• Highly sensitive dye for rapid measurement of calcium flux in cells
  • Large fluorescence increase (typically >100-fold) provides good sensitivity
  • Extensively used for cell-based HTS applications
  • Visible light-excitable dye for less autofluorescence and cellular photodamage issues, and efficient excitation with most laser-based instrumentation, including confocal laser-scanning microscopes and flow cytometers

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.  

Figure 1. Fluo Indicators.


Figure 2: Ca2+-dependent fluorescence emission spectra of fluo-3 (F1240, F3715). The spectrum for the Ca2+-free solution is indistinguishable from the baseline.


Table 1. Comparison of physical and spectroscopic properties for fluo-3 and fluo-4.

Property fluo-3 fluo-4
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.