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Photobleaching

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jross34

Photobleaching

I know there are lots of posts out there about how to correct for photo bleaching but what I haven't seen yet is how to correct for photo bleaching when you have a true signal at some point.

I'm curious to know if there's a plugin/macro out there that will allow me to set the exponent for bleach correction for only a certain window but then apply it to the whole series.

For example, I may image for 100 frames before I evoke a signal change and then continue to image for another 100 frames. Obviously applying a bleach correction for the full 200 frames doesn't work because it will flatten my evoked signal. Instead I would like to set the correction parameters within the first 100 frames and then apply it to all 200 frames.

Anyone have any suggestions?

meeks

Re: Photobleaching

Hi.
Maybe I'm completely off with this....but I had once a similar problem when I used a method called FRET-mediated Protection Against Photobleaching (FPAP; quenching of the excited fluorochrome via FRET). The background problem was (partially) solved by subtracting the previous frame from the next one of a stack. This way the photobleaching was constantly pretty much removed but a signal on a spot where FPAP occurred appeared after a while.

Greetz,

>m

jerie

Re: Photobleaching

have a look at http://fiji.sc/Bleach_Correction.

you find the function in FijI ...image...adjust...bleach correction. Select a method,
set a ROI to a fluorescent region, outside the region that is later photoconverted and try.

Cheers, Jens

Jens Rietdorf Visiting Scientist Fundação Oswaldo Cruz - Ministério da Saúde, Centro de Desenvolvimento Tecnológico em Saúde (CDTS), Rio de Janeiro, Brasil.

jross34

Re: Photobleaching

If I completely understand your suggestion, I'm not sure it would work.

I'm using GCaMPs to assess action potential generation in an area of brain. I begin imaging sometime before I expect action potentials to occur and then continue to image for several seconds. At some point in response to a stimulus, cells will begin to generate action potentials and will have dramatic changes in fluorescence above background. However, because I'm imaging over time, I'm subject to photobleaching meaning that I will have decreasing background fluorescence overtime and the amplitude of each sequential action potential generation will look smaller, which may or may not be true.

So what I'm looking to do is generate an equation to fit the bleaching of the frames before any action potentials generate and then fit it to my entire stack. So far, all I've been able to do is determine an averaged constant for simple subtraction, which doesn't really work, and make an equation off the entire stack, which doesn't work because the higher fluorescence values associated with action potentials pulls my line equation up falsely.

Hopefully the additional information is useful.

Kota Miura

Re: Photobleaching

In reply to this post by jross34

Hi,

I think that's a good feature, so I test added an option to set range for
fitting. Please test:

https://www.dropbox.com/s/baex4obsb1kqpeq/CorrectBleach_-2.0.3-SNAPSHOT.jar?dl=0

Just place this jar file instead of the one in your plugin folder already
(better keep the old version somewhere, of course). If you have comments,
please do so at

https://github.com/fiji/CorrectBleach/issues/2

Cheers,
Kota

On Wed, Sep 2, 2015 at 10:30 PM, jross34 <[hidden email]> wrote:

> I know there are lots of posts out there about how to correct for photo
> bleaching but *what I haven't seen yet is how to correct for photo
> bleaching
> when you have a true signal at some point*.
>
> I'm curious to know if there's a plugin/macro out there that will allow me
> to set the exponent for bleach correction for only a certain window but
> then
> apply it to the whole series.
>
> For example, I may image for 100 frames before I evoke a signal change and
> then continue to image for another 100 frames. Obviously applying a bleach
> correction for the full 200 frames doesn't work because it will flatten my
> evoked signal. Instead I would like to set the correction parameters within
> the first 100 frames and then apply it to all 200 frames.
>
> Anyone have any suggestions?
>
>
>
> --
> View this message in context:
> http://imagej.1557.x6.nabble.com/Photobleaching-tp5014227.html
> Sent from the ImageJ mailing list archive at Nabble.com.
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html
>

--

-------------------------------------------------------------
Dr. Kota Miura <[hidden email]>

Senior Image Analyst / NINS Research Administrator
European Molecular Biology Laboratory
Meyerhofstr. 1, 69117 Heidelberg, GERMANY
Tel +49 6221 387 8404
Mobile +49 160 95001177http://cmci.embl.de
++++++++++++++++++

Dr. Kota Miura <[hidden email]>
Project Associate Professor
National Institutes of Natural Sciences (NINS), Japan
Bonn Office: Ahrstr. 58, 53175 Bonn, Germany
Tel: +4916095001177http://www.nins.jp/english/
-------------------------------------------------------------

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

jross34

RE: Photobleaching

Thanks so much! I've only tried it on a single stack so far but it seems to be doing exactly what I need! I'll keep testing it throughout the day. Again, I really appreciate it!

Sincerely,

Jordan M. Ross

Graduate Student

University of Tennessee Health Science Center

From: Kota Miura [via ImageJ] [ml-node+[hidden email]]
Sent: Thursday, September 10, 2015 7:24 AM
To: Ross, Jordan Marie
Subject: Re: Photobleaching

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