Biology Reference
In-Depth Information
wavelength is the only experimental measurement that is related to [Ca
2
þ
], intensity
changes arising from factors unrelated to changes in [Ca
2
þ
] (e.g., changes in cell
thickness, leakage of indicator from the cell) can confound interpretation of the
intensity data. In contrast, because the Ca
2
þ
-free and Ca
2
þ
-bound forms of ratio-
metric indicators are characterized by spectral peaks at di
V
erent wavelengths,
intensity measurements can be made at two di
erent wavelengths, and the ratio
between these intensities is quantitatively related to [Ca
2
þ
](
Grynkiewicz et al.,
1985
). Obtaining a ratio minimizes the e
V
ect of many artifacts that are unrelated
to changes in [Ca
2
þ
]—for example, a change in cell thickness or indicator loss from
the cell would a
V
ect would
cancel when the two intensities are ratioed. The two commonly used ratiometric
indicators, Fura-2 and Indo-1, require excitation in the ultraviolet (UV) range,
whereas most of the common nonratiometric dyes use visible excitation light.
Although the ratiometric dyes can be calibrated more reliably (
Section V
), some-
times avoiding using UV light for excitation may be necessary (e.g., UV can excite
significant autofluorescence in some biological preparations and can photolyze
photosensitive ''caged'' compounds). Clearly,
V
ect intensities at the two wavelengths equally, so the e
V
in practice,
instrumentation for
using ratiometric indicators is more complex than that
for nonratiometric
indicators.
Quin2 (
Tsien, 1980; Tsien et al., 1982
) is the archetypal tetracarboxylate indicator
listed in
Table I
(structure in
Fig. 1
). Its properties and applications as a nonratio-
metric indicator have been reviewed in detail (
Tsien and Pozzan, 1989
). However,
Quin2 has been superseded by new generations of nonratiometric and ratiometric
indicators. Of the nonratiometric indicators listed in
Table I
, the Fluo and Calcium
Green series as well as Oregon Green 488 BAPTA-2 incorporate fluorescein chro-
mophores and are, therefore, excited at wavelengths typical of fluoresceins. The
Fluo dyes, Calcium Green-2 and Oregon Green 488 BAPTA-2 exhibit the largest
intensity changes in their transition from Ca
2
þ
-free to Ca
2
þ
-bound forms (
100-
fold;
Haugland, 1992; Minta et al., 1989
). This change can be an advantage because,
for a given rise in [Ca
2
þ
], these indicators give a larger increase in brightness
compared to other nonratiometric indicators. Because fluorescence quantum
e
erence between Ca
2
þ
-
bound and Ca
2
þ
-free forms implies that the Ca
2
þ
-free forms of the two indicators
must be only weakly fluorescent. Some researchers find this fact annoying because
cells with relatively low resting [Ca
2
þ
]
i
(cytosolic free Ca
2
þ
concentration) would
have most of the indicator in the Ca
2
þ
-free form and therefore would be quite dim.
Rhod-2, Calcium Orange, and Calcium Crimson are indicators that incorporate
rhodamine-type chromophores and therefore are excited at much longer wave-
lengths than are the Fluo and Calcium Green dyes. When the acetoxymethyl (AM)
ciency
3
can range only from 0 to 1, the large intensity di
Y
V
3
Fluorescence quantum e
Y
ciency, symbolized as
F
F
or Q
F
, is the fraction of total light absorbed
that is emitted as fluorescence. Fluorescence quantum e
ciency may also be thought of as the proba-
bility that a molecule will emit fluorescence after absorbing a photon. Being a probability, the quantum
e
Y
Y
ciency can have a value between 0 and 1.
