I`m doing FRET Acceptor Photobleaching and I`m starting to have doubts on the method we use to calculate Bleach efficiency in a defined ROI (not on the entire image).
I acquire images for donor and acceptor a pre and post-bleach and we use bleach efficiency to correct the final fret ratio we get in order to extrapolate FRET% in an ideal 100% bleaching efficiency.
If you have a suggested plug-in for imagej or fiji I can compare data from different methods.
cheers
Andrea
Hello Andrea,
I used a stepped bleaching method. That helped with extrapolating the 100% value. Briefly, I acquired two-channel images before bleaching and after each of 6 small bleaching. I used the hand tracing tool in Image J to draw a region around the cell, excluding the nucleus. I used the ROI and measurement functions to get the mean fluorescence intensity before bleaching and after each small bleaching step. The Measurement Table can be opened as a CSV file and then saved as an Excel file. Excel spreadsheet. After this, I used Excel to perform the necessary calculations to extrapolate the estimated 100% value. I have attached my paper for your convenience.
I hope that was helpful. If there is other information that would be more helpful, feel free to ask.
Best regards,
Teresa (Terri) Murray, PhD
Director, Integrated Neuroscience and Neuroimaging Lab
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