Fig. 4.

Mean fluorescence intensity (MFI) measurement of cell nuclei using automated region of interest selection based on a signal threshold (Approach 2 (3.2.2)) A. Original confocal image of a section cut through a WT mouse lens capsule at 3 days post cataract surgery (three-color imaging; (pSmad3- Alexa Fluor 568- red), αSMA (FITC- green) & DNA (DRAQ5-blue)). B. pSmad3 channel alone only. C. Adjusting the red channel to select for cell nuclei stained for pSmad3 protein by using the ‘Threshold’ button. D. Nuclei exhibiting pSmad3 staining above ‘Threshold’ are outlined in yellow and are ready for MFI quantitation. E. Click on the ‘Reset’ button to get back to the original image and results of MFI quantitation of pSmad3 levels in selected cell nuclei. C- Lens Capsule, LC- Lens cells, Scale bar- 35 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)