Flow cytometers are essential tools for the analysis of blood and microparticles and are a standard component of modern medical laboratories. However, there remains a strong need to lower the detection limit toward smaller particles, to extract shape-related information from the particles, and to develop improved coincidence correction methods to further enhance measurement accuracy. To measure the absolute size of (sub-)microparticles, the optical parameters of the flow cytometer must be known in advance. A new, simple, and time-efficient method has been developed to determine these parameters, which only requires rough initial estimates. The true values of the optical parameters are determined using Differential Evolution by optimizing the agreement between theoretically calculated and measured scattered light intensity. For the forward direction, this method provides stable results with low variance, whereas the variance in the sideward direction is higher. However, precise validation remains difficult. In contrast to conventional flow cytometers that capture only a single intensity value per detected particle, here the entire transit of a particle through the laser over time is recorded. By evaluating the temporal width of the pulse, coincidences of similarly sized particles can be detected and corrected significantly more accurately than with, for example, the pulse area alone. Using the skewness of the pulse, even coincidences of particles with significantly different sizes could largely be detected—something not possible with previously established coincidence correction techniques. An advantage of this method is the ability to access individual coincidence events and to apply coincidence correction to measurements that do not follow a Poisson distribution.

In addition, a statistical coincidence correction method was developed, based on the exponential waiting time distribution of successive events. From the waiting times of events that exceed a defined cut-off time, the waiting time of the full distribution can be inferred using the self-similarity property of the waiting time distribution—thus enabling determination of the statistically correct number of counting events. A key advantage of this method is that only a single measurement is required for coincidence correction, unlike dilution series which typically requires at least four different dilution steps.

With the development of imaging flow cytometers, it became possible to image scattered light in the sideward direction. For spherical particles, surprisingly, instead of a circular image, several bright spots appear, spaced approximately by the particle diameter. It was demonstrated that these bright spots observed in the sideward direction for spherical polystyrene microparticles correspond to the from other fields known Glare Points. An imaging model based on an ideal lens and the Rayleigh-Sommerfeld diffraction integral was developed to describe this phenomenon. Theoretical investigations of deformed spheres showed that, for high refractive indices such as polystyrene, additional evenly spaced bright spots appear that are not visible in undeformed spheres.

Finally, the suitability of a quadrant detector for distinguishing spherical from non-spherical particles was theoretically investigated. A ratio R was defined as a measure of asphericity. It was found that this ratio is highly suitable, as the order of aspect ratios for different particle types closely matches the order of R over a wide size range. Increasing the numerical aperture also increases the ratio R, which further improves sensitivity to variations in the aspect ratio.