Photobleaching correction for time lapse images

[Farid Jalali]

Hello Group,
I have acquired a series of time lapse data sets and am beginning some simple analysis. The biology I am looking at is accumulation of an EGFP-tagged protein at the site of UV-laser induced DNA damage. The images were acquired using an EM-CCD and neutral density filter attenuated widefield illumination coming from an EXFO 100W Hg lamp. The EGFP signal accumulates in a well defined spot and I am able analyze the rate of accumulation and fold-increase of florescence intensity at several same-sized ROI's using Image J plugins. Baseline fluorescence intensity subtraction is simple enough. I have used a non-lased cell in the same field of view to monitor EGFP photo-bleaching, calculated the slope for this, but am stuck on how I might use this information to develop a correction factor. I would appreciate any thoughts and help in this regard.
Thanks to all.
Farid

--
Farid Jalali MSc
Program Leader- Cellular Imaging Core
Applied Molecular Oncology and Radiation Medicine Program
Princess Margaret Hospital (University  Health Network)
Toronto Medical Discovery Tower
Toronto, Canada
416-581-7754 STTARR at TMDT
416-581-7791 STTARR Microscopy Suite

[Kevin Braeckmans]

Dear Farid,

 

All you need to do is normalisation of the ‘accumulation signal’ to the intensity of your control cell. This has to be done for each image in the time series.

 

In case you find that this procedure increases the noise too much (a division of two measured signals naturally amplifies the noise), you could first do a best fit to the photobleaching time trace of the control cell (a single or double exponential should be fine) and then normalize the accumulation time trace to the fitting result.

 

Best regards,

 

Kevin

 

 

 

Prof. Dr. Kevin Braeckmans

Lab. General Biochemistry & Physical Pharmacy

Ghent University

Harelbekestraat 72

9000 Ghent

Belgium

Tel: [phone]

Fax: [phone]

E-mail: [hidden email]

 

 

 

Van: Confocal Microscopy List [mailto:[hidden email]] Namens Farid Jalali
Verzonden: woensdag 26 november 2008 3:52
Aan: [hidden email]
Onderwerp: Photobleaching correction for time lapse images

 

Hello Group,
I have acquired a series of time lapse data sets and am beginning some simple analysis. The biology I am looking at is accumulation of an EGFP-tagged protein at the site of UV-laser induced DNA damage. The images were acquired using an EM-CCD and neutral density filter attenuated widefield illumination coming from an EXFO 100W Hg lamp. The EGFP signal accumulates in a well defined spot and I am able analyze the rate of accumulation and fold-increase of florescence intensity at several same-sized ROI's using Image J plugins. Baseline fluorescence intensity subtraction is simple enough. I have used a non-lased cell in the same field of view to monitor EGFP photo-bleaching, calculated the slope for this, but am stuck on how I might use this information to develop a correction factor. I would appreciate any thoughts and help in this regard.
Thanks to all.
Farid

--
Farid Jalali MSc
Program Leader- Cellular Imaging Core
Applied Molecular Oncology and Radiation Medicine Program
Princess Margaret Hospital (University  Health Network)
Toronto Medical Discovery Tower
Toronto, Canada
416-581-7754 STTARR at TMDT
416-581-7791 STTARR Microscopy Suite

[Rietdorf, Jens]

Dear Farid,

 

I have put together a small ImageJ macro that does the normalization to a ROI average much like Kevin suggests.  

 

http://www.embl.de/eamnet/html/bleach_correction.html

 

Cheers, jens

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Farid Jalali
Sent: Wednesday, November 26, 2008 3:52 AM
To: [hidden email]
Subject: Photobleaching correction for time lapse images

 

Hello Group,
I have acquired a series of time lapse data sets and am beginning some simple analysis. The biology I am looking at is accumulation of an EGFP-tagged protein at the site of UV-laser induced DNA damage. The images were acquired using an EM-CCD and neutral density filter attenuated widefield illumination coming from an EXFO 100W Hg lamp. The EGFP signal accumulates in a well defined spot and I am able analyze the rate of accumulation and fold-increase of florescence intensity at several same-sized ROI's using Image J plugins. Baseline fluorescence intensity subtraction is simple enough. I have used a non-lased cell in the same field of view to monitor EGFP photo-bleaching, calculated the slope for this, but am stuck on how I might use this information to develop a correction factor. I would appreciate any thoughts and help in this regard.
Thanks to all.
Farid

--
Farid Jalali MSc
Program Leader- Cellular Imaging Core
Applied Molecular Oncology and Radiation Medicine Program
Princess Margaret Hospital (University  Health Network)
Toronto Medical Discovery Tower
Toronto, Canada
416-581-7754 STTARR at TMDT
416-581-7791 STTARR Microscopy Suite

[Farid Jalali]

Thanks very much Kevin and Jens for your help. I really do appreciate the suggestions.
Best Regards
Farid

On Wed, Nov 26, 2008 at 4:38 AM, Rietdorf, Jens <[hidden email]> wrote:

Dear Farid,

 

I have put together a small ImageJ macro that does the normalization to a ROI average much like Kevin suggests.  

 

http://www.embl.de/eamnet/html/bleach_correction.html

 

Cheers, jens

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Farid Jalali
Sent: Wednesday, November 26, 2008 3:52 AM
To: [hidden email]
Subject: Photobleaching correction for time lapse images

 

Hello Group,
I have acquired a series of time lapse data sets and am beginning some simple analysis. The biology I am looking at is accumulation of an EGFP-tagged protein at the site of UV-laser induced DNA damage. The images were acquired using an EM-CCD and neutral density filter attenuated widefield illumination coming from an EXFO 100W Hg lamp. The EGFP signal accumulates in a well defined spot and I am able analyze the rate of accumulation and fold-increase of florescence intensity at several same-sized ROI's using Image J plugins. Baseline fluorescence intensity subtraction is simple enough. I have used a non-lased cell in the same field of view to monitor EGFP photo-bleaching, calculated the slope for this, but am stuck on how I might use this information to develop a correction factor. I would appreciate any thoughts and help in this regard.
Thanks to all.
Farid

--
Farid Jalali MSc
Program Leader- Cellular Imaging Core
Applied Molecular Oncology and Radiation Medicine Program
Princess Margaret Hospital (University  Health Network)
Toronto Medical Discovery Tower
Toronto, Canada
416-581-7754 STTARR at TMDT
416-581-7791 STTARR Microscopy Suite

--
Farid Jalali MSc
Program Leader- Cellular Imaging Core
Applied Molecular Oncology and Radiation Medicine Program
Princess Margaret Hospital (University  Health Network)
Toronto Medical Discovery Tower
Toronto, Canada
416-581-7754 STTARR at TMDT
416-581-7791 STTARR Microscopy Suite