downloads:bleach_corrector
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downloads:bleach_corrector [2015/09/10 14:09] – [Author] kota | downloads:bleach_corrector [2021/10/08 08:10] (current) – [Author] kota | ||
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+ | ====== Bleach Corrector ====== | ||
+ | {{ : | ||
+ | |||
+ | ===== Author ===== | ||
+ | |||
+ | Kota Miura\\ | ||
+ | Bioimage Analysis & Research (BIAR)\\ | ||
+ | Heidelberg\\ | ||
+ | |||
+ | Video: Christophe Leterrier(@chrislet) [[https:// | ||
+ | |||
+ | ===== 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:// | ||
+ | * Code: Kota Miura et al. (2014). ImageJ Plugin CorrectBleach V2.0.2. Zenodo. [[http:// | ||
+ | |||
+ | ===== History ===== | ||
+ | |||
+ | * 10-09-15 First Version | ||
+ | * 12-04-16 Compiled with JRE 1.6.0_20 | ||
+ | ==== recent changes ==== | ||
+ | {{rss> | ||
+ | |||
+ | ===== Source ===== | ||
+ | |||
+ | Released under the [[http:// | ||
+ | |||
+ | ===== Requires ===== | ||
+ | |||
+ | ImageJ ver 1.34j or higher ([[http:// | ||
+ | ===== Installation ===== | ||
+ | |||
+ | This plugin will be a part of Fiji, so you could find it under menu tree [Image > Adjust >] in near future (as of Apr. 18, 2012). | ||
+ | |||
+ | If you want to use it in ImageJ, download [[https:// | ||
+ | |||
+ | ===== Description ===== | ||
+ | |||
+ | This plugin contains three different methods for correcting the intensity decay due to photobleaching. They all work with either 2D or 3D time series. In case of 3D time series, image properties should be appropriately set. If you are not sure, check your image header by [Image -> Properties]. | ||
+ | |||
+ | * Simple Ratio Method: | ||
+ | * Plugin version of [[http:// | ||
+ | * [[http:// | ||
+ | * You need to estimate the base line intensity before using this method. Measure the mean intensity of the region outside the target signal and use that value. | ||
+ | * Exponential Fitting Method: | ||
+ | * Similar to the description in the manual of [[http:// | ||
+ | * MBF-ImageJ suggests to use " | ||
+ | * Figure below is an example of fitting exponential decay equation to the intensity changes over time. This is rather an ideal case example. If you see that the fit quality is not good enough, do not use this method. Beside the evaluation of the fitting quality by eyes, use R^2 (residual) as an indicator of the quality of fit. {{: | ||
+ | * Exponential Fitting Method (base subtraction): | ||
+ | * similar to above, but does two rounds of curve fitting. | ||
+ | * 1st round: estimate the baseline intensity and subtract the base line value | ||
+ | * 2nd round: do the curve fitting again and correct each frame accordingly. | ||
+ | * Histogram Matching Method: | ||
+ | * A brand-new method for bleach correction. | ||
+ | * This algorithm first samples the histogram of initial frame, and for the successive frames, [[http:// | ||
+ | * This method does much better restoration of bleaching sequence for segmentation but not appropriate for intensity quantification. | ||
+ | * See the blog entry, [[http:// | ||
+ | |||
+ | ==== Headless Usage ==== | ||
+ | [[https:// | ||
+ | |||
+ | ==== Q & A ==== | ||
+ | |||
+ | < | ||
+ | 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:// | ||
+ | |||
+ | 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) | ||
+ | </ | ||
+ | \\ | ||
+ | \\ | ||
+ | The reason histogram matching cannot be used for the measurement, | ||
+ | |||
+ | 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 " | ||
+ | - 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 " | ||
+ | - You will then see a plot, fitted by a curve. 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], | ||
+ | - 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.txt · Last modified: 2021/10/08 08:10 by kota