Biology Reference
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fortunately at wavelengths di
erent from those at which the photolabile chelators
show any significant absorbance. However, these dyes are also di
V
cult to calibrate
for absolute levels of [Ca 2 þ ], although changes in [Ca 2 þ ] may be determined fairly
accurately. Fura-red is a ratiometric dye excited by visible light, so it might have
some application in calibrating photolysis. A problem common to all the fluores-
cent indicators is that their fluorescent properties may be altered by the presence of
photolabile chelators, which generally are used at millimolar levels whereas the
indicators are present at 100 m M or less. The photolabile chelators often produce
contaminating fluorescence, which also may be Ca 2 þ -dependent and may partially
quench the fluorescence of the indicators ( Hadley et al., 1993; Zucker, 1992 ). Thus,
the indicators must be calibrated in the presence of photolabile chelator at three
well-controlled [Ca 2 þ ] levels, preferably before and after exposure to the photolysis
flash, before they can be used to measure the e
Y
ects of photolysis on [Ca 2 þ ]( Neher
and Zucker, 1993 ). The low and high [Ca 2 þ ] calibrating solutions may be made
with excess Ca 2 þ or another bu
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V
er such as EGTA or BAPTA, but the intermediate
[Ca 2 þ ] solution is more di
cult to generate, since photolysis of the chelator will
release some Ca 2 þ and change the [Ca 2 þ ] i and pH in this solution unless it contains
a very high concentration of controlling chelator and pH bu
Y
er.
The calibration procedure is generally the same for any combination of chelator
and indicator. A small sample of the mixture is placed in a 1-mm length of micro-
cuvette with a 20- m m pathlength (Vitro Dynamics, Rockaway, New Jersey) under
mineral oil to prevent evaporation. This cuvette is exposed repeatedly to the
photolysis beam or to flashes, which should illuminate the whole cuvette uniformly,
and the [Ca 2 þ ] after each flash or exposure is measured using a microscope-based
fluorescence or absorbance photometer. A small droplet of solution under mineral
oil alone would work, and may be necessary if the photolysis beam is directed
through the microscope and illuminates a very small area, but sometimes the
fluorescent properties of the indicators are a
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ect
would be detected in the procedure for calibrating the chelator-indicator mixture,
but is best avoided using the microcuvettes, in which contact with oil is only at the
edges, the fluorescence or absorbance change of which need not be measured.
In some applications, such as whole-cell patch clamping of cultured cells, using
the cell as a calibration chamber can be easier than any other procedure.
The expected changes in [Ca 2 þ ] depend on the chelator used. The nitr and diazo
chelators should lead to a stepwise rise or fall in [Ca 2 þ ] after each exposure; the
results can be fitted to models of the chelators and their photoproducts, using their
a
ected by the mineral oil. This e
ciencies of free and bound chelators ( Fryer
and Zucker, 1993; Land ` and Zucker, 1989 ). The percentage photolysis of the
chelator in response to each light exposure is the only free parameter, and is varied
until the model fits the results. In the case of the high-a
Y
nities and the relative quantum e
Y
nity DM-nitrophen, little
rise in [Ca 2 þ ] will occur until the total amount of remaining unphotolyzed chelator
equals the total amount of Ca 2 þ in the solution, whereupon the [Ca 2 þ ] will increase
suddenly. Equations relating initial and final concentrations of DM-nitrophen,
Y
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