Photobleaching correction

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Photobleaching correction

Shane Harding-2
Hello,
I have a data set of live cells, imaged over 5 minutes for a GFP  
labelled protein. I would like to correct for the bleaching that  
occurs. Within each image set I've collected I have one cell from  
which I'm gathering measurements, and at least one other cell that I  
can use as a reference for the photobleaching. Any suggestions are  
appreciated.

Thank you,
Shane Harding
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Re: Photobleaching correction

Stephan Preibisch
Hi,

a very simple zero order approximation would be to monitor the average image
intensity avg(timepoint) over time and multiply each pixel intensity in each
time point with its respective avg(tn)/avg(t1). That is of course only valid
if the images do not change too much....do not forget to use 32-bit images
for that.

Btw, the curve of intensity decay should be some exponential decay
function...you could fit the function to the datapoints you get to improve
this estimate.

All the best,
Stephan

-----Original Message-----
From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of Shane
Harding
Sent: Saturday, January 24, 2009 5:27 PM
To: [hidden email]
Subject: Photobleaching correction

Hello,
I have a data set of live cells, imaged over 5 minutes for a GFP  
labelled protein. I would like to correct for the bleaching that  
occurs. Within each image set I've collected I have one cell from  
which I'm gathering measurements, and at least one other cell that I  
can use as a reference for the photobleaching. Any suggestions are  
appreciated.

Thank you,
Shane Harding
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Re: Photobleaching correction

rb-13
In reply to this post by Shane Harding-2
Hello,

there is a tool for exponential bleaching correction:
http://www.embl.de/eamnet/html/bleach_correction.html

Best,
Ralf
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Re: Photobleaching correction

Joris FA Meys
In reply to this post by Shane Harding-2
Hi Shane,

You can use the single or double normalization as proposed by Phair et al (2004). The most easy way to do that is just using the roi manager, and define the bleached area, a reference area, a background area and a whole cell area. These ones you can use then to go through the stack and measure the intensities. Export the measurements, and do the calculation in another program (a simple excel sheet can do that already).

More info available at : http://www.embl.org/cmci/downloads/FRAPmanual.htm

If you care to wait another month or so, I'm working on a plugin to do exactly that and export the normalized data for fitting.

Kind regards
Joris

Shane Harding-2 wrote
Hello,
I have a data set of live cells, imaged over 5 minutes for a GFP  
labelled protein. I would like to correct for the bleaching that  
occurs. Within each image set I've collected I have one cell from  
which I'm gathering measurements, and at least one other cell that I  
can use as a reference for the photobleaching. Any suggestions are  
appreciated.

Thank you,
Shane Harding