Why does the sample stream in flow cytometer need to be aligned in single file?

The purpose of aligning the sample of cells or particles in single file is to increase the accuracy of the data by reducing the data spread. Without any focusing technique, the sample of cells or particles entering the flow cell would be moving in a random and disorganized manner, which may result in two possible consequences:

• (1) Variability may be introduced in the data since cells are travelling at different speeds.
• (2) Coincident events (two cells being analyzed at the same time instead of separately) may be collected because cells are in too close proximity to each other.