Controls for FRET efficiency measurements

In my experiment, I want to evaluate FRET efficiency using emission measurements. I have cell lines expressing FRET-pair, donor-only, and acceptor-only. The problem is that the expression level of the donor is much higher than that for FRET-pair and acceptor, hence I can’t use exactly the same exposure times as it will either saturate the signal of donor-only or FRET sample will be underexposed.
I’m wondering whether it is okay to use different exposure times for measurements of donor cross-talk and bleed-through? Should I compensate for the exposure time difference further in the calculations?

A co-worker of mine Gert-Jan Kremers wrote a nice article about it.

I think if you have different expression levels etc acceptor photobleaching would be the method of choice. It is a more direct measurement of the change in FRET (before and after bleaching) in any case FRET is a technique that is at its most useful if you can compare two situations.

I will mention this post to my co-worker he might react…

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Thank you for the reference. I’ve read that paper. It nicely covers the basics of the different FRET measurement approaches, however, I don’t think it really answers my question.

In my case, I stick to sensitize emission measurements. Acceptor photobleaching is not an option, as I need to measure a time-series, not a single time point.

Maybe I need to clarify my question - I will use a FRET biosensor that has both donor and acceptor, i.e. stoichiometry will be 1:1. However, controls necessary for correction of cross-talks, have different expression levels. Hence, my problem is not in the measurement of FRET itself, but in how to correctly compensate my measurements for cross-talks.

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Ah yes that makes the question different. I havent done FRET myself so I’m not the right person to anwser these questions, hope somebody else can help you.

Hi Oleg,

The cross-talk and bleedthrough values are the ratio of two channels and they should be intensity independent. You may adjust the excitation intensity to get good quality images for the different samples.
I think it is best to use high expressors to calculate the crosstalk factors as you minimize the effects of autofluorescence and other background contributions.