Biology Reference
In-Depth Information
diaphragm, or by conveying the photolysis beam through a tapered quarty fiber
optic filter to the cell surface (
Eberius and Schild, 2001; Godwin et al., 1997
).
Lasers provide an alternative source of light with the advantages of a coherent
collimated beam that is focused much more easily to a very small spot. Pulsed
lasers such as the frequency-doubled ruby laser or the XeF excimer laser provide at
least 200 mJ energy at 347 or 351 nm in 50 and 10 ns, with possible repetition rates
of 1 and 80 Hz, respectively. Liquid coumarin-dye lasers, with up to 100 mJ
tunable energy in the UV and pulse duration, are also available. Inexpensive
nitrogen lasers providing lower pulse energies (0.25 mJ) in 5-ns pulses at 337 nm
also have been developed (
Engert et al., 1996
) and, with appropriate focusing,
might be useful. To date, lasers have found their widest application in studies of
muscle contraction. More information on these laser options is contained in
discussions by
Goldman et al. (1984) and McCray and Trentham (1989)
.
An adaptation of laser photolysis is the two-photon absorption technique (
Denk
et al.,1990
). A colliding-pulse mode-locked Ti:Sapphire laser generating 100-fs
pulses of 630-nm light at 80 MHz is focused through a confocal scanning micro-
scope. Photolysis of UV-sensitive caged compounds requires simultaneous absorp-
tion of two red photons, so photolysis occurs only in the focal plane of the scanning
beam. This behavior restricts photolysis to about 1
m
m
3
in three dimensions, but for
most compounds the photolysis rate is so slow, due to their extremely limited two-
photon cross sections, that several minutes of exposure are required with currently
available equipment. The best results were achieved with azid-1 and NDBF-EGTA
(
Brown et al., 1999; DelPrincipe et al., 1999; Momotake et al., 2006
), as expected
from their high single photon absorbances. Azid-1 could be fully photolyzed in the
two-photon focal volume with a 10-
m
s pulse train of 7 mW average power, with a
retention time of the released Ca
2
þ
in this volume of about 150
m
s. This technique is
expensive and specialized, and is still under development, but may have practical
applications in revealing the precise localization within cells or subcellular orga-
nelles of fixed targets of Ca
2
þ
action or of highly localized Ca
2
þ
bu
V
ers.
ect on most biological tissues, with
the obvious exception of photoreceptors and the less obvious case of smooth
muscle (
Gurney
Near-UV light alone seems to have little e
V
ects of light on
unloaded cells, and on the normal physiological response under study, can be
used to ascertain the absence of photic e
et al., 1987
). Control experiments on the e
V
V
ects.
VII. Calibration
When designing a new optical system or trying a new caged compound, being
able to estimate the rate of photolysis of the apparatus used is important. This
information is necessary to adjust the light intensity or duration for the desired
degree of photolysis, and to insure that photolysis is occurring at all.
In principle, the fraction (F) of a substance photolyzed by a light exposure of
energy J can be computed from the formula e
-(J-J
0
)
(I
F)/0.1, where J
0
¼
is the
