Figure 2.

Parameter optimization during processing of immunolabeled samples

(A) Bandpass filter was applied in the criteria using 50 and 7 pixels for large and small structures. Seed dynamics was set to 3. Intensity threshold was determined by Li’s minimum cross entropy threshold. Peak flooding (in %) was set to 100.

(B) Peak flooding (in %) can be decreased slightly to select punctuate area preciously. Peak flooding (in %) was set to 80.

(C) When bandpass filter was applied in the criteria using 30 for large structures (smaller than 50), the size of H-Watershed segmentation was smaller than (A) image.

(D) When bandpass filter was applied in the criteria using 3 for small structure (smaller than 7), tiny H-Watershed segmentations were detected. Scale bar for the upper and middle images, 5 μm. Scale bar for the bottom images, 2 μm.