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

ABSTRACT 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. We propose that our technique will also be useful in other applications that involve the tracking of microscopic objects in three dimensions. SIGNIFICANCE Tracking objects in 3D is a common task in biology, but typically requires the acquisition of image stacks, which is limited by speed, the depth of field of microscope objectives and by the presence of other objects that obscure the illumination. Here we develop HoloMiP ( Holog raphic Mi croscopy with P arabolic masking), which uses digital holography to reconstruct the three-dimensional images from a single plane allowing tracking of light-scattering objects in 3D. HoloMiP outperforms existing methods in precision, speed, simplicity and tolerance to crowding. We show that it is particularly suitable for fast, multiplexed magnetic tweezer experiments, opening new avenues to high-throughput force spectroscopy.