Chemistry Reference
In-Depth Information
to oxygen measurements in biological systems have been proposed in the past [9-13],
but all of them need continuous development in order to satisfy diverse needs of
biomedical research. Oxygen-dependent quenching of phosphorescence [14], or simply
phosphorescence quenching, is an optical technique, suitable for oxygen measurements
in the physiological pO
2
(partial pressure of oxygen) range. Originally proposed in 1987
for in vitro oxygen sensing [14], phosphorescence quenching soon thereafter was
implemented for local in vivo single-point oxygen measurements [15] and planar
2-D imaging [16]. Currently, high-resolution microscopy [17,18] and 3-D tomography
[19-21] of oxygen are being developed. The characteristic features of phosphorescence
quenching include excellent specificity, submillisecond temporal response, high sen-
sitivity, especially at low oxygen concentrations and relative simplicity of implemen-
tation. This method is being increasingly applied in biological research, driving the
development and continuous improvement of phosphorescent probes.
Phosphorescence quenching relies on the ability of molecular oxygen, which is a
triplet molecule in the ground state (O
2
X
3
S
g
), to react with molecules in their
excited states, quenching their luminescence [22]. Collisional quenching is much less
probable on the time scale of singlet excited states (nanoseconds) than of triplet states
(microseconds to milliseconds), making phosphorescence significantly more sensi-
tive to oxygen than fluorescence. Assuming a large excess of oxygen relative to the
concentration of triplet emitters—a condition typically met in biological environ-
ments—the dependence of the phosphorescence intensity and lifetime on oxygen
concentration follows the Stern-Volmer relationship:
I
0
=
I
¼ t
0
=t ¼
1
þ
K
SV
½
O
2
ð
14
:
1
Þ
where I and
are the phosphorescence intensity and the lifetime at oxygen concen-
tration [O
2
] and in the absence of oxygen (I
0
,
t
t
0
); and K
SV
is the Stern-Volmer
quenching constant. In practice, using lifetime
as the analytical signal for [O
2
]is
more accurate, because the lifetime is independent of the probe distribution and of any
other chromophores present in the biological system.
It is customary to express oxygen content in the units of pressure (mmHg) rather
than concentration (M),
1
since in the majority of biological experiments partial
pressure of oxygen (pO
2
) is the actually controlled experimental parameter. We
assume that theHenry's lawholds in the physiological range of oxygen concentration:
[O
2
]
t
is the oxygen solubility coefficient (M/mmHg) for the bulk
phase. Considering K
SV
¼
k
2
t
0
, where k
2
is the bimolecular rate constant for the
quenching reaction, Eq. 14.1 can be rewritten as
¼a
pO
2
, where
a
1
=t ¼
1
=t
0
þ
k
q
pO
2
ð
14
:
2
Þ
where k
q
¼a
k
2
and has the units of mmHg
1
s
1
.
1
At 298 K and the air pressure of 760mmHg (oxygen fraction in the air is 21% or 159.6mmHg), air-
equilibrated aqueous solutions are 252
MinO
2
[23]. Here for simplicity we neglect the pressure of water
vapor above the solution, although at higher temperatures it will rise and, consequently, the partial pressure
of oxygen (pO
2
) will decrease.
m
