Calcium measurement is critical for numerous biological investigations. Fluorescent probes that show spectral responses upon binding Ca2+ have enabled researchers to investigate changes in intracellular free Ca2+ concentrations by using fluorescence microscopy, flow cytometry, fluorescence spectroscopy and fluorescence microplate readers. Fura-2 and Indo-1 are most commonly used among the UV-excitable calcium indicators. Fura-2 is excitation-ratioable while Indo-1 is emission-ratioable. Fura-2 is preferred for ratio-imaging microscopy, in which it is more practical to change excitation wavelengths than emission wavelengths. Upon binding Ca2+, Fura-2 exhibits an absorption shift that can be observed by scanning the excitation spectrum between 300 and 400 nm, while monitoring the emission at ~510 nm. In contrast, Indo-1 is the preferred dye for flow cytometry, where it is more practical to use a single laser for excitation (usually the 351–364 nm spectral lines of the argon-ion laser). The emission of Indo-1 shifts to 400 nm (when bound to Ca2+) from 480 nm in Ca2+-free environments. Indo-1 AM is cell-permeable version of Indo-1.
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