Separating twin images in digital holographic microscopy using weak scatterers

When using inline digital holographic microscopy (DHM) and placing the hologram plane within a particle suspension, both real and virtual images come into focus during reconstruction, limiting our ability to resolve three-dimensional (3D) particle distribution. Here, we propose a new method to distinguish between real and virtual images in the 3D reconstruction field. This new method is based on the use of weak scatterers, and the fact that the real and virtual images of weak scatterers display distinct intensity distributions along the optical axis. We experimentally demonstrate this method by localizing and tracking 1 µm particles in a 3D volume with a particle concentration ranging from 200 to 6000 p a r t i c l e s / m m 3 . Unlike previous approaches to address the virtual image problem, this method does not require the recording of multiple holograms or the insertion of additional optical components. The proposed method allows the hologram plane to be placed within the sample volume, and extends the capability of DHM to measure the 3D movements of particles in deep samples far away from the optical window.