Digital holography-based 3D particle localization for single-molecule tweezer techniques.

We present a three-dimensional imaging technique for fast tracking of microscopic objects in a fluid environment. Our technique couples digital holographic microscopy with three-dimensional localisation via parabolic masking. Compared with existing approaches, our method reconstructs 3D volumes from single-plane images, which greatly simplifies image acquisition, reduces the demand on microscope hardware, and facilitates tracking higher densities of microscopic particles while maintaining similar levels of precision. We demonstrate utility of this method in magnetic tweezer experiments, opening their use to multiplexed single-molecule force spectroscopy assays, which were previously limited by particle crowding and fast dissociation times. We propose that our technique will also be useful in other applications that involve the tracking of microscopic objects in three dimensions, such as studies of microorganism motility and 3D flow characterisation of microfluidic devices.