(A) The merged, super-resolved image of Dendra2-Pol II (green, PALM) and JF646-β-actin mRNA (magenta, STORM) is presented for a MEF cell chemically fixed 12 min after serum stimulation. (A, left inset) conventional fluorescence image shows two bright gene loci (orange arrows, indicated as “Locus 1” and “Locus 2”) in the same plane of focus for the same cell. In a fixed cell background non-diffusing mRNA may also appear as puncta of lower intensity in both conventional, and STORM images. (A, right insets) Co-localized Pol II clusters are detected on both gene loci. Standard magenta and green are used for β-actin mRNA and Pol II respectively, such that co-localizations appear as white. (B, C) The cumulant graphs for the two co-localized Pol II clusters are represented for Locus 1 and Locus 2, respectively. Note the slope onset (black arrows) from the start of acquisition indicative of stable clusters as expected for fixed cells. Both cumulant graphs feature total counts greater than 200 detections. The estimation of the count of Dendra2-Pol II molecules in the cluster is ~80 molecules for each locus. (D) Cumulant graphs of co-localized Pol II signals from 34 fixed cells are represented. More frequent are Pol II accumulations with relatively low counts of detections (in gray, 29 out of 44 cumulants), and molecule counts consistent with the number of Pol II engaged in β-actin mRNA synthesis (see Figure 3—figure supplement 1H). These may likely represent elongating Pol II. Also featured in the cumulant graphs are large Pol II clusters (in red, 15 out of 44 cumulants) with total counts greater than 200 detections, and molecular counts significantly higher than expected for elongating Pol II on a β-actin gene. These likely represent the rare transient clusters observed in living cells, captured during the fixation process. For the estimated number of Dendra2-Pol II molecules for a selection of Pol II cumulants, see Appendix 4,5 and Figure 3—figure supplement 1. Temporal localization profiles for 44 Pol II clusters co-localized with β-actin gene loci are available in Figure 3—source data 1.