FRET ratio

[Knecht, David]

I have a colleague that would like to measure an average FRET ratio (FRET/CFP image) of all the pixels of cells in a series of images.  Is there a FRET plugin that will allow you to exclude pixels below background (outside of cells) from the calculation.  Slidebook lets you just choose a background value and does this automatically.  I have considered converting the background to zero in the FRET image (numerator), dividing and then thresholding the zero values out before measuring, but wonder if there is an easier way.  Thanks- Dave

David Knecht, Ph.D.
Professor and Head of Microscopy Facility
Department of Molecular and Cell Biology
U-3125
91 N. Eagleville Rd.
University of Connecticut
Storrs, CT 06269
860-486-2200
860-486-4331 (fax)

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ImageJ mailing list: http://imagej.nih.gov/ij/list.html

[Robert Baer]

David,

Normally, I would refrain from talking about something for which I know
so little, but about a year ago, i wrote some notes for myself (and
student) about what FRET tools were available using ImageJ and thought
they might or might not be of interest to your colleague since I can't
quite tell exactly how he is using FRET from your post.  These notes
mostly try to synthesize "best practices" from the various ImageJ plugin
authors, but deal with background corrections (at least indirectly) for
certain types of issues.

My own expertise is negligible, but the references and outline of the
problems in my notes are,  I think, worth looking at.  If interested,
contact me off-line

Rob Baer


On 4/8/2013 9:01 AM, Knecht, David wrote:

> I have a colleague that would like to measure an average FRET ratio (FRET/CFP image) of all the pixels of cells in a series of images.  Is there a FRET plugin that will allow you to exclude pixels below background (outside of cells) from the calculation.  Slidebook lets you just choose a background value and does this automatically.  I have considered converting the background to zero in the FRET image (numerator), dividing and then thresholding the zero values out before measuring, but wonder if there is an easier way.  Thanks- Dave
>
> David Knecht, Ph.D.
> Professor and Head of Microscopy Facility
> Department of Molecular and Cell Biology
> U-3125
> 91 N. Eagleville Rd.
> University of Connecticut
> Storrs, CT 06269
> 860-486-2200
> 860-486-4331 (fax)
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html

--

Robert W. Baer, Ph.D.
rbaer at atsu dot edu
Professor of Physiology
Kirksille College of Osteopathic Medicine
A. T. Still University of Health Sciences
Kirksville, MO 63501 USA

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ImageJ mailing list: http://imagej.nih.gov/ij/list.html

[complex]

HI David:

Normalized FRET can be presented as
1. FRET/CFP image
2. FRET/(CFP*YFP)
3. FRET/Sqrt(CFP*YFP)

The problem here is that lots of the control CFP and YFP images was low background (which they should be) and when you do image arithmetic you will get high pixel intensity due to a divide by zero pixel effect.

I have been advised to set background >200 intensity for example to zero to remove most of the 65535 intensities off. I have not been able to figure it out functionally imageJ as of the moment so if you can supply me with an answer it will be great.

I am using the FRET plugin from Ammasi Periasamy at the WM Keck Center for Cellular Imaging University of Virginia. Their FRET workshops are highly recommended as well.

-Nguyen

Nguyen Phan, Ph.D.
University of North Carolina at Chapel Hill
316 Coker Hall
Chapel Hill, NC