(A) Example observables that can be marked and viewed in optical mapping. (B) labeling schemes of the epigenetic modifications shown in A. (i) labeling of TCGA sequences that contains an unmodified cytosine by the enzyme M.TaqI and a fluorescent cofactor; (ii) two-step labeling of 5-hmC: the enzyme β-GT attaches a modified glucose with an azide group on it from uridine-diphospho-6-azide-glucose (UDP-6-N3-Glu) to the hydroxyl group of the 5-hmC. Then the azide group is reacted with a fluorescently-labeled alkyne via click chemistry; (iii) two-step labeling of DNA damage sites: The damaged lesion is enzymatically excised, and replaced by fluorescent nucleotides; (iv) in vitro labeling of origins of DNA replication: fluorescent nucleotides enter synchronized cells following electroporation. Then, fluorescence is incorporated into the newly replicated DNA and creates symmetrical replication ‘forks’. (C) An exaggerated illustration demonstrating the impact of thermal fluctuations on multi-color marker detection. Sequential color acquisitions capture the same molecule at different off-equilibrium conformations due to fluctuations. This results in an erroneous interpretation of marker locations along the DNA molecule compared to the ground truth.