Genome-wide analysis of the dynamic and biophysical properties of chromatin and nuclear proteins in living cells with Hi-DTo understand the dynamic nature of the genome in real time, the localization and rearrangement of DNA and DNA-binding proteins must be analyzed across the entire nucleus of single living cells. Recently, we developed a new computational light microscopy technique, called high-resolution diffusion mapping (Hi-D), that can accurately detect, classify, and map the types of diffusion and biophysical parameters at a high spatial resolution over the entire genome in living cells. Hi-D combines dense Optical Flow to detect and track local chromatin and protein motion, and Bayesian inference to characterize this local movement at nanoscale resolution. The initial implementation requires some experience using MATLAB (MathWorks) and computational resources, for instance in form of access to a computer cluster, to perform the Hi-D analysis. In addition, this implementation takes approximately 18-24 hours to analyze a typical image series. To avoid these limitations and emphasize high-performance implementation, we present a customized version called Hi-D-Py. The new open-source implementation is written in the open source Python programming language, and options for parallelizing the calculations have been added to the code pipeline. We introduce user-friendly python notebooks and automatically generated reports that summarize and tabulate the results of the analysis. Our efficient implementation reduces the analysis time to less than one hour using a multi-core CPU with a single compute node. We also present different applications of Hi-D for live-imaging of DNA, H2B, and RNA Pol II sequences acquired with spinning disk confocal and super-resolution Structured Illumination Microscopy.
### Competing Interest Statement
The authors have declared no competing interest.
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