Measurement of RNA synthesis in nucleoli. HeLa cells were incubated with 1 mM 5-ethynyluridine for 6 hours at 37°C, fixed and treated with azide-modified Alexa Fluor488. (a) Confocal images were acquired, and the probe image was corrected for background fluorescence (EU, Stat. correction). The Detect light holes and Median filters were applied and segments were overlaid with the median filter image. (b-d) Fluorescence intensities were measured in nucleoli and nuclei of 44 cells. (b) Original data for the nucleolar intensity/area are depicted for each of the 78 nucleoli. Results were sorted smallest to largest value. The X-axis depicts individual nucleoli. (c) The sum of nucleolar pixel intensity/sum of nucleolar area is shown for each nucleus, and (d) the nucleolar/nuclear intensity was calculated for each nucleus. In (c) and (d) the X-axis displays individual nuclei. (e) HeLa cells were incubated with EU as described for part a, and B23 was subsequently located by staining with antibodies. (f) For the demarcation of EU-labeled nucleoli HeLa cells were treated with EU as described for part a, then incubated with antibodies against Pol II and Cy3-labeled secondary antibodies. DNA was stained with DAPI. Top panels show the DAPI and Pol II images; the image obtained after applying the Add function and the EU image. Nucleoli were then identified based on the EU image (with the Detect light holes and Median filter functions) or the DAPI image (Detect dark holes and Median filter). In the bottom panels, Pol II staining (Detect dark holes and Median filter) or a combination of DAPI and Pol II staining (Add function, then Detect dark holes and Median filter operations) served as references to define nucleoli.