I would like to be able to compare fluorescence measurements taken at different times in different samples directly with one another.
From previous experience I know that microscope settings need to vary between samples in order to optimise the image being taken. I use live tissue and variations in dye loading, tissue thickness, etc. prevent keeping the microscope settings (detector gain, amplifier offset, amplifier gain) the same between measurements of different samples. Obviously settings for different measurements made within the same sample are kept the same, and laser power and exposure time are also kept constant for different samples.
As such, is there a way to normalise fluorescence measurements? Ideally I'd like to know If I can normalise my fluorescence measurement of interest (e.g. TMRM) to some cellular autofluorescent species (something that shouldn't vary in my experimental conditions of course). Something essentially akin to the loading control used in Western blotting. I'd measure both my probe of interest and the 'loading control' under the same microscope settings (just varying the excitation and emission wavelengths as necessary). Is this at all possible? Or does having to change excitation and emission wavelengths make this impossible.
Alternatively, could I normalise measurements of the fluorophore of interest from my region of interest (I currently take measurements in my time-course experiments by selecting the nephrons in kidney tissue) to the background fluorescence of the same fluorophore? And if so what counts as the background? Interstitial tissue? The perfusate fluid that surrounds the tissue on the stage?
Any other suggestions? Some kind of physical standard like a fluorescence ruler, or fluorescent beads?
I do not want to manipulate the data with ImageJ or some other software as I think this will somewhat invalidate it.
Many thanks for your time!
Hi Sean,
Several methods may be used to address your issue with more or less accuracy associated with (depending on your requirements). I suggest that you check out these references:
- https://www.google.fr/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&cad=rja&uact=8&ved=0ahUKEwi2vZShrsrUAhUHmBoKHQymAI4QFggmMAA&url=http%3A%2F%2Fwww.springer.com%2Fcda%2Fcontent%2Fdocument%2Fcda_downloaddocument%2F9783540705703-c1.pdf%3FSGWID%3D0-0-45-600701-p173830211&usg=AFQjCNHOCqX30tt60Kck0mFH-qOYxRUgQg
- http://www.imaging-git.com/applications/quality-control-fluorescence-imaging-systems
- https://pdfs.semanticscholar.org/7c3a/f6561c875d645e9f2de833ca6aa3a965dff7.pdf
- Model et al., "A standard for calibration and shading correction of a fluorescence microscope", 2001 - http://booksc.org/dl/168084/0576b8
- https://pdfs.semanticscholar.org/9bb2/2abcb390b96eb2a46364d284f8ec553c3fe5.pdf
Moreover, an overview of selected image processing and analysis methods for fluorescence and electron microscopy is available by following http://bigwww.epfl.ch/publications/kervrann1601.pdf
Best Regards
I doubt you could have a source of autofluorescence that you could just assume to be stable and unchanging. If you have not yet done the experiments, I would suggest using fluorescent beads with wavelengths that are close but not overlapping.
- Badges
- Science topic
- Similar topics
- Biological Science
- Immunology
- Antibodies