Biomedical Engineering Reference
In-Depth Information
Fig. 5.1
The fl uorescence emission and excitation spectra of three different organic synthetic Ca
2+
dyes in solutions containing 0-39.8
μ
M free Ca
2+
. (
a
) The emission at 530 nm of fl uo-3, which is
a typical singlemetric dye, is dependent on free Ca
2+
concentration. The spectrum for the Ca
2+
-free
solution is indistinguishable from the baseline. (
b
) In Fura-2, a ratiometric dye, the ratio of 510 nm
emission emitted by excitation at 340 and 380 nm wavelengths can be used to determine the con-
centration of Ca
2+
. (
c
) Indo-1 is excited by 338 nm light. The peak of the emission fl uorescence
shifts from 400 to 480 nm with Ca
2+
binding. The ratio of the fl uorescence at 480 nm to at 400 nm
indicates Ca
2+
concentration (From The Molecular Probes Handbook)
Some of these technical limitations associated with singlemetric dyes can be
circumvented through the use of ratiometric dyes, although at the expense of requir-
ing a more complicated experimental confi guration. The fl uorescence behavior of
ratiometric dyes also depends on [Ca
2+
]
i
. However, rather than responding to a
change in [Ca
2+
]
i
with a simple change in fl uorescence intensity across the entire
absorption spectrum, ratiometric dyes demonstrate a much more complicated
behavior: the shapes of the absorption spectra of these indicators depends on [Ca
2+
]
i
.
