I’m trying to quantify and compare the intensity of my interested proteins from mutant cells and WT cells. I took pictures with a 40x objective lens and cropped several cells per picture (see sample image). For quantifying the signal in each cell, I created ROI by freehand selections in the green (GFP) channel and applied it in the red channel to perform the measurement. Then I did statistical analysis for obtained mean value or integrated density.
sample.tif (631.2 KB)
I have several questions:
which value should I use for statistical analysis? mean or intDen?
should I use a 20x objective lens to obtain more cells per picture to increase efficiency? Usually, I have many mutant groups and I want to quantify more cells in one group. So far, I just take less than 100 cells from one group.
If I take picture with more cells under the 20x lens, is there any automatic method to trace the outline of the cell? I want to create the ROI of the cells which expressed GFP and measure the intensity of each cell by dividing the total red signal intensity by the number of cells. I know I can create a mask after thresholding, but unfortunately, I don’t want to measure the dividing cells or the cells with multinuclear or on the edge.
Do you have any suggestions?