Coincidence in high-speed flow cytometry: models and measurements

In flow cytometry, the coincident arrival of particles becomes a major problem when high sample rates are required. For the development of our high-speed photodamage flow cytometer (ZAPPER), it was of importance to understand the behavior of cells at flow rates of around 50,000-250,000 event/s. We developed and compared two models that describe the relation between the real cell rate and the detectable single cell rate. Both the Computer Simulation model and the Input/Output Device model show distinct optima for the cell rate. The models were compared to measurements performed on the ZAPPER-prototype. Fits of the two models to the experimental data were excellent for cycle times of 4 and 15 microseconds and acceptable for a 2 microseconds cycle time. A third model (Mercer WB, Rev. Sci. Instr. 37:1515-1521,1966) could be fitted to the experimental data, after the proportionality constant k was adapted to the experimental data. At a yield of detectable single cells of 70%, the maximum cell rates are 180,000, 100,000, and 40,000 cells/s for cycle times of 2, 4, and 15 microseconds, respectively. Based on these results we can now select an optimal cell rate for analysis and sorting based on criteria such as accepted cell loss. In addition, the advantages of reducing the cycle time can now be evaluated with respect to the costs of that modification.