Use the following conventions and guidelines to prepare and acquire FRET data.
*Direct FRET is calculated using the following equations:
Net FRET = (FRET signal) - (FRET due to Donor crosstalk) - (FRET due to Acceptor crosstalk)
FRET Efficiency (%E) = (Net FRET) / (Donor Signal)
Direct FRET involves acquisition and analysis of three types of images:
The Donor Control image is used to quantify the effect of donor crosstalk.
The Acceptor Control image is used to quantify the effect of acceptor crosstalk.
The FRET Experiment image is used to measure FRET.
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Note The Calculate FRET analysis also generates a FRET Results image that is used to quantify Net FRET and FRET Efficiency. |
For each image in the FRET experiment suite, use the following conventions to insure the best analysis path.
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Use this channel |
For |
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1 |
Donor (Donor Excitation, Donor Emission) |
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2 |
Acceptor (Acceptor Excitation, Acceptor Emission) |
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3 |
FRET (Donor Excitation, Acceptor Emission) |
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Note The preferred order of the Donor and Acceptor can be reversed by hand- modifying the configuration file .softworx_hostname/user.swrc to reverse the order for a particular user or by modifying the file /usr/local/softWoRx/config/system.swrc (as the "root" user) to reverse the order for all users. Add the following variable definition to reverse the default Donor/Acceptor order for analysis: SW_FRET_ACCEPTOR_FIRST True |
Prepare the following types of slides:
Acceptor Control: Prepare the slide with the Acceptor probe only.
Donor Control: Prepare the slide with the Donor probe only.
FRET Experiment: Prepare the slide with both Donor and Acceptor probes.
Use the following guidelines to acquire images:
To retain numerical consistency during FRET experimentation, it is critical that exposure times and neutral density filter settings remain the same for all channels.
For a given set of acquisition conditions (atmospheric pressure, temperature, humidity, etc.) you can collect one set of Control images to characterize crosstalk for multiple FRET Experiment images.
Collect Control and Experiment images using standard softWoRx Resolve3D methods, adhering to the channel assignment conventions defined below.
After you acquire images, you will need to process them to prepare them for analysis as follows:
Chromatic Correction: If images have been collected as Z stacks and the images exhibit significant Z shifts between channels, they may be adjusted using the Chromatic Correction tool accessed from the Measure menu of the softWoRx main menu bar. If a channel shift is necessary, it must be applied to all images involved in the experiment.
Intensity Correction: If images are not being deconvolved, use the Correct tool to ensure that the images are properly adjusted according to the photosensor measurements made during acquisition.