I have seen in several places this formula for CTCF: CTCF = Integrated Density – (Area of selected cell X Mean fluorescence of background readings). Is it standard practice to "correct" the mean gray value output from ImageJ to CTCF? What are the benefits of doing so? I am trying to determine if there is a difference in mean fluorescence based on five pictures each before and after a treatment. In your opinion, would it be better to use just the mean gray values or CTCF?
The CTCF method does a background subtraction and also takes the area of the cell that you are measuring into consideration. Check this out:
- https://theolb.readthedocs.io/en/latest/imaging/measuring-cell-fluorescence-using-imagej.html
Hope the clarification provided in the above page provides the info where they talk about the rounded cell and a normal looking cell.
@Sathya Srinivasan
Hi, as Sathya Srinivasan says CTCF is basically background subtraction, so if there's a significant background in your images and the background level can vary from image to image (especially between sample and control), it will help you to get background-independent fluorescence readings. There are a few points to consider when designing your analysis pipeline:
1. The procedure outline in the link cited by Sathya Srinivasan is completely manual and as such not suitable for high throughputs. It can be however quite easily automated using e.g. automatic thresholding to segment cells and the background.
2. CTFC is basically the integrated fluorescence from the cell - background. In some cases it may be more meaningful to use the average pixel value in cells rather than the integrated value. Consider these two scenarios:
a. All cells have approximately the same volume but some cells are flatter than others (the situation discussed in the above cited link). Then the flatter cells occupy larger area and the fluorescence is distributed over more pixels. Thus flatter cells have lower average pixel value even if the total amount of fluorophore was the same in all cells.
b. All cells can be considered similarly flat (or similarly non-flat) but some cells are bigger than others (have larger volume). In this case the mean pixel value would be more meaningful as the integrated intensity will depend on both the fluorophore concentration (florescent protein expression or fluorophore intake) as well as the cell size.
- Badges
- Science topic
- Similar topics
- Physical Sciences
- Physical Phenomena
- Luminescence
- Fluorescence