Introduction:

Quantitative determination of messenger substances like intracellular calcium ions with calcium fluorescent probes is a major technical approach to study the secreting activity of cells. The basic principle is as follows: the calcium ions in the sample are labeled with fluorescent probes, and based on the characteristics of fluorescent probes in the sample, the monochromatic source emits monochromatic light to induce fluorescence. Next, the fluorescence characteristics are detected by a sensor, based on which the calcium ion concentration in the sample can be analyzed. The single-wavelength method and dual-wavelength ratiometric determination are generally employed for measurement. The latter has been widely applied due to its sensitivity and convenience, of which the principle is that the combination of probe and calcium induces not only a change in fluorescence intensity, but also a shift of excitation or emission spectrum. In the case of a shift of the excitation peak, dual-excitation ratiometric fluorescence, such as fura-2, is detected. If the emission peak is shifted, the fluorescence, such as indo-1, is measured through dual-emission ratiometric determination. Generally, two wavelengths sensitive to calcium ions but opposite in fluorescence intensity, such as 340/380 of fura-2, are selected, so as to obtain the maximum ratio. Through determination with a fluorescence spectrophotometer, a fluorescence microscope system, a photomultiplier or an enhanced camera, the calibrated intracellular Ca²⁺ concentration can be obtained. Other indicators have also been used in different experiments.