FRET and Microscopy

[moleculardude]

Hey guys,

has anyone tried doing FRET combined with microscopy?

thanks in advance

Moleculardude

[PeterScherp]

Hi Dude,

sure, what do you need to know???
Some basics: You need a fluorescence microscope (ideally one for Live cell imaging) with the appropriate filter set (for CFP and YFP FRET). Then you need to tag your protein of interest with CFP and YFP. The best way to do FRET is that you have two controls, one is your cells with CFP only and the second with YFP only. CFP only and YFP only are used to calculate the bleed-through of your fluorescent channels. This is more or less a one-time thing and is then saved on you computer (at least Olympus is doing it that way). Then you are ready to do your FRET experiments. Typically you will do a timelapse experiment where you have you cells happily sitting in their medium. Then you capture images for a few minutes. Then you might want to add a chemical that changes the protein structure due to whatever intracellular interaction you expect. Then, your cells hopefully fret and you capture images for the remainder of the experiment.
The actual data analysis is done with the software (FRET module) that is hopefully included in you microscope software.
Hope that helped a little. Let me know when you have further questions!!

Peter