Calcium ions are important second messengers in cells. They are involved in muscle contraction, nerve signal transmission, immune cell activation, hormone secretion etc. Fluorescent calcium indicators selectively bind to calcium ions and show a spectral response upon binding Ca2+, i.e. fluorescence intensity or wavelength are changing. This shift of fluorescence wavelength and change of intensity upon calcium chelation can be monitored using fluorescence microscopy. Fura-2, Fluo-3, Rhod-2 and Indo-1 (as cell-impermeable salts or cell-permeable AM esters) as well as their low-affinity derivatives Fura-2FF, Fluo-3FF, Indo-1FF which are particularly useful for certain applications

Fluorescence-linked detection is best used for high-resolution studies. In addition, some fluorochromes have emission spectra that do not overlap, so two fluorochromes can be observed on the same sample. This allows the study of two different antigens in the same specimen even when they have identical subcellular distributions. Flurorescein, rhodamine, and Texas Red are the most commonly used fluorochromes. Fluorescein emits a yellow-green light that is easily detected, but it is prone to rapid photobleaching. Rhodamine emits a red color and is not as prone to fading as fluorescein, but the rhodamine conjugates are more hydrophobic and therefore yield higher