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INNOVATIVE METHODOLOGY

Deconvolution of confocal images of dihydropyridine and ryanodine receptors in developing cardiomyocytes

¹Cardiac Membrane Research Laboratory, Simon Fraser University, Burnaby V5A 1S6; ²Cardiovascular Sciences, British Columbia Research Institute for Children and Women's Health, Vancouver V5Z 4H4; and ³Department of Physiology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada

Submitted 28 January 2004 ; accepted in final form 1 April 2004

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 by immunocytochemistry and confocal microscopy. We now report on the effects of deconvolution (using a maximum-likelihood estimation algorithm) on the calculation of colocalization indexes. 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 (full-width half-maximum) pre- and postdeconvolution 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 days (62 ± 2% before deconvolution, 43 ± 3 after deconvolution) to 20 days old (79 ± 1% before deconvolution, 63 ± 2% after deconvolution). The data demonstrate that confocal images should be deconvolved before any quantitative analysis, such as colocalization index determination, to minimize the detrimental effects of out-of-focus light in coincident voxels.

cardiac excitation-contraction coupling; neonates; ontogeny; three-dimensional imaging

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