Digital in-line holography with numerical reconstruction: 4D tracking of microstructures and organisms

Using holographic microscopy we have been able to visualize submicron-sized. bacteria in-vivo. A simple holographic method enables us to capture as a single data set the trajectories of micron size objects suspended in water. By subtracting consecutive holograms of a particle suspension and then adding these difference holograms, a final data set is constructed that contains the time evolution of the particle trajectories free from spurious background interference effects. Temporal and spatial resolution at the sub-second and sub-micron levels can easily be achieved. The method is illustrated by recording the motion in 3-D of 5μm diameter latex spheres subject to gravity and electrostatic fields to visualize their micro-fluidic flow. Another example is the 3-D motion of a collection of algae, protozoa and bacteria in water.