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Correcting drift in FRAP experiments

7 bytes removed, 14:54, 31 July 2014
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Load the 4 different time series in Fiji.
The next step will be to unite the 4 movies in a single one. For this I use the '''Concatenator plugin''' for ImageJ which can be found here (http://rsbwebimagej.nih.gov/ijnet/plugins/concatenator.html). Once you have done this, you should carefully look at your specimen. If it has moved during image acquisition, so will have your bleaching ROI. You can minimize this by using the [[Linear Stack Alignment with SIFT]] plugin designed by Stephan Saalfeld which basically compares a frame with its previous one and tries to compensate eventual movement by rigidly moving the whole image so that they better overlap. This way you are not playing around with the fluorescence intensity values and you do not have to manually adjusting your ROI frame by frame. Of course, this plugin does not always work - if your sample moved a lot or if the signal does not allow for efficient comparison, the resulting movie will be pointless.
Once you have an aligned movie you can draw the bleaching ROI and run '''Plot Z-axis profile''' (under ''Images > Stacks''). This will not only show you a graph with the mean intensity values over time but will also open a measurement window with raw data. Copy these values to an excel spreadsheet. You still have to normalize these values - the acquisition of images also bleaches somehow the sample and this effect has to be minimized. For that, all you need to do is draw a non-bleached ROI and take out all its values using the above mentioned method.
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