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Behaviors of platonic bacteria individuals are profoundly influenced by their interplay. However, probing such interplay still remains a challenge since identification and tracking of bacterial individuals becomes difficult as they come close and interact with each other. Herein, we report 3D tracking of the motions of multiple bacteria by using digital holographic microscopy (DHM), where the subtle 3D behaviors can be characterized as bacteria approach and run away from each other. An algorithm was developed to identify and recover the gap between 3D trajectory segments raising by the interruption from other bacteria through lateral image recognition and axial loalization utilizing cost function. We value the performance of the algorithm in terms of the statistics in trajectory length and correct rate. The study clearly shows how the interplaying Escherichia coli alter their motions.

Improving the axial resolution for multiparticle three-dimensional (3D) holographic tracking is crucial but challenging. Here we study the impacts of incident light power, uniformity of the illumination as well as image pixel size on the axial tracking resolution for digital holographic microscopy (DHM). We demonstrate that the resolution highly depends on the image pixel size and the uniformity of the illumination. A 3D localization algorithm based on local-intensity-maxima searching and a Gaussian fit to the integrated intensity of the reconstructed lateral images along the axial direction proves a robust strategy to enhance the axial resolution for colloids and bacteria within a wide depth of field over several tens of micrometers.

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