Segmenting Nuclei

This panel completes the nuclear segmentation by watershed on the filtered image from the previous step, using the nuclear centres identified previously as seed points. The Threshold for Nuclear Segmentation determines the intensity threshold that will be used to segment all nuclei from background. The nuclei are segmented from each other using one of two methods:

Nuclear Volume Marker

Dividing lines between nuclei are constructed based solely on distance from seed points. Each resulting boundary line is equidistant from the seed points in the nuclei that it bounds. This is implemented using Thomas Boudier’s Watershed Segmentation.

Nuclear Membrane Marker

The construction of dividing lines takes into consideration both the distance to the nearest seed points and the local intensity variations in the nuclear signal. In theory, the dividing lines constructed using this method should more closely align with sharp gradients between nuclei, if they exist. How much weighting is attributed to local intensity versus distance to seed points is tunable via the Distance Weighting parameter - a larger value will result in segmentations that are more heavily based on distance to seed points alone. This method is implemented using MorphoLibJ’s marker-controlled watershed. Bear in mind that this approach is quite computationally intensive and can take some time to execute - be patient!

Preview Segmentation

Clicking Preview will calculate and display the resultant segmentation:

If you wish, you can render the resultant segmentation using FIJI’s in-built 3D viewer:

Proceed to Segmenting Cells

Go back to Background Subtraction Prior to Nuclear Segmentation