A trigger channel threshold artifact in nanoparticle analysis

The analysis of individual nanoparticles by flow cytometry involves the measurement of dim signals that are near the detection limits of the instrument. Discriminating the signal from particles of interest from that of background particles in buffers and from optical and electronic noise can be challenging, and requires careful consideration of the measurement approach, control experiments, and scrutiny of the resulting data. In applying this scrutiny, we have come to recognize an artifact that results from the inappropriate selection of the trigger channel threshold that might not be obvious to the casual user. When measuring dim signals close to the noise or background levels, it is intuitive and common for the operator to adjust the trigger threshold to minimize the "false triggers" acquired by the system, and then to run the unknown sample, interpreting the events detected above the background as measurements of individual particles. We show here that when this approach is used to measure particles whose signals fall below the trigger threshold, only coincident events are detected, producing erroneous measurements of both particle number and brightness. We suggest that in many cases, the analysis of dim nanoparticles is best achieved using a fluorescence channel for the trigger.