HEKA Elektronik EPC 10 USB, Hardware Manual, Page: 59 / 81

9. Patch Pipettes
9.1 Glass Capillaries
Procedures for fabricating pipettes are presented in some detail in the paper by Hamill et al. We will summarize
the procedure and present some tips that we have found helpful. The main steps in pipette fabrication are to form
a smooth tip on the pipette (to allow seals to be formed without damaging the cell membrane) and to coat the
pipette with a suitable insulating coating to reduce the background noise.
Figure 9.1: Sources of glass pipettes
Pipettes can be made from many different types of glass. Our impression is that different types of glass work better
on different cell types. Glass capillaries are available from soft (soda glass, flint glass) or hard glasses (borosilicate,
aluminosilicate).
Soft-glass pipettes have a lower melting point (800
°
C vs. 1200
°
C), are easily polished, and can be pulled to have a
resistance of 1 − 2 M Ω. They are often used for whole cell recording, where series resistance rather than noise is the
limiting criterion. The large dielectric relaxation in soft glass sometimes results in additional capacitive-transient
components that interfere with good capacitance compensation. Hard-glass pipettes often have a narrow shank
after pulling and consequently a higher resistance. Hard glasses tend to have better noise and relaxation properties,
however: the important parameter here is the dielectric loss parameter, which describes the AC conductivity of
the glass. Although the DC conductivity of most glasses is very low, soft glasses in particular have a conductivity
around 1 kHz; that is sufficiently high to become the major source of thermal noise in a patch clamp recording. We
find that Kimax glass is a good compromise for whole cell recording. Borosilicate and, especially, aluminosilicate
glasses (Rae and Levis, 1984) have low dielectric loss and are desirable for the lowest noise recordings. They do not
necessarily form the best seals, however; this might be due to evaporation of metal onto the glass surface during
the high-temperature pulling and polishing steps.
9.2 Pulling
Pipettes are pulled in two stages: the first to thin the glass to 200
−
400 µm at the narrowest point over a 7-10 mm
region, and the second to pull the two halves apart, leaving clean, symmetrical breaks. Both halves can be used.
The length of the first pull and the heat of the second pull are the main determinants of the tip diameter of the final
pipette. A number of commercial pullers can be used to make pipettes. For reproducibility, however, a regulated
current supply to the heater coil is best. A mechanical stop to set the length of the first pull is also important for
reproducibility.
Note: HEKA is offering a gravitational force two-stage vertical puller, the PIP 6 .