I would say there are two main things to be aware of when using subcellular detections for counts.
- If you sum up the areas of subcellular detections, you will usually get a lower number than the estimated spot count because spots below the size of a single spot will still count as 1 spot.
- Each spot or spot cluster is an object. The estimated number of spots in a cluster is the area of the cluster divided by the estimated spot size you chose.
If you chose 1 square micron for your spot size, the estimated spot count for a subcellular cluster will equal the area of that cluster. The estimated spot size of a spot that is smaller than one micron will still be one.
Example assuming the estimated spot size is 1.0.
One cell has two spots and one cluster.
Spot 1 is 0.5 um^2
Spot 2 is 1.0 um^2
Subcellular cluster 1 is 2.5 um^2
There are 4 detections in this example, one cell and it’s three children.
The total area if you summed the areas of those objects would be 4um^2.
In the cell’s measurement list:
The estimated spot count (“Subcellular: DAB: Num spots estimated” ) would be 1+1+2.5 = 4.5 estimated spots.
Num single spots would be 2
Num clusters should be 1, but upon testing it does not seem to be registering clusters for me. That might have been lost after the change to the object type for subcellular objects.
In the annotation measurement list, you would get
You cannot tell the number of total estimated spots from the annotation measurement list, since you do not know what each cluster represents (it could be 2.5 spots, it could be 50).