Accurate automatic object 4D tracking in digital in-line holographic microscopy based on computationally rendered dark fields

Building on Gabor seminal principle, digital in-line holographic microscopy provides efficient means for space–time investigations of large volumes of interest. Thus, it has a pivotal impact on particle tracking that is crucial in advancing various branches of science and technology, e.g., microfluidics and biophysical processes examination (cell motility, migration, interplay etc.). Well-established algorithms often rely on heavily regularized inverse problem modelling and encounter limitations in terms of tracking accuracy, hologram signal-to-noise ratio, accessible object volume, particle concentration and computational burden. This work demonstrates the DarkTrack algorithm—a new approach to versatile, fast, precise, and robust 4D holographic tracking based on deterministic computationally rendered high-contrast dark fields. Its unique capabilities are quantitatively corroborated employing a novel numerical engine for simulating Gabor holographic recording of time-variant volumes filled with predefined dynamic particles. Our solution accounts for multiple scattering and thus it is poised to secure an important gap in holographic particle tracking technology and allow for ground-truth-driven benchmarking and quantitative assessment of tracking algorithms. Proof-of-concept experimental evaluation of DarkTrack is presented via analyzing live spermatozoa. Software supporting both novel numerical holographic engine and DarkTrack algorithm is made open access, which opens new possibilities and sets the stage for democratization of robust holographic 4D particle examination.