Dye exclusion artifact in flow cytometers.

Abstract

Cells exclude their own volume of dye solution in the sample flow which carries them through the flow chamber of the flow cytometer, thereby affecting the otherwise constant signal arising from the fluorescence of this solution. Under certain conditions, this phenomenon may significantly influence the fluorescence signal of the cells. Using the slit scan technique, we studied this phenomenon as observed for monodisperse polystyrene particles in fluorescein solution. The measurements show that dye solution accumulates just in front of the particle and just behind it, with a relative void in between. This phenomenon is most likely caused by the rapid constriction of the flow as it enters the orifice of the nozzle or flow chamber, giving rise to a pulse of fluorescence which adds to that of the particle or cell itself. The magnitude of this artifact depends on the design and dimensions of the nozzle/flow chamber as well as on the rate of sample flow. The dye exclusion artifact may affect measurements of cells when they are in a dye solution having a fluorescence per unit volume which is significant compared to that of the cells, especially at low sample flow rates.