near-Field Scanning Fluorescence Microscopy Study of Ion Channel Clusters in Cardiac Myocyte Membranes

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DOIResolve DOI: http://doi.org/10.1529/biophysj.104.046383
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TypeArticle
Journal titleBiophys. J.
Volume87
Issue5
Pages35253535; # of pages: 11
AbstractNear-field scanning optical microscopy (NSOM) has been used to study the nanoscale distribution of voltage-gated L-type Ca2+ ion channels, which play an important role in cardiac function. NSOM fluorescence imaging of immunostained cardiac myocytes (H9C2 cells) demonstrates that the ion channel is localized in small clusters with an average diameter of 100 nm. The clusters are randomly distributed throughout the cell membrane, with some larger fluorescent patches that high-resolution images show to consist of many small closely-spaced clusters. We have imaged unstained cells to assess the contribution of topography-induced artifacts and find that the topography-induced signal is <10% of the NSOM fluorescence intensity. We have also examined the dependence of the NSOM signal intensity on the tip-sample separation to assess the contributions from fluorophores that are significantly below the cell surface. This indicates that chromophores >[~]200 nm below the probe will have negligible contributions to the observed signal. The ability to quantitatively measure small clusters of ion channels will facilitate future studies that examine changes in protein localization in stimulated cells and during cardiac development. Our work illustrates the potential of NSOM for studying membrane domains and protein localization/colocalization on a length scale which exceeds that available with optical microscopy.
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AffiliationNational Research Council Canada; NRC Steacie Institute for Molecular Sciences
Peer reviewedNo
NPARC number12327194
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Record identifier04b358b3-fd44-4318-9f03-be544e1b2e41
Record created2009-09-10
Record modified2016-05-09
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