Any fluid stream ejected into air will break up into droplets but this is not a
stable process; the distance from the orifice that the stream begins to break up
will depend on many factors such as the orifice size, the pressure of the sheath
fluid, the ambient temperature, and the viscosity of the fluid. However, if a stationary
wave of vibration of known frequency and amplitude is applied to the
fluid stream, it is possible to stabilize the break-off, and for a given set of conditions,
the size of the droplets and the distance between drops will also stabilize.
In a flow sorter, this vibration is produced by a transducer, which is generally a
piezo-electric crystal acoustically coupled to the nozzle. As cells are ejected
from a nozzle, they pass through one or more laser beams and at this point – the
moment of analysis – information is gathered about the cell or particle (Figure 1).
The distance, and therefore time, between the point of analysis and the point at
which the cell breaks off from the solid stream in a droplet is constant and under
given conditions can be calculated. This distance between the laser intercept
and the break-off point is measured in drop equivalents and is often referred
to as the drop delay. The calculation and monitoring of this is critical and is the
factor that makes a sort successful or not. The drop break-off can be observed
microscopically under stroboscopic illumination to allow the break-off point to
be monitored. The drop delay is calculated by determining how many drops are
in the distance between the analysis point and the break-off point; drops will
start to form as soon as the stream emerges from the nozzle but will be coalesced
until the break-off point. There are several ways of measuring the drop delay:
by counting the number of drops formed in a known distance, by sorting beads
onto slides at varying drop delays and checking microscopically, or by viewing
fluorescent beads in sorted side streams. The precise way of calculating drop
delay will vary with the type of sorter used.