Izu L T, Wier W G, Balke C W
Department of Medicine, University of Maryland School of Medicine, Baltimore 21201, USA.
Biophys J. 1998 Sep;75(3):1144-62. doi: 10.1016/s0006-3495(98)74034-2.
A difficulty of using confocal microscopy to study Ca2+ sparks is the uncertainty of the linescan position with respect to the source of Ca2+ release. Random placement of the linescan is expected to result in a broad distribution of measured Ca2+ spark amplitudes (a) even if all Ca2+ sparks were generated identically. Thus variations in Ca2+ spark amplitude due to positional differences between confocal linescans and Ca2+ release site are intertwined with variations due to intrinsic differences in Ca2+ release properties. To separate these two sources of variations on the Ca2+ spark amplitude, we determined the effect changes of channel current or channel open time--collectively called the source strength, alpha--had on the measured Ca2+ spark amplitude histogram, N(a). This was done by 1) simulating Ca2+ release, Ca2+ and fluo-3 diffusion, and Ca2+ binding reactions; 2) simulation of image formation of the Ca2+ spark by a confocal microscope; and 3) using a novel automatic Ca2+ spark detector. From these results we derived an integral equation relating the probability density function of source strengths, f alpha (alpha), to N(a), which takes into account random positional variations between the source and linescan. In the special, but important, case that the spatial distribution of Ca(2+)-bound fluo-3 is Gaussian, we show the following: 1) variations of Ca2+ spark amplitude due to positional or intrinsic differences can be separated, and 2) f alpha (alpha) can, in principle, be calculated from the Ca2+ spark amplitude histogram since N(a) is the sum of shifted hyperbolas, where the magnitudes of the shifts and weights depend on f alpha (alpha). In particular, if all Ca2+ sparks were generated identically, then the plot of 1/N(a) against a will be a straight line. Multiple populations of channels carrying distinct currents are revealed by discontinuities in the 1/N(a) plot. 3) Although the inverse relationship between Ca2+ spark amplitude and decay time might be used to distinguish Ca2+ sparks from different channel populations, noise can render the measured decay times meaningless for small amplitude Ca2+ sparks.
利用共聚焦显微镜研究Ca2+火花的一个困难在于线扫描位置相对于Ca2+释放源的不确定性。即使所有Ca2+火花的产生方式完全相同，线扫描的随机放置预计也会导致测量的Ca2+火花幅度（a）分布广泛。因此，由于共聚焦线扫描与Ca2+释放位点之间的位置差异导致的Ca2+火花幅度变化，与由于Ca2+释放特性的内在差异导致的变化相互交织。为了区分Ca2+火花幅度变化的这两个来源，我们确定了通道电流或通道开放时间（统称为源强度，α）的变化对测量的Ca2+火花幅度直方图N(a)的影响。这是通过以下步骤完成的：1）模拟Ca2+释放、Ca2+和荧光素-3扩散以及Ca2+结合反应；2）用共聚焦显微镜模拟Ca2+火花的图像形成；3）使用一种新型的自动Ca2+火花探测器。从这些结果中，我们推导出一个积分方程，将源强度的概率密度函数fα(α)与N(a)联系起来，该方程考虑了源与线扫描之间的随机位置变化。在特殊但重要的情况下，即Ca(2+)结合的荧光素-3的空间分布是高斯分布时，我们展示了以下内容：1）由于位置或内在差异导致的Ca2+火花幅度变化可以被分离，并且2）原则上可以从Ca2+火花幅度直方图计算出fα(α)，因为N(a)是移位双曲线的总和，其中移位的大小和权重取决于fα(α)。特别是，如果所有Ca2+火花的产生方式完全相同，那么1/N(a)对a的绘图将是一条直线。1/N(a)绘图中的不连续性揭示了携带不同电流的多个通道群体。3）尽管Ca2+火花幅度与衰减时间之间的反比关系可用于区分来自不同通道群体的Ca2+火花，但对于小幅度Ca2+火花，噪声会使测量的衰减时间变得毫无意义。
