22
World Precision Instruments
Calcium Measurements with Fura-2
Fura-2 is a commercially-available calcium fl uorophore. It is a dual-excitation,
single-emission dye that fl uoresces when excited with either 340nm or 380nm of
light. Fura-2 can be used to make ratiometric measurements, because the intrinsic
absorbance of the dye is different in calcium-free state than it is in the calcium-
bound state. However, in either state, the maximum emission of Fura-2 is still at
510nm.
When Fura-2 is excited with light at 340nm, and its fl uorescence is measured at
510nm, then the emission intensity of the dye increases as the amount of calcium
binding to Fura-2 increases. Therefore, the increase in fl uorescence is an indicator
of the increase in the calcium concentration in the fi bers. On the other hand,
when Fura-2 is excited with light at 380nm, and its fl uorescence is measured at
510nm, then the emission intensity of the dye decreases as the amount of calcium
binding to Fura-2 increases. By alternating the excitation of the dye at these two
wavelengths (340nm and 380nm), the changes in the fl uorescence intensity
determine the concentration ratio of the bound and free calcium in the fi bers.
Percent Signal
0
20
40
60
80
100
Wavelength [nm]
300
350
400
450
500
550
600
650
LED [emission]
Fura-2 Calcuim-Bound [excitation]
Fura-2 Calcium-Free [excitation]
Fura-2 Calcium-Bound [emission]
Fura-2 Calcium-Free [emission]
510nm Filter [transmission]
340nm
380nm
Fig. 40— Excitation, emission and fi ltering of Indo-1 with the SI-BF-100 Biofl uorometer.
In Fig. 40, calcium-bound Fura-2 is identifi ed in red and calcium-free Fura-2 is
identifi ed in blue. The intrinsic absorbance of Fura-2 is represented by dashed
lines. The emission spectra are represented by solid lines. The optimal sites for
the calcium-bound and calcium-free fl uorophore excitation at 340nm and 380nm
occur at the peak absorbencies of the two states of the dye. The solid gray areas
represent the bands of the 340nm and 380nm LED modules used to excite Fura-2.
The bandpass spectrum of the emission fi lter for either state of Fura-2 is represented
by the other shaded area.
As indicated in Fig. 1, the fl uorescence of Fura-2 increases with increasing calcium
concentrations. Because more calcium is bound to the dye, the calcium-bound dye
is excited more effectively by shorter wavelengths of light. However, when excited
by longer wavelengths of light, the fl uorescence of Fura-2 increases with decreasing
