Biology Reference
In-Depth Information
tungsten or quartz halide beams used for viewing. For more detail on these filling
procedures, see
Gurney (1991)
. Other methods of loading cells, used primarily with
other sorts of caged compounds, are discussed by
Adams and Tsien (1993)
.
VI. Light Sources
Photolysis of caged Ca
2
þ
chelators requires a bright source of near UV light.
If speed is unimportant, an ordinary mercury or xenon arc lamp may be used.
Mercury lamps have a convenient emission line at 366 nm. Exposure can be
controlled with a shutter, using MgF-coated Teflon blades for particularly bright
sources. Lamps of 100-150 W power with collimating quartz lenses provide su
Y-
25% of caged Ca
2
þ
compounds in
cient energy to photolyze
2 s. Bulbs of larger
power only generate bigger arcs, with more energy in a larger spot of similar
intensity. With additional focusing, photolysis can be achieved in one-tenth the
time or even less. These light sources are the appropriate choice in applications
using reversible [Ca
2
þ
]
i
elevation with DM-nitrophen.
Fast events require the use of a laser or xenon arc flashlamp. The xenon lamps
are less expensive and cumbersome; convenient commercial systems are available
from Chadwick-Helmuth (El Monte, California), Rapp Optoelektronik (Hamburg,
Germany), TILL Photonics (Gr
¨
felting, Germany), and Cairn Research (Faver-
sham, UK). These flashlamps discharge up to 200-300 J electrical energy across
the bulb to provide a pulse of
1 ms duration with up to 300 mJ energy in the
330
380-nm band. The Chadwick-Helmuth unit includes only a power supply and
lamp socket, so a housing with focusing optics must be constructed (see
Rapp and
Guth, 1988
). Focusing can be accomplished with a UV-optimized elliptical reflec-
tor or with quartz refractive optics. The reflector can be designed to capture more
light (i.e., have a larger e
V
ective numerical aperture), but reflectors have greater
physical distortion than well-made lenses. In practice, the reflector generates a
larger spot with more total energy, but somewhat less intensity, than refractive
methods. One advantage of reflectors is that they are not subject to chromatic
aberration—focusing is independent of wavelength—so the UV will be focused in
the same spot as visual light. This is not true of refractive lenses. To focus and aim
them accurately at the sample, a UV filter must be used to block visual light and the
beam must be focused on a fluorescent surface for visualization. Both types of
housing are available from Rapp Optoelektronik. Using either system, photolysis
rates approaching 80-90% in one flash are achievable. This rate may be reduced by
imposing neutral density filters or reducing discharge energy, but the relationship
between electrical and light energy is not linear and should be measured with a
photometer. Flashlamps can be reactivated only after their storage capacitors have
recharged, setting the minimal interval between successive maximal flashes at
several seconds or more.
F1ashlamps are prone to generating a number of artifacts. The discharge causes
electrical artifacts that can burn out semiconductors and op amps, and reset or
