Principles of Fluorescence Spectroscopy

630 FLUORESCENCE SENSING
Figure 19.14. Oxygen sensing using a platinum complex in a polymeric
film. Revised from [39].
octaethylporphyrin ketone in a polymer film, which displays
an unquenched lifetime in the range of 40–60 µs. The
excitation source is an amplitude-modulated LED. The
electronics measures the phase angle of the emission relative
to the excitation, using separate detectors for the excitation
and emission. The phase angles are highly sensitive
to the oxygen concentration, and are stable over long periods
of time. Reasonable amounts of photobleaching or continuity
drifts would not affect the phase angle measurements.
Wavelength-ratiometric measurements are usually not
possible with collisonally quenched probes. One interesting
exception is the platinum complex shown in Figure 19.14.
This compound displays both a singlet emission near 560
nm and a triplet emission near 670 nm, with lifetimes of 0.5
ns and 14 µs, respectively. 39 The emission intensity of the
long-lived emission is sensitive to oxygen and the short life-
time emission is not sensitive to oxygen (lower panel). A
simple solid-state device can be used to measure the ratios
of emission intensities at 560 and 670 nm, and thus the oxygen
concentration.
19.4.5. Lifetime Imaging of Oxygen
Molecules that display dual emission (Figure 19.14) are
highly unusual, and there are not many opportunities for
wavelength-ratiometric oxygen sensing. One method to
make measurements that are mostly independent of intensity
is to use fluorescence lifetime imaging microscopy
(FLIM), 40–41 which is described in Chapter 22. In FLIM the
contrast in the image is based on the lifetime at each point
in the sample and not on the emission intensity. Figure
19.15 shows images of bronchial epithelial cells labeled
with [Ru(bpy) 3 ] 2+ , which in the oxygen-free samples displays
a lifetime near 600 ns. 42–43 The intensity image on the
left reveals the local concentration and/or quantum yield of
the probe in the cells. The image on the right shows the lifetimes
in each region of the sample. The lifetimes are essentially
constant which indicates the oxygen concentration is
constant throughout the cells. These lifetime images were
calculated from images taken with a gated image intensifier
and CCD camera.
Figure 19.15. Fluorescence intensity and lifetime images of human
bronchial epithelia cells labeled with [Ru(bpy) 3 ]Cl 2 . Lifetime images
were obtained using a gated image intensifier and a CCD camera.
From [43].