Analyze Beta-Catenin with CyteSeer’s Membrane Segmentation Algorithm
These pages demonstrate how to analyze beta-catenin fluorescent image – with a DAPI nuclear counterstain – using CyteSeer's membrane segmentation algorithm. Please refer to the relevant application note and references for more detailed scientific discussion as appropriate.
Membrane Segmentation Settings for Beta-Catenin
- Select "Membrane Segmentation" for CyteSeer's "Algorithm to RUn"
- For this example, we used a sensitivity setting of 125% and a nuclear size value of 7. These will vary with respect to imaging conditions.
- Click "Run" to execute the analysis on the checked wells.
In general, if CyteSeer creates masks that under-represent certain parts of the cell, the sensitivity is too low. If the mask is over-represented, the sensitivity is too large. In routine use, CyteSeer will likely work best with a sensitivities of 25% to 400%.
The size values most often refer to nuclear size. If CyteSeer incorrectly identifies a large proportion of clearly distinct single nuclei into multiple nuclei, the size value is too low. If there are many cases of two nearby nuclei being counted as one, the size value is too large. In routine use, CyteSeer will likely work best with a nuclear sizes from 3 to 30.
Image Viewer: Unprocessed Fluorescent Images
Use CyteSeer's Image Viewer to view the raw fluorescent images. (green = beta-catenin; blue=DAPI)
Image Viewer: Membrane Segmentation Results
- Use the "Set Images" dialog to navigate between wells
- Use the "Set Colors" dialog to set which parts of the raw image or image masks to show. Note that each cell has a unique ID and that the membrane segmentation is overlaid in yellow on the original raw image.
Negative Control
The negative control here shows a near complete lack of membrane structure.
Report Membrane Intensity
Once CyteSeer has created image masks well, many shape, size, intensity and colocalization are automatically generated as part of the algorithm.
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