Electrophysiological Assessment of Cerebral Vasospasm - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 477

Microscope . The type depends on the type of experiments either an
upright or an inverted microscope can be used for patch-clamping.
Generally, an inverted microscope is good for cultured or acutely
dissociated cells. In contrast, an upright microscope is necessary
for acute or cultured brain slices. Currently, all the major manufac-
tures sell upright microscopes specifi cally designed for patch-
clamping on slices. The special upright microscopes are equipped
with a fi xed stage and long working distance water immersion
objectives (20× or 40×). Unlike the traditional upright microscope
where the stage moves for focusing, in these microscopes the
objective moves for focusing so that the experimenter can change
the view of the slice without disrupting an already established patch
recording. To visualize the cells in the brain slices, the microscope
needs to be equipped with appropriate optics, such as differential
interference contrast (DIC) combined with an infrared charge-coupled
device (CCD) camera. For experiments using patch-clamping
combined with fl uorescence ion imaging, such as Ca 2+ imaging, the
microscope needs to be equipped with fl uorescence components,
such as fi lter systems, shutter control and appropriate light source.
Amplifi er and data acquisition software . The electric currents or
voltage changes generated by ion channels and receptors are usu-
ally small, and need to be amplifi ed. Several major manufactures
make amplifi ers used for patch-clamping. They included but are
not limited to Molecular Devices (originally known as Axon
Instruments, Silicon Valley, CA, USA), HEKA Electronik (Lambrecht/
Pfalz, Germany) and many other companies. The newest versions
of patch-clamp amplifi ers are knob-less computer-controlled devices,
which are easier to use. The amplifi ers usually come with a data
acquisition software package, which is used to control all operations
of the patch-clamping, data acquisition and storage and offl ine data
analysis following the acquisition.
Micromanipulators . To place patch pipettes on cells, it is very
important to be able to precisely control the movement of the
pipette. There are many types of commercially available microma-
nipulators, including mechanical, hydraulic and motorized ones.
Motorized micromanipulator tend to be more stable and precise,
but more expensive as well. The manipulators usually are mounted
on the fi xed stage of the microscope. At least one manipulator is
needed to hold the headstage for patch-clamping, which is the
electronic probe connected to the amplifi er and holding the
patch pipette. If electrical stimulation, for example, for studying
the neurotransmission of neurons in a brain slice, a second micro-
manipulator is needed to hold the stimulating electrode from the
electrical stimulator.
Electrical stimulator : Stimulators may be analogue, digital with
knobs or knob-less computer-controlled digital stimulators. The
latter is favourable because of its capacity to integrate into the data
acquisition program.