Principles of Fluorescence Spectroscopy

632 FLUORESCENCE SENSING
Perhaps more importantly, SPQ is quenched by free amines,
which can distort measurements in amine-containing
buffers. In fact, the Stern-Volmer quenching constant of
SPQ in aqueous solution is 118 M –1 , whereas in cells the
quenching constant is near 13 M –1 . This decrease has been
attributed to quenching of SPQ by non-chloride anions and
proteins in cells. 51 Quenching of SPQ by amines was turned
into an opportunity, by using the quenching caused by the
amine buffers as an indicator of pH. 50 As the pH increases,
more of the buffer is in the free amine form, resulting in a
decrease in the intensity of SPQ. A disadvantage of the
chlorine probes is that they are not ratiometric probes.
Some of the probes leak out of cells, decreasing the intensity
and preventing accurate measurements of the chloride
concentration. The quinoline probes have been made into
wavelength-ratiometric probes by linking them to a chloride-insensitive
fluorophore using dextran or a flexible
chain. 52–53
19.4.7. Lifetime Imaging of
Chloride Concentrations
The need for covalently linked chloride-sensitive and -
insensitive probes can be avoided by lifetime imaging.
Since chloride is a collisional quencher, the decreases in
lifetime are proportional to the decreases in intensity (Figure
19.17). FLIM of the chloride probe 6-methoxy-quinolyl
acetoethyl ester (MQAE) was used to determine the concentrations
of chloride in olfactory epithelium. 54 In this tissue
the olfactory sensory neurons penetrate through supporting
epithelial cells, terminating in dendritic knobs (Figure
19.19). The transduction mechanism for olfactory signal
transduction involves an influx of calcium and an efflux
of chloride. For this to occur the olfactory dendrites must
accumulate higher concentrations of chloride than the surrounding
tissue. Lifetime measurements of MQAE in this
tissue revealed a shorter lifetime of MQAE in the dendrite
knob than in the supporting cells, which indicates a higher
chloride concentration in the knobs. Lifetime images were
obtained using laser scanning microscopy and two-photon
excitation (Chapter 18). Intensity decays were recorded by
TCSPC at each point in the image. A calibration curve for
the lifetime at various chloride concentrations was determined
using a similar tissue and ionophore to control the
intracellular chloride concentrations. Using this calibration
it was possible to use the lifetime image to create a chloride
concentration image in the olfactory tissue. This image
shows higher chloride concentrations in the dendrites than
Figure 19.19. Top: Intensity decays of MQAE in the ends of the neuronal
sensing cells (dendritic knob) or in the epithelial supporting
cells. Bottom: The lower panels show the intensity (left) and chloride
concentration image (right) of the olfactory epithelium. Reprinted
with permission from [54]. Copyright © 2004, Journal of
Neuroscience.
in the surrounding tissue. This experiment was made possible
by the advances in TCSPC (Chapter 4) and multiphoton
microscopy.
19.4.8. Other Collisional Quenchers
A wide variety of molecules can act as quenchers (Chapter
8), and they permit developments of sensors based on collisional
quenching. Benzo(b)fluoranthene was found to be
highly sensitive to sulfur dioxide. 55 Oxygen interfered with
the measurements but was 26-fold less efficient as a
quencher than SO 2 . Halogenated anesthetics are known to
quench protein fluorescence and can be detected by collisional
quenching of anthracene and perylene. 56 Carbazole is
quenched by a wide variety of chlorinated hydrocarbons. 57
NO, which serves as a signal for blood vessel dilation, is