Automated Single-Molecule Imaging To Track DNA Shape

We describe a straightforward, automated line tracking method to visualize linear macromolecules as they rearrange shape by Brownian diffusion and under external fields such as electrophoresis. The analysis, implemented here with 30 ms time resolution, identifies contour lines from one end of the molecule to the other without attention to structure smaller than the optical resolution. There are three sequential stages of analysis: first, feature finding to discriminate signal from noise; second, line tracking to approximate those shapes as lines; and third, temporal consistency check to discriminate reasonable from unreasonable fitted conformations in the time domain. Automation makes it straightforward to accumulate vast quantities of data while excluding the unreliable parts of it. We implement this analysis on fluorescence images of A-DNA molecules in agarose gel to demonstrate its capability to produce large data sets for subsequent statistical analysis.