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Recent extensive studies revealed that the intracellular concentration of magnesium ions (Mg2+) is one of the important factors to regulate cellular functions. To evaluate the impact of Mg2+ concentration changes on intracellular signals or events, simultaneous imaging of Mg2+ with those phenomena is a powerful technique. The present protocol describes the synthesis and evaluation of near-infrared (NIR) fluorescent Mg2+-selective probes, named KMG-500 series, and the application to simultaneous imaging of the corresponding intracellular signal transductions and molecular events. The present protocol for multicolor imaging using fluorescent probes in the NIR and visible ranges is highly useful to reveal how multiple molecular events are correlated each other in each single cell.