Calculate_ratiometric_FRET_Images_with_the_FRET-Image_Script_Step_by_step_v1.3

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Tutorial

Calculate Ratiometric FRET-Images with the FRET-Image-Script

Summary

This tutorial shows step-by-step, how the “FRET Image” script of SymPhoTime 64 can be used to calculate pixel-by-pixel the average FRET efficiency in an image containing several cells transfected with Cerulean/YFP constructs. To calculate the average FRET efficiency, a ratiometric approach based on the intensities in the donor and the acceptor channel is used.

Step-by-Step Tutorial

1. Start SymPhoTime 64 software.
2. Open the “Samples” workspace via “File\open Workspace” from the main menu.

1. Response: The files of the sample workspace are displayed in the workspace panel on the left side of the main window.

• Highlight the file “CENP-labelled _cells_for_FRET.ptu” by a single mouse click.

1. Select the “Analysis” tab and in there, open the drop down menu “Imaging”.

1. Start the “FRET Image” script by clicking on “Start”.

• Response: The FRET image script is applied to the file “CENP-labelled _cells_for_FRET.ptu”. Thereby, a new Window opens:

• Set the pixel “Binning” to 2 points.
• As data channels, check only Channel 1 and 2, as in these channels the relevant photons are recorded.

• Press “Calculate”.
• Response:
  • Automatically, the two active detector channels are assigned to the donor and acceptor channel. By default, detector channel 2 is considered the donor channel and detector channel 1 is assigned as acceptor channel.

• A false color image is calculated, in which the intensity of detector 1 and 2 are plotted in green and red, respectively.

• Invert the color assignment to display the acceptor channel in red and the donor channel in green. Therefore, click on the bar next to the image to open the display options and invert the channel selection.



• Response:The image is adapted, the acceptor is now plotted in red.

1. Set the Min. Photons per FRET event to “700” Cnts (counts).
2. Click on “more” to open the additional options for entering the correction parameters.



• As parameters, enter “70” for “Gamma” and “7” for “Cross Talk”.

• Click “Calculate FRET”.

• Response:
  • The FRET image is calculated, and the corresponding histograms are calculated. In the FRET image the FRET efficiency is only calculated for pixels with intensities higher than the threshold.
  • The FRET efficiency color scale is automatically set from 0 to 100% FRET efficiency.
  • The FRET efficiency histogram and the Distances histogram are shown.

• It can be seen that a few fluorescent dots on the lower right are over the set intensity threshold. As these signals are not in an area of interest, place the mouse cursor over the FRET image, activate the context menu with a right mouse click and select the “Rectangle ROI” tool.

1. Now it is possible to remove the unwanted area by constantly pressing the “CRTL” button and the left mouse button, draw a rectangle over these areas and release mouse and keyboard. Do this repetitively until all unwanted signals are removed.

1. Press “Calculate FRET” again to update the histograms.
2. Response:The FRET efficiency and distance histograms are updated. This can be seen clearly at the small peak around 20% FRET efficiency which disappears now.

1. Store the result by clicking on “Save Result.

1. Response: A result file is generated and linked to the raw data file.

1. The image analysis is now finished. There are several possibilities to continue:
   1. To adapt the histogram plots, change the plot parameters on the right of the histogram to the desired values and click “Recalculate”. To save these plots, click “Save Results” again.
   2. To export certain analyses, place the mouse cursor over the area of interest, activate the context menu with a right mouse click and choose the export function of interest to export the data.
   3. In case one wants to analyze many images with the same FRET pair, it is possible to save the starting values for the fits by clicking on “Save Defaults” and store the starting values as a User Profile by entering the main menu (“Settings\Save User Profile as”). The software will need to restart to adapt the changes.
2. If you don't want to do anything more, just close the result window.