FRET in FIJI

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There are two methods that can be easily implemented in FIJI and on our microscope to calculate FRET ratios in fixed or live cell images. The methods, and FIJI scripts to automate their calculation, are provided below. For both methods you need to collect images in a specified order:

  1. Idd (intensity of the donor when the donor is excited)
  2. Ida (intensity of the acceptor when then donor is excited)
  3. Iaa (intensity of the acceptor when the acceptor in excited)

For the robust method, you will also need these images acquired on samples where only the donor or acceptor label are present. These are used to calculate channel cross-talk and to address photobleaching.

Fluorophore Pairs

It is critical that you use appropriate fluorophore pairs that are also compatible with the microscopes excitation and emission lines. If using the MNI Widefield Microscope you need to use fluorophore that match the available emission and excitation wavelengths. Emission and excitation wavelengths are not linked on this system, allowing large stoke-shift dyes to be used. Available lines are from the Chroma Sedat Quad set:

Excitation Wheel 1 Excitation Wheel 2 Emission Wheel
AT350/50 430 470/24
ET490/20 500/40 535/50
ET555/25 490 605/52
ET645/30 705/72

A number of fluorophore FRET pairs as possible, but pairs with higher R0 × QYA will provide superior signal. These values can be calculated at FPbase. Proven pairs are listed below in regular text; untested but likely useful pairs are in italics.

Donor Acceptor R0 × QYA
FITC or Alexa 488 ORB 31
Cy3 ATTO 647N 30
eCFP eYFP 32
eCFP mVenus 33
ATTO490LS ATTO647N 38

Method 1 - Uncorrected Donor Quenching

This is based off of Joachim Goedhart's approach, and works best for fixed images, or for live-cell acquisitions where there is minimal photobleaching. In this approach, the loss of donor excitation (quenching) due to excitation energy being transferred to the acceptor, is measured. Idd, and Ida must be collected, and must be the first and second channel of the image respectively. Collection Iaa and DIC images is optional but recommended.

  1. Acquire Idd, Ida and Iaa in samples labelled with both the donor and acceptor fluorophore. Note that the same exposure setting need to be kept for Idd and Ida, and that Idd and Iaa shuld be configured to give bright donor and acceptor signals without saturation.
  2. Open the resulting images. Image should be in the format of a multi-channel stack.
  3. Selection a region of interest (ROI) that lacks signal in the Idd and Ida channel. This will be used for background subtraction. For live-cell images, make sure that no cells migrate into this region during the time lapse.
  4. Run the UDC-FRET macro (available below). This macro will:
    1. Separate the Idd and Ida channels
    2. Background subtract each channel, using the mean background found in the ROI from above.
    3. Calculate the ratio of Ida/Idd.
    4. Mask the resulting image using the Idd channel. Note: the macro could easily be modified to mask using Iaa, if this region is of more interest.
    5. Generate output figures.

Note: This macro is intended to be run on time series, and may need modification to work on single still images. Before the first run, you must install the Temporal Colour Code script into FIJI:

  1. Download macro from https://forum.image.sc/uploads/short-url/9o65DNmyl4G1TYLE5fSYMTyyPn8.ijm
  2. Copy macro into <fijifolder>/plugins/Scripts/Image/Hyperstacks
  3. Restart FIJI

Method 2 - Corrected Sensitized Emission

This is a superior method that can account for bled-through of the fluorophores across filters, and photobleaching. It requires that Idd, Ida, and Iaa be capture, and it also requires that the Idd, Ida and Iaa be captured of cells singly-labelled with the donor and acceptor. These later samples are used to address bleed-through. In theory, you can re-use these values for any experiment using the same fluorophores, but the most robust data will be generated when these controls are run in every experiment. This approach is based on the work of van Reehan et al[1].

Calculating Correction Factors

These values need to be calculated for every fluorophore-pair, but ideally should be determined for each independent experiment.

Donor-Only Image

  1. Label your cell with the donor fluorophore only, at the same density and under the same conditions as your planned samples.
  2. Capture Idd and Ida (Iaa is optional, and should lack any signal). Idd and Ida must be the first and second channel, respectively.
  3. Select a region of interest lacking any signal in either channel.
  4. Run CSE-Dcorr macro, which will:
    1. Background subtract Idd and Ida.
    2. Auto-generate an ROI using auto-thresholding.
    3. Calculate β (channel cross-talk; defined as Ida/Idd).

Note: The script only runs on single images.

Acceptor-Only Image

  1. Label your cell with the acceptor fluorophore only, at the same density and under the same conditions as your planned samples.
  2. Capture Idd, Ida and Iaa. These channels must be collected in this order, and be the first three channels in the image
  3. Select a region of interest lacking any signal in all three channels.
  4. Run CSE-Acorr macro, which will:
    1. Background subtract all three channels.
    2. Auto-generate an ROI using auto-thresholding.
    3. Calculate α (donor cross-excitation; defined as Idd/Iaa).
    4. Calculate γ (acceptor cross-excitation; defined as Ida/Iaa).
    5. Calculate δ (FRET cross-talk; defined as Idd/Ida).

Note: The script only runs on single images.

Acquisition and Calculating FRET

  1. Acquire Idd, Ida and Iaa in samples labelled with both the donor and acceptor fluorophore. Note that the same exposure setting need to be kept for Idd and Ida, and that Idd and Iaa should be configured to give bright donor and acceptor signals without saturation.
  2. Open the resulting images. Image should be in the format of a multi-channel stack.
  3. Selection a region of interest (ROI) that lacks signal in the Idd, Ida, and Iaa channels. This will be used for background subtraction. For live-cell images, make sure that no cells migrate into this region during the time lapse.
  4. Run the CSE-FRET macro (available below). This macro will:
    1. Background subtract the Idd, Ida, and Iaa channels.
    2. Request the values of α, β, γ, and δ.
    3. Calculate the FRET efficiency as:
      FRET equasion.png
  5. Mask the resulting image using the Idd channel. Note: the macro could easily be modified to mask using Iaa, if this region is of more interest.
  6. Generate output figures.


Note: This macro is intended to be run on time series, and may need modification to work on single still images. Before the first run, you must install the Temporal Colour Code script into FIJI:

  1. Download macro from https://forum.image.sc/uploads/short-url/9o65DNmyl4G1TYLE5fSYMTyyPn8.ijm
  2. Copy macro into <fijifolder>/plugins/Scripts/Image/Hyperstacks
  3. Restart FIJI

Scripts

While we have done everything we can to ensure that these scripts work as described, we cannot guarantee their accuracy or whether they will work with your samples or microscope setup. As such, these scripts are offered without warranty or guarantee that the results produced by these scripts will be accurate or scientifically sound.

UDC-FRET

Download Link, Version 1.0

CSE-Dcorr

// Script not yet available

CSE-Dcorr

// Script not yet available

CSE-FRET

// Script not yet available

Citations

  1. van Rheenen J, Langeslag M, Jalink K. Correcting confocal acquisition to optimize imaging of fluorescence resonance energy transfer by sensitized emission. Biophys J. 2004 Apr;86(4):2517-29. https://www.sciencedirect.com/science/article/pii/S0006349504743076