Biology Reference
In-Depth Information
energy needed to photolyze 90% of the substance and is given by J
0
¼
hcA/Q
e
l
,
where h is Planck's constant, c is the speed of light, A is Avogadro's number, Q is
the quantum e
is the
wavelength of the light. In practice, however, this equation is rarely useful for the
following reasons.
1. Measuring the energy of the incident light on a cell accurately is di
Y
ciency,
e
is the decadic molar extinction coe
Y
cient, and
l
Y
cult,
especially for light of broad bandwidth with varying intensity at di
V
erent
wavelengths.
2. The quantum e
ciency, although provided for all the photolabile chelators, is
not such a well-defined quantity. The value depends critically on how it is measured,
which is not always reported. In particular, the e
Y
ciency for a
pulse of light of moderate duration (e.g., from a flashlamp) is often greater than that
of either weak steady illumination or a very brief pulse (e.g., from a laser), because
of the possibility of multiple photon absorptions of higher e
V
ective quantum e
Y
ciency by photochem-
ical intermediates. This phenomenon has been noted to play a particularly strong
role in nitr-5 photolysis (
McCray and Trentham, 1989
). Thus, apparent di
Y
V
erences
in quantum e
Y
ciencies between di
V
erent classes of chelators may be mainly the
results of di
erent measurement procedures.
3. Finally, the quantum e
V
Y
ciency is a function of wavelength, which is rarely
given.
A more practical and commonly adopted approach is mixing a partially Ca
2
þ
-
loaded photolabile chelator with a Ca
2
þ
indicator in a solution with appropriate
ionic strength and pH bu
ering, and measuring the [Ca
2
þ
] change in a small
volume of this solution, the net absorbance of which is su
V
Y
ciently small to
minimize inner filtering of the photolyzing radiation. Suitable indicators include
fura-2, indo-1 (
Grynkiewicz et al., 1985
), furaptra (
Konishi et al., 1991
), f1uo-3,
rhod-2 (
Minta and Tsien, 1989
), Calcium Green
TM
, Orange
TM
, and Crimson
TM
(
Eberhard and Erne, 1991
), arsenazo III (
Scarpa et al., 1978
), and fura-red
(
Kurebayashi et al., 1993
). The choice depends largely on available equipment.
Fura-2, indo-1, and furaptra are dual-excitation or-emission wavelength fluores-
cent dyes, allowing more accurate ratiometric measurement of [Ca
2
þ
], but they
require excitation at wavelengths that photolyze the photolabile Ca
2
þ
chelators
and are subject to bleaching by the photolysis light. The former problem may be
minimized by using low intensity measuring light with a high sensitivity detection
system. Furaptra is especially useful for DM-nitrophen, because of its lower Ca
2
þ
a
nity. Fluo-3 and rhod-2 were designed specifically for use with photolabile
chelators (
Kao et al., 1989
), being excited at wavelengths di
Y
erent from those
used to photolyze the chelators, but they are not ratiometric dyes and are di
V
Y
cult
to calibrate accurately. Calcium Green, Orange, and Crimson su
er the same
limitation, but they are often used because of their fast kinetics and bright intensity,
allowing the accurate tracking of fast changes in [Ca
2
þ
]
i
. Arsenazo and antipyr-
alazo are metallochromic dyes that change absorbance on binding Ca
2
þ
,
V
