Principles of Fluorescence Spectroscopy

PRINCIPLES OF FLUORESCENCE SPECTROSCOPY 649
Figure 19.50. Excitation (Fura-2) and emission spectra (Indo-1, Calcium Green-1 and Magnesium Orange) of Ca 2+ and Mg 2+ probes in the presence
of various concentrations of the ion. Revised from [142] and [144].
concentration to be determined from the lifetimes. 160–161
One of the first calcium probes, Quin-2, displays a tenfold
increase in lifetime when bound to calcium. 162–163 However,
Quin-2 requires UV excitation and displays a low quantum
Figure 19.51. Excitation spectra of the coumarin benzothiazole-based
indicator (BTC) lithium salt in Ca 2+ solutions with concentrations
ranging from 1.3 to 100 µM CaCl 2 . Revised from [156].
yield, so that it is now used less frequently. Recently, Ca 2+
probes based on squaraines have been reported, allowing
excitation wavelengths as long as 635 nm. 164–165
While the use of the calcium probes seems straightforward,
calibration is difficult when such probes are located
within cells. 166–170 When used as intracellular indicators, the
calcium probes are typically calibrated in the presence of
other intracellular ions at their expected concentrations. 171
It is difficult to maintain nanomolar Ca 2+ concentrations in
the calibration solutions, and the probes themselves can
alter the overall Ca 2+ concentration. For this reason, calcium
buffers have been developed and are commercially
available. And, finally, the probe may interact with intracellular
macromolecules, or by phototransformation during
illumination in the microscope, resulting in altered behavior
compared to the calibration data. 166,172
Calcium probes have also been developed using azacrown
ethers as the chelator rather than BAPTA. 173–177
However, these probes have been mostly studied in organic
solvents and used to study the effects of Ca 2+ on electron
transfer. Magnesium-sensitive probes are available (Table
19.4), 176–184 and some have been characterized as lifetime
probes. 184 These probes typically have the APTRA chelator,
rather than BAPTA, as can be seen for the calcium probe