Hello,
I have a question about quantitative fluorescent microscopy. Maybe is an obvious one, but I would like to be sure about that. I am using fluorescent images to realize quantitative measurements. I stained mitochondria in my experiments; now I am using this imagess to measure the correlation with the red channel (mitochondria) and the green one (cytoplasm), with Cell Profiler. My question is: Will I modify the intensity of the pixels within the channels by improving the contrast or/and the brightness of the images previous to analysis? In other words, could this kind of image processing altering my data?
Thank you
Hi Chiara,
You must not change the intensities before analyzing colocalization. When you change the brightness and contrast of an image, you can easily saturate the displayed pixel intensities, i.e. many pixels will contain values corresponding to the maximum displayable value. Changing the appearance of the image (i.e. the look-up table, LUT) is allowed before colocalization analysis.
Besides these considerations, you must also make sure that the microscope detectors weren't saturated either when you recorded your images.
Best wishes,
Peter
You can also try procedure of histogram equalization that allow to see variations within mitochondria that appeared uniform in the original image and to check the amount of non-mitochondrial staining. This is not useful for quantative imaging.
- Badges
- Science topic
- Similar topics
- Biological Science
- Physiology
- Physiological Processes
- Stress