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Open Access Highly Accessed Methodology article

Computer-based fluorescence quantification: a novel approach to study nucleolar biology

Mohamed Kodiha, Piotr Bański and Ursula Stochaj*

Author Affiliations

Department of Physiology, McGill University, 3655 Promenade Sir William Osler, Montreal, H3G 1Y6, Canada

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BMC Cell Biology 2011, 12:25  doi:10.1186/1471-2121-12-25

Published: 3 June 2011

Additional files

Additional file 1:

Multiple processing steps are applied to identify the nucleolar compartment. Examples of the settings for different operations are shown. (a) If fluorescence signals in nucleoli are below the pixel intensities in the nucleoplasm, the Detect dark holes filter can identify nucleoli using the DAPI or probe image. (b) The Median filter reduces noise; regions within the yellow box were magnified 2.5-fold and contrast was increased to show changes generated by the operation. Note that all of the filters are applied only to define the nucleolar compartment; none of the operations affects the fluorescence of the probe image for which pixel values will be measured.

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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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Additional file 3:

Nucleoli can be detected in heat-stressed cells. HeLa cells were incubated for 1 h at 45.5°C, fixed and stained with antibodies against Pol II and RPA194, a subunit of RNA polymerase I. RPA194 was chosen as a marker for nucleoli, because it is less affected by heat than B23 and fibrillarin (unpublished data). Original images are shown in the top panels. Nucleoli are identified based on the Pol II staining (panels in the middle). This requires the Detect dark holes (Holes) and Median filter operations. Alternatively, nucleoli are demarcated by combining the information of DAPI and Pol II staining. To this end, the Add function, Detect dark holes and Median filter operations are performed.

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Additional file 4:

The effect of heat stress on EU staining. For all experiments, HeLa cells were kept with EU for 6 hours, and EU was labeled with Alexa Fluor488 as described in the Methods section. In the top panels, cells were grown with EU at 37°C for 6 hours and fixed. Panels in the middle show cells that were first heat-shocked for 1 h at 45.5°C, then treated for 6 hours with EU at 37°C and fixed. For the bottom panels, cells were incubated with EU for 6 hours at 37°C, then transferred to medium without EU and stressed for 1 h at 45.5°C. Following heat shock, samples were immediately fixed and processed to visualize EU incorporation. Size bar is 20 μm.

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Additional file 5:

Quantification of nucleolar fluorescence with the multi wavelength cell scoring module. Details are shown for a protocol that identifies nucleoli with the DAPI image and measures florescence intensities in the probe image. (a) The flowchart provides an overview over the operations that are applied to the DAPI and probe images before nucleolar fluorescence is measured with the multi wavelength cell scoring module. The nucleolar compartment is delimited by applying first the Detect dark holes and subsequently the Median filter. The probe image is corrected for nonspecific background fluorescence; this generates the statistical correction image for which nucleolar fluorescence will be quantified. (b) Individual steps of the analysis are compiled into a journal which will automatically carry out all of the different operations and save data in an Excel sheet. (c) In order to run the analysis with the multi wavelength cell scoring module, several parameters are required to define nucleoli and quantify fluorescence signals. The parameters include size constraints for the compartment and fluorescence intensities above background for both the DAPI image (left panel) and probe image (right panel). Based on this information, the software generates segments that colocalize with nucleoli. Segments are overlaid with the statistical correction image, and fluorescence intensities are measured in the statistical correction image for the areas defined by the segmentation.

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Additional file 6:

Comparison of the Erode/dilate and Median filter function to define nucleoli with the DAPI image. (a) Original DAPI and probe images on which the analysis was carried out. (b) Nucleoli are identified with the Detect dark holes filter as in Figure 3, and noise is reduced with Erode and Dilate filters. Segments generated for the nucleolar compartment are overlaid with the Dilate, DAPI or probe image, which was corrected for background fluorescence (Statistical correction). (c) For comparison, the holes image is processed with the Median filter to reduce noise. Note that with the Erode/dilate operation there is a smaller number of false positives. However, as compared to the Median filter operation, some of the nucleoli will be missed (white arrows).

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