Leybaert L, Sneyd J, Sanderson M J
Department of Physiology and Pathophysiology, University of Ghent, B-9000 Ghent, Belgium.
Biophys J. 1998 Oct;75(4):2025-9. doi: 10.1016/S0006-3495(98)77644-1.
Calcium-sensitive dual excitation dyes, such as fura-2, are now widely used to measure the free calcium concentration ([Ca2+]) in living cells. Preferentially, [Ca2+] is calculated in a ratiometric manner, but if calcium images need to be acquired at high temporal resolution, a potential drawback of ratiometry is that it requires equally fast switching of the excitation light between two wavelengths. To circumvent continuous excitation switching, some investigators have devised methods for calculating [Ca2+] from single-wavelength measurements combined with the acquisition of a single ratiometric pair of fluorescence images at the start of the recording. These methods, however, are based on the assumption that the concentration of the dye does not change during the experiment, a condition that is often not fulfilled. We describe here a method of single-wavelength calcium imaging, in which the dye concentration is estimated from ratiometric fluorescence image pairs acquired at regular intervals during the recording period, that furthermore includes a correction for the changing dye concentration in the calculation of [Ca2+].
钙敏双激发染料，如fura-2，如今被广泛用于测量活细胞中的游离钙浓度（[Ca2+]）。优先以比率法计算[Ca2+]，但如果需要以高时间分辨率获取钙图像，比率法的一个潜在缺点是它需要在两个波长之间同样快速地切换激发光。为了避免连续的激发光切换，一些研究人员设计了从单波长测量结合记录开始时获取的一对比率荧光图像来计算[Ca2+]的方法。然而，这些方法基于染料浓度在实验过程中不变的假设，而这一条件往往无法满足。我们在此描述一种单波长钙成像方法，其中染料浓度是根据记录期间定期获取的比率荧光图像对来估计的，并且在计算[Ca2+]时还包括对变化的染料浓度的校正。
