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1 Cardiac Membrane Research Laboratory, Simon Fraser University, Burnaby, BC, Canada; Cardiovascular Sciences, BC Research Institute for Children and Women's Health, Vancouver, BC, Canada
2 Department of Physiology, University of British Columbia, Vancouver, BC, Canada
* To whom correspondence should be addressed. E-mail: tibbits{at}sfu.ca.
Colocalization of dihydropyridine (DHPR) and ryanodine (RyR) receptors, a key determinant of Ca2+-induced Ca2+ release, was previously estimated in 3, 6, 10 and 20-day old rabbit ventricular myocytes using immunocytochemistry and confocal microscopy. We now report on the effects of deconvolution (using a Maximum Likelihood Estimation algorithm) on the calculation of colocalization indices. Clusters of DHPR and RyR can be accurately represented as point sources of fluorescence, which enables a model of their relative distributions to be constructed using images of point spread functions to simulate their fluorescence inside a cell. This model was used to investigate the effects of deconvolution on colocalization as a function of separation distance. Deconvolution resulted in significant improvements in both axial and transverse resolutions, producing significant increases in clarity. Comparisons of intensity profiles (FWHM) pre- and post-deconvolution showed decreased dispersion of the fluorescent signal and a corresponding decrease in false colocalization as determined by fluorescence modeling. This hypothesis was extended to physiological data previously collected. The number of colocalized voxels was quantified after deconvolution and the degree of colocalization of DHPR with RyR decreased significantly after deconvolution in all age groups; 3 day (62 ± 2% before deconvolution, 43 ± 3 after deconvolution) to the 20-day old (79 ± 1% before deconvolution, 63 ± 2% after deconvolution). The data demonstrate that confocal images should be deconvolved prior to any quantitative analysis, such as colocalization index determination, to minimize the detrimental effects of out-of-focus light in co-incident voxels.
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