Microscopic analysis of pressure ejection of drugs from micropipettes.

Ejection of drugs from micropipettes by pressure is widely used for chemical stimulation of cells in electrophysiological experiments, but little is known about the dynamics of the processes involved. Several aspects of the formation of liquid droplets were studied by photographing them under a microscope using brief light flashes. The observations led to the following conclusions: 1. The characteristics of the micropipette tip strongly influence both the amount of liquid and the shape of the droplet. Micropipettes were divided according to their tip diameter. Those with a 3-8 microns diameter tip produce small droplets that remain near the tip; those with a tip diameter of 9-15 microns produce conical droplets that extent some distance from the tip; droplets ejected from micropipettes with tip diameter greater than 15 microns are mushroom-shaped and reach distances several hundred microns from the tip. 2. Back-flow of the bath medium, due to capillary force, diminishes the amount of the drug ejected from the micropipette. This effect is very prominent when brief (< 100 ms) pressure pulses are used, and to overcome it priming of the micropipettes several seconds before use is required. 3. Flow of the bath medium can divert the droplet from the expected trajectory. This point was verified during intracellular recordings of responses to pressure-ejected substance P in myenteric neurons. The recommended parameters for optimal use of pressure ejection are: tip diameter of 10-15 microns, pressure of 80-100 kPa, pulse pressure duration over 100 ms and priming by pressure pulse of 1-2 s duration; liquid column of about 5 cm can minimize back-flow.