Noise-Free Visualization of Microscopic Calcium Signaling by Pixel-Wise Fitting
Noise-Free Visualization of Microscopic Calcium Signaling by Pixel-Wise Fitting
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DOI：
10.1161/circresaha.112.266403
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发表时间：
2012-06
影响因子：
20.1
通讯作者：
Qinghai Tian;L. Kaestner;P. Lipp
中科院分区：
文献类型：
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作者：
Qinghai Tian;L. Kaestner;P. Lipp
基本原理：我们对生理学和病理生理学心脏兴奋-收缩耦合的见解极大地受益于光学技术的重大进步，如高速共聚焦显微镜。这将像素停留时间推到纳秒的时域，导致低信噪比，这限制了数据分析和解释。目的：线扫描成像在高速共焦记录中一直是并且仍然是主要的。它只允许分析一小部分细胞的横截面（1.5%），但欣赏时空的兴奋-收缩耦合的细节是有助于进一步了解病理机制。我们的目的是提供一种新的分析工具，以提取否则隐藏的心脏兴奋收缩耦合的细节，从高速二维共焦图像系列。方法和结果：我们证明，高速2维共焦数据（150帧/秒）可以定量分析的像素方式拟合的方法，使用数学形式主义的现象学描述局部钙瞬变。这样的方法产生几乎无噪声的荧光数据源自分钟体积（0.025毫微微升），并允许提取详细的和最重要的定量和机械上的微观钙信号和兴奋收缩耦合的新信息，在一个强大的方式。结论：逐像素拟合为心脏兴奋-收缩耦合提供了新的见解。具体而言，它揭示了微观钙交替的水平上的个别耦合网站。微观钙交替是细胞交替的早期前体，因此将更多地揭示导致心律失常的机制。
Rationale: Our insights into physiological and pathophysiological cardiac excitation-contraction coupling has greatly benefited from significant advancement in optical technologies such as high-speed confocal microscopy. This has pushed pixel dwell times into the time domain of nanoseconds, resulting in low signal-to-noise ratios, which have limited data analysis and interpretation. Objective: Line scan imaging has been and still is dominant in high speed confocal recording. It allows analysis only of a small fraction of a cell's cross section (1.5%), but the appreciation of spatiotemporal fine details of excitation-contraction coupling is instrumental for the further understanding of pathological mechanisms. We aim to provide a novel analysis tool to extract otherwise hidden fine details in cardiac excitation-contraction coupling from high-speed 2-dimensional confocal image series. Methods and Results: We demonstrate that high-speed 2-dimensional confocal data (150 frames/s) can be analyzed quantitatively by a pixel-wise fitting approach, using a mathematical formalism to phenomenologically describe local calcium transients. Such an approach produces virtually noise-free fluorescence data originating from minute volumes (0.025 femtoliter) and allows extraction of detailed and most importantly quantitative and mechanistically novel information on microscopic calcium signaling and excitation-contraction coupling in a robust manner. Conclusions: Pixel-wise fitting provides novel insights into cardiac excitation-contraction coupling. Specifically, it revealed microscopic calcium alternans on the level of individual coupling sites. Microscopic calcium alternans is an early precursor of cellular alternans and as such will shed more light onto this mechanism leading to cardiac arrhythmia.