In flow cytometry, why might distinguishing populations of cells be more complex in heterogeneous samples?

Distinguishing populations of cells in heterogeneous samples is often more complex due to similar light scattering properties. In flow cytometry, cells are analyzed based on their light scattering characteristics and fluorescence intensity. When different cell types have similar size and granularity, they can scatter light in comparable ways, making it challenging to differentiate between them solely based on these properties. This overlap can result in ambiguous data, where distinct populations are not readily discernible.

To address this issue, flow cytometrists often employ additional markers or fluorescent dyes that can bind specifically to different cell types, enhancing the resolution of the populations based on their differential staining profiles. However, when the cells themselves exhibit similar physical characteristics, the differentiate becomes fundamentally difficult, thus complicating the analysis even further.