Sensitised FRET protocol. FRET results in a decrease in donor emission whilst there is a simultaneous increase in acceptor emission, which is known as sensitised FRET. Unlike acceptor photobleach, there are 4 corrections which need to be made: Emission crosstalk, eg donor fluorescence into acceptor. Concentration of donor Concentration of acceptor Presence of FRET and non-FRET components. Acceptor photobleach MicroFRET Acceptor in FRET channel (Co-efficient A) = Mean intensity of Acceptor using FRET set Mean intensity of Acceptor using acceptor set Donor in FRET channel (Co-efficient B) = Mean intensity of Donor only using FRET filter set Mean intensity of Donor only using Donor filter set Place correction factors into FRET Efficiency equation: FRET Specimen - (A * FRET Specimen using Acceptor filter set) - (B * FRET Specimen using Donor filter set) Protocol Use 3 samples Acceptor Only Donor Only FRET Sample Collect 5 images Acceptor Only using Acceptor filter set Acceptor Only using FRET filter set Donor Only using Donor filter set Donor Only using FRET filter set FRET Specimen Only using FRET filter set Correct for background before proceeding any further Calculating Co-efficient A Acceptor Only Using FRET filter set (Threshold in Red) Acceptor Only Using Acceptor Only Filter set (Threshold in Red ) Threshold areas of interest and then calculate the average intensity (grey values) A = 6.74 56.16 A = 0.12 Image Image Calculating Co-efficient B Donor Only using FRET filter set (Threshold in Red) Donor Only using Donor filter set (Threshold in Red) Threshold areas of interest and then calculate the average intensity (grey values) B = 8.13 51.42 B = 0.15 Image Image Image The values for A and B can now be used in the FRET equation Average FRET The average FRET % was calculated using average thresholded grey values from the above images to give the equation below Average FRET % = 99.92 - (0.12 * 56.16) - (0.15 * 51.42) = 85.48 Images were 8 bit so had a maximum grey value of 255, so average FRET % = (85.48/255) * 100 NOTES: Values of between 0.1 - 0.2 for co-efficient A give good results Values of between 0.1 - 0.4 for co-efficient B give good results It is a good idea to check your FRET results using the Acceptor photobleach method at the end of the experiment, they should be in reasonably close agreement For FRET, it's best to use 12 bit images Acceptor photobleach Useful References Gordon et al, 1998 Biophysical Journal; 74: 2702-2713 Erickson et al, Neuron 2001; 31: 973-985 This article was published on 2024-06-17
