Increasing the Sensitivity of a FACScan Flow Cytometer to Study Oceanic Picoplankton

Existing limitations on the sensitivity of commercially available flow cytometers do not permit the detection of extremely dim picoplankton cells, particularly Prochlorococcus marinus in nearsurface oligotrophic ocean waters. This problem was overcome by making some simple modifications to a FACScan (Becton Dickinson) flow cytometer to increase its sensitivity by about fivefold. The fluorescence and side scatter sensitivity was increased by replacing the existing focusing lens and beamsteering mirror with a $3\times$ expansion telescope and a slightly shorter focusing lens, resulting in a smaller laser spot size at the interrogation point. Forward scatter sensitivity was increased by replacing the existing photodiode with a photomultiplier tube, which has a background noise level about an order of magnitude lower. The fluorescence sensitivity of the modified instrument, measured with a quantitative fluorescence microbead standards kit, is 74 molecules of equivalent soluble fluorechrome for fluorescein. The instrument can now detect extremely dim P. marinus from open-ocean surface waters.