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downloads:bleach_corrector [2012/05/22 11:28] kotadownloads:bleach_corrector [2021/10/08 08:10] (current) – [Author] kota
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 ====== Bleach Corrector ====== ====== Bleach Corrector ======
 +
 +{{ :downloads:bleach_correction.mp4 |}}
  
 ===== Author ===== ===== Author =====
  
 Kota Miura\\  Kota Miura\\ 
-Centre for Molecular and Cellular Imaging (CMCI)\\ +Bioimage Analysis & Research (BIAR)\\ 
-EMBL Heidelberg\\  +Heidelberg\\  
-miura at embl dot de\\  + 
-Tel+49 6221 387 404+Video: Christophe Leterrier(@chrislet) [[https://twitter.com/christlet/status/1445658436276867081|See here for the twitter presenting this video]] 
 + 
 +===== Citing the Plugin ===== 
 + 
 +  *  Paper: Miura K. Bleach correction ImageJ plugin for compensating the photobleaching of time-lapse sequences [version 1]. F1000Research 2020, 9:1494 [[https://doi.org/10.12688/f1000research.27171.1]] 
 +  *  Code: Kota Miura et al. (2014). ImageJ Plugin CorrectBleach V2.0.2. Zenodo. [[http://dx.doi.org/10.5281/zenodo.30769|10.5281/zenodo.30769]]
  
 ===== History ===== ===== History =====
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   * 12-04-16 Compiled with JRE 1.6.0_20   * 12-04-16 Compiled with JRE 1.6.0_20
 ==== recent changes ==== ==== recent changes ====
-{{rss>https://github.com/cmci/CorrectBleach_/commits/master.atom 10 date 48h}} +{{rss>https://github.com/fiji/CorrectBleach/commits/master.atom 10 date 48h}} 
  
 ===== Source ===== ===== Source =====
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     * See the blog entry, [[http://cmci.embl.de/blogtng/2010-05-04/photobleaching_correction_3d_time_series|a bit more detail on this issue]] and [[http://cmci.embl.de/blogtng/2010-05-06/bleach_correction_2|here is some more notes.]]     * See the blog entry, [[http://cmci.embl.de/blogtng/2010-05-04/photobleaching_correction_3d_time_series|a bit more detail on this issue]] and [[http://cmci.embl.de/blogtng/2010-05-06/bleach_correction_2|here is some more notes.]]
  
-==== headless usage ==== +==== Headless Usage ==== 
-[[https://github.com/cmci/ijmacros/blob/master/headless/bleachCorHM.js|This script]] demonstrates the headless usage. +[[https://github.com/cmci/ijmacros/blob/master/headless/bleachCorHM.js|This script]] demonstrates the headless usage. 
 + 
 +==== Q & A ==== 
 + 
 +<blockquote> 
 +One of our users is making timelapse experiments to track a GFP marker in cell cultures. GFP signal is very dim and background is quite strong (so SNR very poor). Over the time, background intensity decreases while specific signal keeps more or less the same so it becomes gradually more visible. He really expects the GFP to increase over the time, and he would like to quantify this increase in GFP signal over time. To compensate background bleaching he is using your bleach_corrector plugin in FIJI. He obtains the best visualization of what he expects with the Histogram Matching Method. The thing is that, as you mention in your blog's entry (http://cmci.embl.de/downloads/bleach_corrector,  http://cmci.embl.de/blogtng/2010-05-06/bleach_correction_2 ), with this method you cannot quantify intensities.  
 + 
 +Why?  
 + 
 +Can you recommend us an alternative method to be able to quantify changes in the GFP signal over time? 
 + 
 +Other thing is at this moment it is difficult to know is wherther everything is bleached (so GFP signal kept constant reflects an increase) or wherther bleaching affects only the medium (so GFP is really constant and is not increasing, which is not what he expects...). We will make test to address this issue... 
 + 
 +Xavier Sanjuan (ALMU, Parc de Recerca Biomèdica de Barcelona), \\ 
 +on behalf of Diego Barcena (Mark Isalan group, CRG, Barcelona) 
 +</blockquote> 
 +\\ 
 +\\ 
 +The reason histogram matching cannot be used for the measurement, to explain in your case, is because the algorithm assumes that the histogram shape is always constant (which also means that the average intensity is constant over time). 
 + 
 +On the other hand, you know that the signal should increase if its background is constant, which means that you must assume that histogram shape does change over time, contradicting with the assumption that histogram matching is based on.  
 + 
 +One way that I can suggest to do the correction is as follows:  
 + 
 +  - Estimate the baseline intensity level.  
 +    - select a region (ROI) in the background, and do [Image > Stack > Plot z-axis profile...]. This will create a plot, expected to be decaying. Click "List" button which will then create a table with frame number and average intensity. Select All and Copy them all.  
 +    - Do [Analyze > Tools > Curve Fitting]. Curve fitting interface appears, so paste the copied value in the field (you probably need to delete the default values). Then fit the values using "Exponential with offset"  
 +    - You will then see a plot, fitted by a curve. the value "C" will be the estimate of baseline. Keep the value.  
 +  - Go back to your image stack, select a background region (ROI) again. This will be a region where the decay is measured.  
 +  - DO [Bleach correction], and select "Simple ratio" 
 +    - you will be asked for a background value. In put the value you got in 1.3. Click OK 
 +    - you will see the stack corrected by simple ratio method.  
downloads/bleach_corrector.1337686136.txt.gz · Last modified: 2016/05/24 12:46 (external edit)

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