Analysis of image from Fret (CFP-YFP) based calcium imaging

[aget]

Hi,
Could anyone recommemd a plugin for analysis of timelaps images from FRET based calcium indicator imaging?
I acquired images from neurons that will fire, triggering FRET, so that signal from channel one undergo reduction in fluorescence, signal from channel two is enhanced. How could i process the temporal images in order to know which neuron fires (out of hundred of them)? Thanks

Yajie

[Jacob Keller]

I would make a stack for each channel, then use
Process-->ImageCalculator-->divide one stack/channel by the other. Then
you'll have images of the fluorescence ratios, and the nerves should light
up / change when they fire.

Jacob Keller

On Fri, Sep 21, 2012 at 8:27 AM, aget <[hidden email]> wrote:

> Hi,
> Could anyone recommemd a plugin for analysis of timelaps images from FRET
> based calcium indicator imaging?
> I acquired images from neurons that will fire, triggering FRET, so that
> signal from channel one undergo reduction in fluorescence, signal from
> channel two is enhanced. How could i process the temporal images in order
> to
> know which neuron fires (out of hundred of them)? Thanks
>
> Yajie
>
>
>
> --
> View this message in context:
> http://imagej.1557.n6.nabble.com/Analysis-of-image-from-Fret-CFP-YFP-based-calcium-imaging-tp5000156.html
> Sent from the ImageJ mailing list archive at Nabble.com.
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html
>

--
*******************************************
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: [hidden email]
*******************************************

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

[yuansheng sun]

Dear Yajie,

If it is a type of biosensor, meaning that the FRET donor and acceptor are
at the 1:1 fixed stoichiometry ratio,  you can use the ratiometric FRET
method and calculate the ratios between the two channels using ImageJ
builtin function - "ImageCalculator".  If the donor and the acceptor are
expressed independently,  you will need to use an algorithm-based software
to correct for variant spectral bleedthroughs to quantify FRET signals and
efficiencies.  We developed ImageJ plugins software (known as PFRET) for
various intensity-based FRET methods, including the ratiometric FRET,
acceptor photobleaching FRET, and all kinds of bleedthrough corrections for
FRET quantification based on the acceptor sensitized emission.

Please contact Dr. Ammasi Periasamy ([hidden email]), if you are
interested in getting a copy of the PFRET software.

Best regards,
Sheng
Yuansheng Sun, PH.D.
Research Scientist
Keck Center for Cellular Imaging,
University of Virginia

We teach FRET every March (check it out) -
http://www.kcci.virginia.edu/workshop/workshop2013/index.php



On Fri, Sep 21, 2012 at 9:27 AM, aget <[hidden email]> wrote:

> Hi,
> Could anyone recommemd a plugin for analysis of timelaps images from FRET
> based calcium indicator imaging?
> I acquired images from neurons that will fire, triggering FRET, so that
> signal from channel one undergo reduction in fluorescence, signal from
> channel two is enhanced. How could i process the temporal images in order
> to
> know which neuron fires (out of hundred of them)? Thanks
>
> Yajie
>
>
>
> --
> View this message in context:
> http://imagej.1557.n6.nabble.com/Analysis-of-image-from-Fret-CFP-YFP-based-calcium-imaging-tp5000156.html
> Sent from the ImageJ mailing list archive at Nabble.com.
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html
>

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

[aget]

Thanks Sheng and Jacob, i now used combination of ImageJ and Igor for analysis of wave-like data, which is not bad, though time consuming. Best  Yajie