3D Tracking of Weak Phase Objects by Digital Holographic Microscopy *

In this work, we improve the measurement accuracy of Digital Holographic Microscopy (DHM) for tracking the three-dimensional (3D) motions of Weak Phase Objects (WPO). Particles with a small size and a refractive index similar to the surrounding medium, such as bacteria and transparent particles, are classified as WPO. First, to maximize the signal-to-noise ratio of the hologram, we place the hologram plane in the middle of the sample volume. As a result, both real and virtual images are reconstructed from the hologram. We distinguish between the two images based on the axial intensity profiles of WPO, and recover the original 3D particle distribution. Second, we address the low axial localization accuracy in DHM. We find a shorter axial elongation of WPO image in the reconstructed field consisting of both scattered and incident waves, and utilize them to reduce the uncertainty of the particle position. Finally, we test and demonstrate our methods by tracking the 3D Brownian motions of micro-particles, as well as the swimming of marine bacteria. The particle concentration exceeds 5000 particles/mm³. Our method can be applied to understand bacterial movement, bacteria-wall interactions, and the mechanism of biofilm formation.