Sample image and/or code
This is a mouse brain implanted with tumor, the immune cells were engineered with Green fluorescence protein or red fluorescence protein, and we stained the nuclei with blue. So there are green, red, and blue in the slice. We need to quantify the green cells and red cells. The histology images were acquired using a machine named NanoZoomer, the raw data are. NDP files, we review the images with NDP viewer, calculate the area of the tumor, make a screen shot, then quantify the cells from the screenshots with FIJI.
Quantify Green and red cells
We draw the outline of the tumor in FIJI, then Edit-Clear outside-Subtract Background with 4.0 Pixels-Separate channels to red, green, and blue. For the Green or Red separated images, we do Pluggins-Filter-Otsu Thresholding, then we go to Process-Binary-Convert to Mask. Usually the background will become white when we run Convert to Mask, but with this IMAC (it is a new computer, we installed FIJI, Java, Otsu thresholding pluggin) it doesnot work. I ran the same steps, it works on my Macpro book.
Thank you for the help in advance!