Quantifying the geometry of micropipets.

Accurate knowledge of the internal diameter (id) of micropipet tips is important, because the ability to study many different aspects of biological membranes is a very sensitive function of tip size. The authors examined two methods used to characterize pipet tips: the digital manometric method (DMM) and bubble number method (BNM). For DMM, the authors compared the ability of Laplace's equation (model I) and a modified form of his equation (model II), which accounts for adhesion between the test fluid and glass. Pressure measurements were made with a digital manometer, and ids at the tip were measured using scanning electron microscopy (SEM). The micropipet tips showed a slight asymmetry in id, with a approx 5% difference between maximum and minimum id. On average, model I overestimates the largest id by 2%. Model II overestimates the smaller id by 2%. For micropipet tips ranging from 1.00 to 5.00 microm, the corresponding uncertainties range from 20 to 100 nm. Making the normally hydrophilic glass surface hydrophobic strongly reduced threshold pressures when tested in water, but not 100% methanol. Compared to BNM, DMM was insensitive to changes in atmospheric pressure: BNM can be corrected for changes in atmospheric pressure. Convergence angle(s) can be determined from measurements of the pressure and the axial distance of the meniscus from the tip. The accuracy and precision of digital manometry approaches that of SEM. DMM should be particularly useful in selecting micropipets for patch clamp studies of small vesicles (< 10 microm), and may enable systematic selection of micropipets for many other experiments.