Fig. 2
Comparison of traditional surface creation with hue-saturation-brightness surface creation. Traditional surface creation: For a three color image acquired in the red, green, and channels, it is necessary to first identify the different possible combinations of triple positive and double-positive cells using the co-localization channel for each combination. Cell surfaces of the triple and double-positive cells are created using image analysis software, surfaces are merged and used to mask the relevant cells in the original red, green, and blue channels to generate single-positive cells. Each of the individual channels is then used for surface creation and statistics extraction. Hue-saturation brightness surface creation: Hue, saturation, and brightness channels will be generated from the original image using the HSB algorithm. Any cell that expresses a marker will be captured in the brightness channel, thus making it possible to generate cell surfaces for all the cells in one channel. Triple positive cells can be identified using the saturation channel, and information from the hue channel can then be used to render the surfaces in their native hue. Scale bar, 5 µm
