I want to determine the colocalization of a transmembrane protein along with a membrane marker, could it be possible to obtain the result with fluorescence microscopy alone or it is must to have a confocal microscopy analysis?
Hi Manisha Shahu
Yes, it can be done. Acquire images at 40x or higher if needed and then measure the colocalization rate using ImageJ.
Thanks
Hi there Manisha Shahu ,
Caution should be taken when performing colocalization on fluorescence microscopes.
If you are working with a cell monolayer, then the results shouldn't be very different. However, if you work with tissue sections (unless they are extremely thin), you can easily run into false positives.
In a confocal microscope you will be only collecting light from a single focal plane, while in a fluorescence microscope you collect out-of-focus photons.
This means that the pixel values that you get in your final images will be somehow "contaminated" by fluorescence that is not in the same plane that your object or region of interest in which you measure colocalization.
So, if you have one available, is way better to perform this kind of experiments in a confocal microscope.
Hope this helps,
Cheers,
J
To add, please check this paper
https://www.nature.com/articles/s41598-020-75835-7
In figure 2 and Figure 11 is well illustrated how objects in different position in a z stack could yield colocalization artifacts when the Max proj is rendered. You could imagine a similar scenario for Fluorescence microscopy.
Thank you all for the valuable input.
J. Ramirez-Franco I am using HEK cell line so its monolayer.
The issue is the focal depth. Even if two points seem in focus, the focal depth of a widefield microscope can be quite broad, and the lower the magnification of the objective, the wider the focal depth. It can be microns in distance. Confocal is a much narrower focal depth, and determined by the pinhole size and objective magnification, but can still be broad enough that true colocalization is questionable. The best option is to use a FRET pair between the two proteins of interest, with corresponding FRET filter pairs, and if FRET occurs you know it is within Angstroms of each other (potentially 50-100 A), rather than microns.
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