I’ve been using FRAP tests on a confocal microscope to test local diffusion rates and analyzing the image stacks in FIJI. For now, I’ve just been pulling ROI intensity data from FIJI to analyze in excel (normalizing bleach region against background/reference) and using FIJI’s curve fitting feature for exponential recovery to generate a graph, but I know there are at least a couple plug ins out there which might make this process easier. Could anyone recommend a plug in that would be beneficial for generating diffusion-rate related data from FRAP image stacks?
Specifically, what do you expect the plug-in to do?
We usually recommend FRAP analyser.
It’s not a ImageJ plugin, but a standalone solution.
It offers different options for fitting and normalisation of your measurememts.
I’m hoping to look at diffusion rates or fluorescence recovery time. I’m comparing chocolate model systems with different treatments to look at tightness of crystal structures and oil migration within the matrix, which I hope to use FRAP to quantify locally. I’ve tried a couple different plugins but they all seem to yield different results or be specifically meant for use with cells. So really anything you could recommend that would give me some information on recovery time/diffusion rates from FRAP would be helpful. My files are all currently czi if that matters.
I can not imagine your data because I do not know the “chocolate model system”…
However, it probably doesn’t seem to make a big difference in the measurement method, but what is the part of the tools for cells that feel inconvenient?
You may get hints from various people when you say what you want to do.
It can select flexible ROI or line, record the positions of ROI, auto tracking or registration, calculate the background, and so on.
*In my case, I used my own plug-in that I created for another analysis.It can measure the average intensity of clicked position of Rectangle or Circle.And the value are recorded in the table which can save as tsv file. And it measure all of channels when the click by same ROI.In other case, I used another plugin which can measure the intensity on the line ROI.It can also record the ROIs position and save them. The line ROI can set the width, so it can also measure the Area. However, both plugins can not display the graph of recovery curve and so on. So, Recovery curve was calculated and drawn by R or Excel.
The plugins for cells seem to work, but each one yields different results. Most of them don’t have guides (at least that I could find) so I’m not sure if I’m using them correctly or in some cases, what the data means. (I’ve tried FRAP profiler and simFRAP)
I have a circular ROI for bleaching area and can measure average intensity over the experiment. I guess one issue is there’s no defined “cell”, since the whole frame is part of the sample. For the plugins requiring defined cell area, I’ve just been drawing a larger ROI around the bleaching ROI. This has been giving me results, although since I also don’t have a clear “background”, things might be skewed. I suppose I could run another experiment at the edge of the sample where I could label the blank slide as background.
Thank you for the suggestion! I’ve been trying to chase down a computer with the right operating system to run this software since it’s incompatible with my laptop.
I tried using simFRAP, but I thought it was a fairly usable plug-in.(There was also a Doc file)
The plug-in has flexible roi settings and seems to be able to display graphs and save its data.
In calculating recovery speed and other value, it would be better if we could know how and where the intensity was used.
Whatever the calculation, it will analyze the relative differences compared to the control.
I’m sorry I couldn’t provide much useful information about the FRAP plugin.