Re: Frap analysis

Posted by Kevin Braeckmans on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Frap-analysis-tp591758p591765.html

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Dear Anchal,

It would be helpful if you could give some more details - what are you
trying to measure, what kind of sample are you using, do you want to obtain
quantitative data and if so, which FRAP method would you like to use etc.

Very generally, the difficulty is not the image processing, which usually is
just averaging the fluorescence intensity in some ROI and normalizing that
to a reference area and the fluorescence before bleaching. If you want to
obtain quantitative data, the real challenge is to perform the experiment
according to the limits implied in the FRAP model (which are not always
clearly discussed in articles). Only then one can expect to obtain
reasonable values from a best fit of the model to the recovery curve
(obtaining a value does not necessarily mean it is a correct one).

If you are starting from scratch, I would advise the following:
1. Define your experiment, the sample geometry being the most important
factor (3D extended sample, sample with limited thickness, sample of limited
volume)
2. Choose a FRAP method which is suitable for your experiment and which can
be performed by your instrument.
3. Practice on some reference solutions of known diffusion coefficient to
see if the method works correctly (each FRAP method has some limits which
are implied in the mathematical derivation - make sure to perform your
experiment accordingly)
4. Apply the method to your actual samples.

You also might want to check out the archives since this topic has been
discussed several times on this list.

Best regards,

Kevin


Kevin Braeckmans, Ph.D.
Lab. General Biochemistry and Physical Pharmacy
Ghent University
Harelbekestraat 72
9000 Ghent
Belgium
Tel: [phone]
Fax: [phone]

> -----Oorspronkelijk bericht-----
> Van: Confocal Microscopy List [mailto:[hidden email]]
> Namens Anchal Chandra
> Verzonden: donderdag 24 januari 2008 18:31
> Aan: [hidden email]
> Onderwerp: Frap analysis
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Hello all,
>        I am new FRAP user.CAN ANYONE HELP ME WITH ANALYSIS FRAP DATA
> USING
> IMAGE J AND IGOR PRO.aLSO HOW CAN i quantify my data.It will be nice if
> someone can tell me step by step procedure to analyse frap data .I use
> leica
> sp5 for collecting frap data.
> Hope to hear from you all soon
> Thanks
> Anchal