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:

  1. Emission crosstalk, eg donor fluorescence into acceptor.
  2. Concentration of donor
  3. Concentration of acceptor
  4. 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

  1. Acceptor Only
  2. Donor Only
  3. FRET Sample

Collect 5 images

  1. Acceptor Only using Acceptor filter set
  2. Acceptor Only using FRET filter set
  3. Donor Only using Donor filter set
  4. Donor Only using FRET filter set
  5. FRET Specimen Only using FRET filter set

Correct for background before proceeding any further

Calculating Co-efficient AAcceptor 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
Sensitised Emission FRET
Sensitised Emission FRET
Calculating Co-efficient BDonor 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
Sensitised Emission FRET
Sensitised Emission FRET
Sensitised Emission FRET
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

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