Deconvolution of confocal images of dihydropyridine and ryanodine receptors in developing cardiomyocytes
Deconvolution of confocal images of dihydropyridine and ryanodine receptors in developing cardiomyocytes
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DOI：
10.1152/japplphysiol.00089.2004
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发表时间：
2004-09-01
影响因子：
3.3
通讯作者：
Tibbits, GF
中科院分区：
文献类型：
--
作者：
Sedarat, F;Lin, E;Tibbits, GF
二氢吡啶（DHPR）和Ryanodine（RyR）受体的共定位，钙离子诱导的钙释放的关键决定因素，以前估计在3-，6-，10-，和20日龄的兔心室肌细胞免疫细胞化学和共聚焦显微镜。我们现在报告反卷积（使用最大似然估计算法）对共定位指数计算的影响。DHPR和RyR的簇可以准确地表示为荧光的点源，这使得能够使用点扩散函数的图像来构建它们的相对分布的模型，以模拟它们在细胞内的荧光。该模型被用来研究作为分离距离的函数的去卷积对共定位的影响。去卷积导致轴向和横向分辨率的显着改善，产生显着增加的清晰度。强度曲线（全宽半最大值）的比较前和后反卷积显示减少的荧光信号的分散和相应的减少假共定位确定的荧光建模。这一假设被扩展到以前收集的生理数据。共定位体素的数量在去卷积后定量，并且在所有年龄组中，DHPR与RyR的共定位程度在去卷积后显著降低：3天（去卷积前62 +/- 2%，去卷积后43 +/- 3）至20天（去卷积前79 +/- 1%，去卷积后63 +/- 2%）。这些数据表明，共焦图像应在任何定量分析之前进行去卷积，例如共定位指数确定，以最大限度地减少重合体素中的失焦光的有害影响。
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.