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FRET calculation .

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Sasmita Rath

FRET calculation .

Hello All,
I'm a new user of imageJ. I'm writting some macros in order to accomplish my
calculations.I do pixel by pixel FRET calculation.I found the plugin named"FRET
calc v3.0 plugin for analysis of FRET by acceptor photobleaching" written by
David Stepensky.

Could I use that program to do my analysis ?I'm asking because I tried to use
that and I found some error message.I know little about java programs.Is it a
good way to write my own macros in java or is it ok to use the programs already
available.

I'm a bit confused.I appreciate any suggestion.

George Sharonov

Re: FRET calculation .

I'm working with FRET too. I'm measuring stimulated acceptor emission within
time stacks. There are two plugins for measurement of stimulated emission,
but they are not intended to process stacks. Therefore I'm writing my own
plugin that allows to process stacks (working version is available if you
need).

I'm not familiar with macro language but I believe that it is almost
impossible to use FRET plugins's methods in macros. Thus it is better to use
plugin as it is. If you need to modify plugin you need to modify its source
code.

What kind of FRET experiments do you have, photobleaching or stimulated
emission?

On Mon, May 26, 2008 at 2:32 AM, Sasmita Rath <[hidden email]> wrote:

> Hello All,
> I'm a new user of imageJ. I'm writting some macros in order to accomplish
> my
> calculations.I do pixel by pixel FRET calculation.I found the plugin
> named"FRET
> calc v3.0 plugin for analysis of FRET by acceptor photobleaching" written
> by
> David Stepensky.
>
> Could I use that program to do my analysis ?I'm asking because I tried to
> use
> that and I found some error message.I know little about java programs.Is it
> a
> good way to write my own macros in java or is it ok to use the programs
> already
> available.
>
> I'm a bit confused.I appreciate any suggestion.
>

George Sharonov,
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry
Moscow, Russia

Sasmita Rath

Re: FRET calculation .

In reply to this post by Sasmita Rath

Thank you very much for your reply.I do stimulated acceptor emission(Tag protein with GFP-YFP and excite using laser to find the FRET ) .Yes you are right .Its not possible to use FRET plugins's methods in macros .I tried and failed.

Could you please let me know the name of the plugins and the (working version to process stacks) you mentioned.

Sasmita

----- Original Message -----
From: "George Sharonov" <[hidden email]>
To: [hidden email]
Sent: Monday, June 2, 2008 2:21:14 AM GMT -06:00 US/Canada Central
Subject: Re: FRET calculation .

I'm working with FRET too. I'm measuring stimulated acceptor emission within
time stacks. There are two plugins for measurement of stimulated emission,
but they are not intended to process stacks. Therefore I'm writing my own
plugin that allows to process stacks (working version is available if you
need).

I'm not familiar with macro language but I believe that it is almost
impossible to use FRET plugins's methods in macros. Thus it is better to use
plugin as it is. If you need to modify plugin you need to modify its source
code.

What kind of FRET experiments do you have, photobleaching or stimulated
emission?

On Mon, May 26, 2008 at 2:32 AM, Sasmita Rath <[hidden email]> wrote:

George Sharonov,
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry
Moscow, Russia

George Sharonov

Re: FRET calculation .

There are two plugins for measurement of stimulated emission:

PixFRET by Jerome Feige et al: http://www.unil.ch/cig/page16989.html
FRET and Colocalization analyzer by Olivier Marchal and Noel Converset:
http://rsb.info.nih.gov/ij/plugins/fret-analyzer/fret-analyzer.htm

Both of them have comprehensive manuals.

As for my plugin, it is in development stage. It has no documentation, do
not support some undesired user behavior, and not tested on different
systems/ImageJ versions/JAVA versions. I run it on Windows on ImageJ 1.39
and 1.4 and on Java 1.6. I found that it does not work on "ImageJ by Tonny
Collins" (http://www.uhnresearch.ca/facilities/wcif/imagej/) perhaps due to
conflict with some other plugins.

The benefits of my plugin:
- can process stacks;
- can process >8 bit grayscale images;
- it takes into account bleed-through from acceptor channel to donor channel
and vice-versa. I have not seen this feature in any FRET plugins or even in
commercial software (by Leica). If spectra of your fluorochromes are well
separates this feature is not necessary, but if you deal with large spectral
overlap (such as CFP/YFP or even more - GFP/YFP) or your optical spectral
separating system is not perfect you need to consider this bleed-through.

I will send you my plugin by e-mail. I'm not guaranteeing you immediate
support of this plugin but will try to help you as far as I could.
If you will have some experience with PixFRET the interface of my plugin
should be familiar for you.

George.

On Mon, Jun 2, 2008 at 6:08 PM, Sasmita Rath <[hidden email]> wrote:

> Thank you very much for your reply.I do stimulated acceptor emission(Tag
> protein with GFP-YFP and excite using laser to find the FRET ) .Yes you are
> right .Its not possible to use FRET plugins's methods in macros .I tried
> and failed.
>
> Could you please let me know the name of the plugins and the (working
> version to process stacks) you mentioned.
>
> Sasmita
>
>
>
>
>
>
> ----- Original Message -----
> From: "George Sharonov" <[hidden email]>
> To: [hidden email]
> Sent: Monday, June 2, 2008 2:21:14 AM GMT -06:00 US/Canada Central
> Subject: Re: FRET calculation .
>
> I'm working with FRET too. I'm measuring stimulated acceptor emission
> within
> time stacks. There are two plugins for measurement of stimulated emission,
> but they are not intended to process stacks. Therefore I'm writing my own
> plugin that allows to process stacks (working version is available if you
> need).
>
> I'm not familiar with macro language but I believe that it is almost
> impossible to use FRET plugins's methods in macros. Thus it is better to
> use
> plugin as it is. If you need to modify plugin you need to modify its source
> code.
>
> What kind of FRET experiments do you have, photobleaching or stimulated
> emission?
>
> On Mon, May 26, 2008 at 2:32 AM, Sasmita Rath <[hidden email]> wrote:
>
> > Hello All,
> > I'm a new user of imageJ. I'm writting some macros in order to accomplish
> > my
> > calculations.I do pixel by pixel FRET calculation.I found the plugin
> > named"FRET
> > calc v3.0 plugin for analysis of FRET by acceptor photobleaching" written
> > by
> > David Stepensky.
> >
> > Could I use that program to do my analysis ?I'm asking because I tried to
> > use
> > that and I found some error message.I know little about java programs.Is
> it
> > a
> > good way to write my own macros in java or is it ok to use the programs
> > already
> > available.
> >
> > I'm a bit confused.I appreciate any suggestion.
> >
>
> George Sharonov,
> Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry
> Moscow, Russia
>

--

George Sharonov,
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry
Moscow, Russia

Sasmita Rath

Re: FRET calculation .

Thanks a lot.I'll try and if find problem I may ask you again.
Sasmita