Fig. 2. Imaging sites (marked by BrRNA) using ESI. HeLa cells were permeabilised, nascent transcripts extended in BrUTP, cells fixed and BrRNA indirectly immunolabelled with gold particles; after sectioning (70 nm), elemental maps were collected by ESI. Arrows indicate clusters of particles. (A-D) Four views of one section. The zero-loss image reveals little other than the cluster of particles marking BrRNA, but P and N maps are complex; in the merge, the grey scale for the zero-loss image is inverted, and particles (now white) mark a N-rich structure. (E-I) Similar merges of other structures labelled with particles; all structures are N-rich. Scale bars: 100 nm. (J) After run-on ± actinomycin D, the number of lone (brown bars) and clustered particles (blue bars) in 15 images (total area 44 µm²) as in D were counted; the inhibitor reduces the number of particles indicating that most mark nascent RNA. As a cluster typically contains 2.9 particles (123 sites analysed), the 2D density of sites marked by clusters is 1.2 sites/µm², which is equivalent to a 3D density of 10.3 sites/µm³. Errors bars represent s.d.