Hello, I am an undergraduate student working this summer with my mentor to streamline the analysis (quantification) of NFATs inside the nucleus of mouse lung epithelial cells. Images are obtained using confocal microscopy, then are transferred to Fiji where we enhance the contrast of the channels and compile into one image. The green shows the lung epithelial cells (club cells), blue DAPI stain for the nuclei, and the red punctate staining shows the NFAT proteins.
Our goal is to use CellProfiler to analyze the amount of NFAT inside the nuclei to streamline and enhance our image analysis process focusing on quantification of punctate staining in the nucleus.
My mentor and I are both unfamiliar with the CellProfiler program, and I am hoping for some assistance in the process of creating a pipeline. We believe we will need one similar to the example pipeline, “Speckle Counting” and will need to use ImageMath (mentioned in forum post below) to subtract out the green club cell in order to focus on NFATs located in the nucleus only.
From our research thus far, we believe our pipeline will resemble the following:
Import/convert .nd2 files
uniform image adjustment (i.e. contrast)
define ROI (i.e. nucleus)
quantify pixel density in ROI
export graph and tables
As an undergraduate, I have little experience with this type of work and I am unfamiliar with this program. I do however, have interest in research and am excited to build upon my knowledge by learning a new program! Any and all advice, assistance, or resources you may be able to provide would be very helpful and appreciated. Thank you for your time!