Centre Optical Instrumentation Laboratory

Sensitised Emission FRET

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

MicroFRET

Acceptor in FRET channel (Co-efficient A) =
Average intensity of Acceptor only using FRET set
Average intensity of Acceptor only using acceptor set

Donor in FRET channel (Co-efficient B) =
Average intensity of Donor only using FRET filter set
Average 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 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

Acceptor with FRET Settings Acceptor With Acceptor Settings

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
Donor With FRET Settings Donor With Donor Settings

The values for A and B can now be used in the FRET equation

FRET Image Calculations

Pseudocolour FRET image

FRET Efficiency =

FRET Image

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, its best to use 12 bit images