Quantification of nucleolar fluorescence in HeLa cells synthesizing a GFP-tagged reporter protein that localizes to nucleoli and the nucleoplasm. The DAPI image is employed to locate nucleoli. Individual operations necessary to identify nucleoli and quantify pixel intensities in the nucleolar compartment are shown. (a) Confocal images for DAPI and GFP. (b) Identification of nucleoli based on DAPI-staining of the DNA. Dark holes in the DAPI image are detected with the Detect dark holes filter, and noise is removed with the Median filter. (c) The median filter and probe images are analyzed with the multi wavelength cell scoring module. The median filter image serves as a compartment image to identify nucleoli, and the software generates segments which colocalize with nucleoli. The overlay of segments with DAPI and probe images verifies that nucleoli are identified correctly. (d) Areas of the nucleolar compartment in μm2 and pixel intensities measured for the probe image (GFP) are depicted. Results for individual nucleoli labeled with numbers are listed in the table.