Factors governing the flow cytometric analysis and sorting of large biological particles

We have investigated the suitability of large flow cell tips for the flow cytometric analysis and sorting of large biological particles, including plant cells (pollen) and protoplasts. Using flow tips ranging in diameter from 79-204 micron, we have optimized conditions for the establishment of a stable hydrodynamic flow leading to accurate droplet production. We describe instrument modifications required for large particle sorting and demonstrate the use of these experimental conditions for the sorting to high purity of pollen and viable plant protoplasts possessing diameters as large as 95 micron. Our experiments have revealed a complex interaction among sorting efficiency, particle diameter, flow cell tip diameter and bimorphic crystal drive frequency. This interaction can be satisfactorily explained in terms of interference effects owing to phase differences between the particle-induced disturbance and the undulation driven by the bimorphic crystal.