[1] It is used to measure Ca2+ inside living cells in flow cytometry, and confocal laser scanning microscopy using visible light excitation (compatible with argon laser sources operating at 488 nm).

Fluo-3 is an essentially nonfluorescent compound, but upon binding of Ca2+ its fluorescence increases sharply with an emission maximum at 525 nm suitable for conventionally used detectors designed for fluorescein isothiocyanate (FITC) measurements.

This large change in fluorescence coupled with a good yield of photons provides very high contrast which allowed the detection of microscopic Ca2+ release events inside cells called "Calcium sparks".

[2] Whereas the salts of fluo-3 are unable to penetrate cells, loading can be achieved using its acetoxymethyl (AM) ester derivative.

Once inside the cell, unspecific esterases cleave the ester effectively trapping fluo-3.