# Controls for FRET efficiency measurements **URL:** \ **Category:** Theory\ **Created:** [March 1, 2021, 3:05am UTC](https://forum.microlist.org/t/controls-for-fret-efficiency-measurements/1306 "2021-03-01T03:05:04Z")\ **Posts on this page:** 5\ **Page:** 1
**Author:** ![Oleg](https://avatars.discourse-cdn.com/v4/letter/o/ebca7d/32.png) [@Oleg](https://forum.microlist.org/u/Oleg)\ **Post date:** [March 1, 2021, 3:05am UTC](https://forum.microlist.org/t/controls-for-fret-efficiency-measurements/1306/1 "2021-03-01T03:05:04Z")
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? ---
**Author:** ![JASlotman](https://sea2.discourse-cdn.com/flex020/user_avatar/forum.microlist.org/jaslotman/32/331_2.png) [@JASlotman](https://forum.microlist.org/u/JASlotman)\ **Post date:** [March 1, 2021, 7:51am UTC](https://forum.microlist.org/t/controls-for-fret-efficiency-measurements/1306/2 "2021-03-01T07:51:20Z")
A co-worker of mine Gert-Jan Kremers wrote a nice article about it. > **[Fluorescent protein FRET: the good, the bad and the ugly](https://www.sciencedirect.com/science/article/abs/pii/S0968000407001910?via=ihub)** > > Dynamic protein interactions play a significant part in many cellular processes. A technique that shows considerable promise in elucidating such inter… 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… ---
**Author:** ![Oleg](https://avatars.discourse-cdn.com/v4/letter/o/ebca7d/32.png) [@Oleg](https://forum.microlist.org/u/Oleg)\ **Post date:** [March 3, 2021, 7:13am UTC](https://forum.microlist.org/t/controls-for-fret-efficiency-measurements/1306/3 "2021-03-03T07:13:00Z")
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. ---
**Author:** ![JASlotman](https://sea2.discourse-cdn.com/flex020/user_avatar/forum.microlist.org/jaslotman/32/331_2.png) [@JASlotman](https://forum.microlist.org/u/JASlotman)\ **Post date:** [March 3, 2021, 9:06am UTC](https://forum.microlist.org/t/controls-for-fret-efficiency-measurements/1306/4 "2021-03-03T09:06:55Z")
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. ---
**Author:** ![joachimgoedhart](https://sea2.discourse-cdn.com/flex020/user_avatar/forum.microlist.org/joachimgoedhart/32/26_2.png) [@joachimgoedhart](https://forum.microlist.org/u/joachimgoedhart)\ **Post date:** [March 5, 2021, 6:47pm UTC](https://forum.microlist.org/t/controls-for-fret-efficiency-measurements/1306/5 "2021-03-05T18:47:28Z")
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. Cheers, Joachim.