Additional file 2.
Identification of nucleoli with Methods 1-4. (a) Original confocal images show the distribution of the DNA marker DAPI (blue) and Pol II (red) as well as a GFP-tagged protein (green, probe image, Stat. correction). (b) Method 1 identifies nucleoli based on the DAPI image. (c) Method 2 employs the Pol II staining to delimit nucleoli. (d) Method 3 demarcates nucleoli by using the DAPI and Pol II images (Add image). It should be noted that reliance on the DAPI image only may result in identification of false positive that should be eliminated upon visual inspection. By contrast, some nucleoli could be missed when Pol II staining serves as the only reference. Using the Add image, a combination of the DAPI and Pol II staining, increases the accuracy of the identification process. This method is preferable when visual inspection is not permitted, as in experiments designed for HTS assays. (e) For Method 4 cells were incubated with antibodies against hsc70 and Cy3-labeled secondary antibodies (red), DNA was stained with DAPI (blue). Nucleoli with pixel intensities lower than the nucleoplasm are identified with the probe image (Method 4). Dark holes that represent nucleoli in the probe image are detected with the Detect dark holes filter. The Median filter will then reduce noise and improve the identification of nucleoli. None of these operations affects pixel values in the original probe image. Once the original probe image has been corrected for nonspecific background staining, the resulting Statistical correction image (Stat. correction) is used to quantify fluorescence signals. Based on the identification of nucleoli, the software measures pixel intensities for nucleolar segments (yellow) in the Statistical correction image.
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Kodiha et al. BMC Cell Biology 2011 12:25 doi:10.1186/1471-2121-12-25