2D vs 3D tracking in bacterial motility analysis

Digital holographic microscopy provides the ability to observe throughout a large volume without refocusing. This capability enables simultaneous observations of large numbers of microorganisms swimming in an essentially unconstrained fashion. However, computational tools for tracking large 4D datasets remain lacking. In this paper, we examine the errors introduced by tracking bacterial motion as 2D projections vs. 3D volumes under different circumstances: bacteria free in liquid media and bacteria near a glass surface. We find that while XYZ speeds are generally equal to or larger than XY speeds, they are still within empirical uncertainties. Additionally, when studying dynamic surface behavior, the Z coordinate cannot be neglected.