Sensitised Emission
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.
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 ) | |||||
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Threshold areas of interest and then calculate the average intensity (grey values)
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| Calculating Co-efficient B | Donor Only using FRET filter set (Threshold in Red) | Donor Only using Donor filter set (Threshold in Red) | |||||
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Threshold areas of interest and then calculate the average intensity (grey values)
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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 theAcceptor 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
Useful References
Gordon et al, 1998 Biophysical Journal; 74: 2702-2713
Erickson et al, Neuron 2001; 31: 973-985