Single-Cell Calcium Imaging for Studying the Activation of Calcium Ion Channels

Single cell Ca²⁺ imaging is essential for the study of Ca²⁺ channels activated by various stimulations like temperature, voltage, native compound and chemicals et al. It primarily relies on microscopy imaging technology and the related Ca²⁺ indicator Fura-2/AM (AM is the abbreviation for Acetoxymethyl ester). Inside the cells, Fura-2/AM is hydrolyzed by esterases into Fura-2, which can reversibly bind with free cytoplasmic Ca²⁺. The maximum excitation wavelength shifts from 380nm to 340nm (when saturated with Ca²⁺) upon binding. The emitted fluorescence intensity is quantitatively related to the concentration of bound Ca²⁺. By measuring the 340/380 ratio, the Ca²⁺ concentration in the cytoplasm can be determined, eliminating errors caused by variations in the loading efficiency of the fluorescent probe among different samples. This technology allows for real-time, quantitative, and simultaneous monitoring of Ca²⁺ changes in multiple cells. The results are stored in ".XLSX" format for subsequent analysis, which is fast and generates intuitive change curves, greatly improving the detection efficiency. From different experimental perspectives, this article lists the use of this technology to detect Ca²⁺ signals in cells with endogenous or overexpressed channel proteins. Meantime, different methods for activating cells were also showed and compared. The aim is to provide readers with a clearer understanding of the usage and applications of single cell Ca²⁺ imaging.