Biology Reference
In-Depth Information
clear digital memory in other nearby equipment. Careful electrostatic shielding,
wrapping inductors with paramagnetic metal, power source isolation, and using
isolation circuits in trigger pulse connections to other equipment prevent most
problems, which are also diminished in pulsed mercury lamps (
Denk, 1997
). The
discharge generates a mechanical thump at the coil used to shape the current pulse
through the bulb; this thump can dislodge electrodes from cells or otherwise
damage the sample. Mechanical isolation of the o
ending coil solves the problem.
Lamp discharge also produces an air pressure pulse that can cause movement
artifacts at electrodes, which can be seen to oscillate violently for a fraction of a
second when videotaped during a flash. This movement can damage cells severely,
especially those impaled with multiple electrodes. Small cells sealed to the end of a
patch pipette often fare better against such mistreatment. To reduce this source of
injury, the light can be filtered to eliminate all but the near UV. Commercial Schott
filters (UG-I, UG-Il), coated to reflect infrared (IR) light, serve well for this
purpose, but can cut the 330
V
380-nm energy to 30% or less. Liquid filters to
remove IR and far UV also have been described (
Tsien and Zucker, 1986
).
Removing IR reduces temperature changes, which otherwise can exceed 1
Cper
flash, whereas removing far UV prevents the damaging e
ects of ionizing radia-
tion. Chlorided silver pellets and wires often used in electrophysiological recording
constitute a final source of artifact. These components must be shielded from the
light source or they will generate large photochemical signals.
To simply aim and focus the light beam directly onto the preparation is easiest.
If isolating the lamp from the preparation is necessary, the light beam may be
transmitted by a fiber optic or liquid light guide, with some loss of intensity. If a
microscope is being used already, the photolysis beam may be directed through the
epifluorescence port of the microscope. The lamp itself, or a light guide, may be
mounted onto this port. Microscope objectives having high numerical aperture
and good UV transmission will focus the light quite e
V
V
ectively onto a small area,
which can be delimited further by a field stop aperture. With the right choice of
objectives and proper optical coupling of the lamp to the light guide and the guide
to the microscope port, light intensities 25 times greater than those obtained by
simply aiming the focused steady lamp or flashlamp can be achieved—su
cient to
half-photolyze DM-nitrophen in 25 ms of steady bright light. TILL Photonics
make a xenon arc spectrophotometer (the Polychrome) with e
Y
cient optical
coupling to several commercial epifluorescence microscopes. Half reflective mir-
rors can be used to combine the photolysis beam with other light sources, such as
those used for [Ca
2
þ
]
i
measurement. However, as the optical arrangement becomes
more complex, photolysis intensity inevitably decreases.
The newest development in light sources is the high intensity light-emitting diode
(
Bernardinelli et al.,2005
). This rapidly evolving and inexpensive technology can
already produce 365-nm UV light at 50 mW/cm
2
(with LEDs made by Prizmatix,
Modi'in Ilite, Israel, e.g.), or about 20%of the intensity of a collimated xenon arc lamp.
It is often important to restrict photolysis to one region of a cell (
Wang and
Augustine, 1995
). With epi-illumination, this may be done with a field stop
Y
