Fig. 1

Visualizing isolated chromosomes in intact nuclei with dilution labeling. a Schematic of the “dilution labeling” strategy to visualize individual chromosomes. Human HCT-116 cells arrested at the G1/S transition (top left) are exposed to EdU during a single round of DNA replication, allowing EdU incorporation in newly synthesized DNA strands (green lines). As a result, each of the 46 chromosomes carries a single DNA strand with EdU modified nucleotides (1st division). Cells are then allowed to undergo multiple replication cycles in absence of EdU (2nd, 3rd,... divisions). Owing to semi-conservative replication of the DNA double helix, the number of chromosomes comprising an EdU-carrying strand is halved, on average, after each division, but EdU-carrying strands are expected to remain intact (in absence of sister chromatid exchange, see b below). After m cell divisions, cells are expected to contain only one entire EdU-carrying chromosome with probability p₁ = 46 × 2^−m(1 − 2^−m)⁴⁵ and no EdU-carrying chromosomes with probability p₀ = (1 − 2^−m)⁴⁶, and on average <n >  = 46 × 2^−m labeled chromosomes (bottom right). Click chemistry with Alexa 647 dyes (red) is used to fluorescently label EdU-carrying strands after cell fixation, enabling imaging (bottom left). b Sister chromatid exchange (SCE) can compromise the integrity of EdU-carrying strands and lead to incompletely labeled chromosomes. c Predicted size distribution of EdU-labeled chromatin regions after six divisions, taking into account the measured rate of SCE (Additional file 1: Fig. S2). d Widefield images of EdU and A647-labeled chromosomes taken immediately after EdU incubation (0 days), after 3 days and after 7 days. As the delay between EdU incorporation and click chemistry is increased, the fluorescent signal becomes confined to a small number of micrometer-sized patches, as expected for single chromosomes