Biology Reference
In-Depth Information
inside a loop of tungsten wire across the prongs of an outlet plug. We control the
heat transiently with a rheostat and observe the pipette through an inexpensive,
low power, dissecting scope mounted horizontally on a boom arm. The Sylgard is
precured to a viscous consistency at room temperature when it is first made and
then stored in small 1 ml aliquots in the freezer. We apply it with a bent hypoder-
mic needle by taking up a dollop onto the needle, touching the dollop to the
pipette, and slowly winding it on to the glass by turning the pipette from below.
Care must be taken to avoid touching Sylgard to the heating/curing wire, or the
Sylgard vapors will quickly coat the tip. You will know when you get Sylgard too
close to the tip because when you try to polish it, the Sylgard contracting around
the thin glass walls at the tip literally wrinkles the glass.
Everybody has their own favorite glass that they believe forms the tightest seals,
but their relative intrinsic noise can be tested empirically using the Sylgard hemi-
spheres. The lowest noise is produced by quartz glass capillaries ( Levis and Rae,
1993 ), but they require a special laser puller to create the pipettes. On real cells, the
size and shape of the tip also influence success. It is extremely di
Y
cult to make
pipettes that will routinely make
10 G O seals when you cannot clearly see the tip
while you polish it. An extra long working distance (ELWD) objective with at least
40
>
magnification is essential but they are expensive, so they must be mounted
below the polishing element that is heated to prevent cracking the lens from
repeated heating. Our polishing strategy is illustrated in Fig. 2 . We find that it is
also critical to melt a small bead of the pipette glass onto the apex of the heating
element, usually a small loop of platinum wire, presumably because it prevents the
tip from being coated with metal. We find that pipettes with bullet-shaped tips and
initial resistances between 3 and 5 M O make the best seals.
Filling the pipettes also requires some attention to detail. All pipette solutions
should be filtered through 0.2 or 0.45- m m filter disks but do not use filter disks
prepared with wetting agents. To avoid bubbles and washing the dirt inside the
capillaries down into the tip, both of which reduce seal success, one must first fill
the tip separately by immersing it in a small vial of pipette solution and allowing
the first 20-50 m m to fill by capillary action. Then the rest can be backfilled.
Usually bubbles are visible to the naked eye, and they can be removed by gently
flicking the pipette while it is held between the thumb and the forefinger.
C. Making Seals
To reliably get seals over 10 G O , all the precautions in Table III are important. In
addition you have to be fast. It should take only 3-5 min from filling the pipette to
touching down onto the cell, including mounting the pipette in the holder, manip-
ulating it into the chamber, zeroing out the o
set, measuring the resistance, finding
it in the microscope, and manipulating it just above the cell without touching the
bottom or another cell. This takes practice. It also helps to run a little solution
through the chamber before lowering the pipette into the bath to clear any debris
that accumulates on the surface. To minimize debris from collecting on the patch
V
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