Ca²⁺ sparks are instrumental to understand physiological and pathological Ca²⁺ signaling in the heart. High-speed two spatially dimensional (2D) confocal imaging (>120 Hz) enables acquisition of sparks with high-content information, however, owing to a wide variety of different acquisition modalities the question arises: how much they reflect the “true” Ca²⁺ spark properties. To address this issue, we compared a fast point and a 2D-array scanner equipped with a range of different detectors. As a quasi-standard biological sample, we employed Ca²⁺ sparks in permeabilized and intact mouse ventricular myocytes and utilized an unbiased, automatic Ca²⁺ spark analysis tool, iSpark. Data from the point scanner suffered from low pixel photon fluxes (PPF) concomitant with high Poissonian noise. Images from the 2D-array scanner displayed substantially increased PPF, lower Poissonian noise and almost 3-fold increased sign-to-noise ratios. Noteworthy, data from the 2D scanner suffered from considerable inter-pinhole crosstalk evident for the permeabilized cells. Spark properties, such as frequency, amplitude, decay time and spatial spread were distinctly different for any scanner/detector combination. Our study reveals that the apparent Ca²⁺ spark properties differ dependent on the particular recording modality and set-up employed, quantitatively.

Ca ²⁺火花有助于了解心脏中的生理和病理性 Ca ²⁺信号传导。高速二维 (2D) 共焦成像 (>120 Hz) 能够采集具有高含量信息的火花，但是，由于各种不同的采集方式，问题出现了：它们在多大程度上反映了“真实”Ca ²⁺火花属性。为了解决这个问题，我们比较了一个快速点和一个配备了一系列不同探测器的二维阵列扫描仪。作为准标准生物样品，我们在透化和完整的小鼠心室肌细胞中使用 Ca ²⁺火花，并利用无偏的自动 Ca ²⁺火花分析工具，iSpark。来自点扫描仪的数据受到低像素光子通量 (PPF) 和高泊松噪声的影响。二维阵列扫描仪的图像显示 PPF 显着增加，泊松噪声降低，信噪比增加近 3 倍。值得注意的是，来自 2D 扫描仪的数据遭受了明显的透化细胞的针孔间串扰。对于任何扫描仪/探测器组合，火花特性，例如频率、幅度、衰减时间和空间扩展都明显不同。我们的研究表明，明显的 Ca ²⁺火花特性在数量上因特定的记录方式和设置而不同。