Principles of Fluorescence Spectroscopy

PRINCIPLES OF FLUORESCENCE SPECTROSCOPY 307
Figure 8.49. Structure and spectra of a wavelength-ratiometric chloride
probe. Revised from [129].
Knowledge of the mechanism of chloride quenching
has allowed the design of wavelength-ratiometric chloride
probes. 127–129 This was accomplished by linking two fluorophores,
one which is quenched by chloride and one which
is not sensitive to chloride (Figure 8.49). This probe contains
two quinoline rings. The methoxy-substituted ring on
the left readily accepts electrons and is quenched by chloride.
The ring on the right, with the amino group, does not
accept electrons from chloride and is not quenched. The
lower panel shows the emission spectra of MQxyDMAQ in
the presence of increasing chloride concentrations. The
shorter-wavelength emission is quenched, but the longerwavelength
emission is not sensitive to chloride. Examination
of the spectra in Figure 8.49 suggests that there will be
RET from the MQ to the DMAQ ring. In this probe some
energy transfer occurs, which reduces the lifetime of the
MQ ring and makes it less sensitive to quenching by chloride.
However, the RET efficiency is modest, so that the
MQ ring remains sensitive to chloride.
Figure 8.50. Structure and intracellular calibration of the chloride
probe bis-DMXPQ. Revised from [129].
When used in an intracellular environment, the sensitivity
of a probe can change from its sensitivity in a pure
solution. For this reason the chloride-sensitive probes were
calibrated in cells by changing the intracellular chloride
concentration. This is accomplished by exposing the membrane
permeability of the 3T3 fibroblast cells using valinomycin
and other similar compounds, followed by exposing
the cells to different chloride concentrations (Figure 8.50).
The intensity of the short-wavelength MQ emission at 450
nm is decreased as the chloride concentration increases.
The intensity of the long-wavelength emission at 565 nm is
not affected.
The wavelength-ratiometric chloride probes were used
to measure local chloride concentrations in CHO cells (Figure
8.51). This was accomplished by collecting the intensity
images at 450 and 565 nm. The intensity at 450 nm will
be sensitive to the local chloride concentration. 129 However,
the image at 450 nm alone does not allow the chloride concentrations
to be determined because the concentration of
the probe can be different in each region of the cell. The
concentration of chloride can be determined from the ratio
of the emission intensities. These images show that the
chloride concentrations in these cells is rather uniform, with
a somewhat lower chloride concentration in the nuclei.
8.14.3. Chloride-Sensitive GFP
Green fluorescent protein (GFP) and its mutants are widely
used to study gene expression and as sensors. 130–131 A yellow
variant of GFP (YFP) is known to be sensitive to chloride.
132–133 The intensity of YFP-H148Q decreases progressively
in response to added chloride in a manner and concentration
range that suggests collisional quenching (Figure