The Ca2+ indicator fluo-3 (Figure 1) was developed by Tsien and colleagues for use with visible-light excitation sources in flow cytometry and confocal laser-scanning microscopy. Since being introduced in 1989, fluo-3 imaging has revealed the spatial dynamics of many elementary processes in calcium signaling. Fluo-3 has also been extensively used for flow cytometry; for experiments involving photoactivation of “caged” chelators, second messengers, and neurotransmitters; and for cell-based pharmacological screening.
The most important properties of fluo-3 in these applications are an absorption spectrum compatible with excitation at 488 nm by argon-ion laser sources, and a very large fluorescence intensity increase in response to Ca2+ binding (Figure 2). Fluo-3, fluo-4 and their derivatives all exhibit large fluorescence intensity increases on binding Ca2+. Unlike the ultraviolet light-excited indicators fura-2 and indo-1, there is no accompanying spectral shift. The fluorescence intensity increase on Ca2+ binding is typically >100-fold. A comparison of physical and spectroscopic properties for fluo-3 and fluo-4 is shown in Table 1.
|Kd (Ca2+)*||325 nM||345 nM|
|Absorption maximum (Ca2+-bound) †||506 nm||494 nm|
|max (Ca2+-bound) †||100,000 cm-1M-1||88,000 cm-1M-1|
|488 nm (Ca2+-bound) †||43,000 cm-1M-1||77,000 cm-1M-1|
|Emission maximum (Ca2+-bound) †||526 nm||516 nm|
|QY (Ca2+-bound) †, ‡||0.15||0.14|
|Fmax /Fmin §||>100||>100|
* Dissociation constant for Ca2+ determined at 22° C in 100 mM KCl, 10 mM MOPS, pH 7.2, 0 to 10 mM CaEGTA. Kd = 390 nM for fluo-3 reported in Molecular Probes’ The Handbook: A Guide to Fluorescent Probes and Labeling Technologies, Tenth Edition (2005).
† Value determined at 22° C in 100 mM KCl, 10 mM MOPS, pH 7.2 containing 39.8 μM free Ca2+.
‡ QY = fluorescence quantum yield. QY = 0.18 for fluo-3 reported in J Biol Chem 264, 8171 (1989).
§ Fluorescence intensity increase on binding Ca2+ in a solution assay.
For Research Use Only. Not for use in diagnostic procedures.