Quantitative assessment of the size and concentration measurement in a novel flow cytometer

Single particle light scattering has gained popularity due to its methodological simplicity, short measurement times, high precision, and suitability for high-throughput particle analysis. This work quantitatively validates two flow cytometry prototypes with regard to particle size and concentration determination based on qualitative studies conducted previously. First, the influence of the interrogation volume on the distribution of the measured scattering signal was examined. The broadening of the scattering signals was found to depend on the diameter of the sample stream, with smaller diameters leading to less broadening. To detect particle sizes ranging from 100 to 1000 nm, an intensity range of at least five orders of magnitude must be detectable. The new device uses two linear amplifiers to enable coverage of six orders of magnitude. Validation of particle size measurements was performed using certified reference standards, resulting in a bias of 8.4 nm and a relative standard deviation of 0.27%. A working range of 30 to 1600 nm, depending on the material, was established. The analysis of a multimodal size distribution was demonstrated. Finally, particle concentrations in the range of 105 to 108 particles per ml could be accurately determined. The amount of fine and coarse fractions in multimodal systems could also be reliably identified. Thus, not only did the validation show excellent results but also offers opportunities for future research on highly complex multimodal systems with widely distributed properties, which has been made possible due to these new technical developments.