Multidimensional detection and analysis of Ca2+ sparks in cardiac myocytes
Multidimensional detection and analysis of Ca2+ sparks in cardiac myocytes
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DOI：
10.1529/biophysj.106.089359
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发表时间：
2007-06-01
影响因子：
3.4
通讯作者：
Parker, Kevin Kit
中科院分区：
文献类型：
--
作者：
Bray, Mark-Anthony;Geisse, Nicholas A.;Parker, Kevin Kit
研究钙火花的形态及其与心肌细胞结构的关系为理解兴奋-收缩耦合机制提供了直接的手段。传统的共焦线扫描获得了优良的时间火花分辨率，但以牺牲垂直维度的空间信息为代价。为了解决这个问题，我们开发了一种方法来识别和分析通过二维共焦或电荷耦合设备显微镜获得的火花。该技术包括非线性减去背景荧光，根据噪声水平对数据进行阈值处理，然后通过广义极值检验定位火花峰，同时注意检测和分离相邻峰。在这篇文章中，我们描述了该算法，将其性能与先前验证的火花检测算法进行了比较，并通过将其应用于合成复制品和在钙瞬变期间显示火花的二维各向同性肌细胞单层的实验制备来演示该算法。我们发现，在时间非均匀背景荧光的情况下，我们的多维算法比传统方法具有更好的灵敏度，并且当火花密度较高时，峰分割的包含降低了假阴性率。我们的算法是稳健的，可以有效地用于不同的成像模式，并允许在亚细胞、细胞和组织准备中进行火花识别和量化。
Examining calcium spark morphology and its relationship to the structure of the cardiac myocyte offers a direct means of understanding excitation-contraction coupling mechanisms. Traditional confocal line scanning achieves excellent temporal spark resolution but at the cost of spatial information in the perpendicular dimension. To address this, we developed a methodology to identify and analyze sparks obtained via two-dimensional confocal or charge-coupled device microscopy. The technique consists of nonlinearly subtracting the background fluorescence, thresholding the data on the basis of noise level, and then localizing the spark peaks via a generalized extrema test, while taking care to detect and separate adjacent peaks. In this article, we describe the algorithm, compare its performance to a previously validated spark detection algorithm, and demonstrate it by applying it to both a synthetic replica and an experimental preparation of a two-dimensional isotropic myocyte monolayer exhibiting sparks during a calcium transient. We find that our multidimensional algorithm provides better sensitivity than the conventional method under conditions of temporally heterogeneous background fluorescence, and the inclusion of peak segmentation reduces false negative rates when spark density is high. Our algorithm is robust and can be effectively used with different imaging modalities and allows spark identification and quantification in subcellular, cellular, and tissue preparations.