Biology Reference
In-Depth Information
2. Microelectrode Pulling and Silanization
MEs can be prepared ''on-demand'' and in ''batch'' methods. The first method
consists in preparing (i.e., pulling and silanizing) MEs on the experimental day,
which has the obvious advantage of having ''freshly'' prepared electrodes and of
being able to manufacture as many as desired a day. Protocols have been described
(reviewed in
Ammann, 1986
). The batch method that we use consists of preparing
a batch of MEs that can be kept for several days in a dry, dust-proof container.
MEs are pulled on a programmable horizontal stage puller (Model P80 PC or
comparable, Sutter Instruments, USA). By trial-and-error, and according to the type
of biological preparations studied, a satisfactory shape ofME can be found. However,
several aspects of ME pulling have to be taken into account when designing its shape.
As ion-selective MEs have an intrinsically high resistance (i.e.,
50-100 G
O
), the
signal-to-noise ratio has to be minimized. This can be achieved in two ways: the first,
and most obvious, is to increase the tip diameter, within certain limits, which are
dictatedby the size of the cell type. Inour case, we impale cardiac cells inwholemuscle.
Cell length is typically between 50 and 150
m
m and tip diameters less than 0.5
m
mare
needed. However, there is a trade-o
>
between tip diameter and detection limit of the
electrode (see below). That is, increasing tip diameter improves electrode response (in
terms of detection limit and speed of response), but is more likely to damage the cell
during impalement. Another simple way to decrease the resistance is to decrease the
length of the ME shank. This further helps to reduce capacitative artifacts, that are
encountered when the level of physiological solution fluctuates in the experimental
bath (
Vaughan-Jones and Kaila, 1986
), and also helps electrolyte filling (see below).
In contracting muscular preparations, the shank should also possess some flexibility
to avoid dislodging of the ME during a contraction. Again, by trial-and-error, an
adequate shape that fulfills all these requirements can be found. Their shape was
designed to reduce their resistance by making them steeply tapered (having a shank
length of approximately 150
m
m and diameter of 20
m
mat10
m
m above the tip).
Under lightmicroscopical observation, the tip diameter was estimated tobe
V
0.5
m
m.
MEs are dehydrated, tip up in an aluminium block, at 200
C for 12 h. Our
experience, and of others (
Vaughan-Jones and Wu, 1990
), has been that a better
silanization and longer lifetime of the MEs are achieved this way.
The silanization protocol consists of spritzing 300
m
lofN,N-dimethyltrimethyl-
silylamine (Fluka) onto the aluminum block and rapidly placing a glass lid on top
of the dish. Care must be taken not to inhale vapor from the silane vial or during its
introduction in the dish. In our hands, once opened, the silane vial can be kept for
at least 2 months without losing its properties. Silanization procedure lasts for
90 min. The lid is then removed and the MEs are baked for another 60 min, which
drives o
the excess silane vapor. The aluminum block and the MEs are then
placed into an air-tight plastic container, also containing desiccant. We advise not
to keep the MEs more than a week, because repetitive openings of the container
and insu
V
cient seal quality will progressively make the electrodes lose their
hydrophobicity.
Y
