TITLE: Quantification of Gel Bands by an Image J Macro, Band/Peak Quantification Tool
AUTHORS: Kenji Ohgane, Hiromasa Yoshioka

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol offers an Image J macro/plugin that enable easy quantification of bands on western blots, dot blots, and fluorescent gels etc., by simply selecting bands with rectangle or oval tools as region of interest (ROI), and running this macro. This macro automatically estimates background level based on mean or median intensity around ROI boundary, and should help you speed up your routine quantification tasks. Note that the quantification algorithm is similar to that used in Image Studio Lite software (freely available from LI-COR Biosciences), which is widely used for quantification purpose in many publications.</div></div>

[STEPS]
?. [Installation of ImageJ and the macro]
Down load the following ImageJ macro file (_BandPeakQuantification.ijm) and place in a directory under the ImageJ plugins directory.
For example, save the ijm file in“Applications> ImageJ > plugins”. With the“_”character at the start of the filename, ImageJ automatically recognize this macro file at startup, so that you can see the macro listed in the “Plugins” menu. Alternatively, you can do "Help > Update Menu" to make the newly installed macro in the menu.
Within ImageJ's plugin directory, you can use subdirectory (or folder) to store multiple macros in an organized manner.
?. [Quantification of a dot blot with traditional lane-profile method]
Make ROI for the first lane of the gel in this case, I used horizontal rectangle). Press Cmd + 1 (or Ctrl +1 or Analyze > Gels > Select First Lane).
?. [Quantification of an western blot image]
?. [Quantification of dot blot with this macro]
Open your image. Here, in this example, we use a sample dot blot image provided with ImageJ software. Open the image from "File > Open Samples > Dot blot".
?. [Quantification of dot blot with this macro]
Place a rectangle (or circle) on the band, and register the ROI (regionof interest) to the ROI Manager by “Command + t” (forMac) or “Ctrl + t” (for Windows) or click "Add" on the ROI Manager window. Note that you can display the ROIs with numbering by checking .
Note that you can change image contrast ("Image > Adjust > Brightness/Contrast" or "Process > Enhance Contrast") to make it easier to select ROIs. However, do not click "Apply" in the Brightness/contrast window, or do not check "normalize" in the Enhance Contrast window, as these operations alter image itself.
Note that this macro can quantify single selection of a band without using ROI Manager. Simply select your band, and run the macro, and you can get the quantification result in the Results window.
?. [Quantification of dot blot with this macro]
Select next band, and register the ROI again. Repeat for all the band you want to quantify.
With this macro, the size of the ROIs needs not to be the same, as this macro quantify integrated intensity above the background. So you can define appropriate ROI of different size for each band.
?. [Quantification of dot blot with this macro]
Run the “BandPeakQuantification”macro from Plugins > BandPeakQuantification on the menu bar. Within the Band/Peak Quantification Tool window, you can set several options, as described in the following notes.
"Background with (pixels)" means how much you expand the ROI for background estimation (see also Guidelines section).
You can select all, top/bottom, or sides for the "Estimate background from" option. If set to "all", background ROI is made by expanding the ROI by background width pixels. If set to "top/bottom", background is estimated only top side and bottom side of the ROI (only applicable for rectangle ROIs). If set to "sides", background ROI is placed on both sides of the ROI.
You can select "median" or "mean" for how background is estimated from the region around the ROI. "Median" (default) can be more robust, as it is less sensitive to outliers.
The reset scale option is checked by default. This option is required only when the image scale (µm/pixel) is set. Note that by running this macro, the scale will be reset.
?. [Quantification of dot blot with this macro]
All the ROIs listed in the ROI Manager will be quantified. You can save the results as a CSV file from right click on the result window, or copy & paste to Excel or other softwares.
Note that "signal" column denotes the background corrected integrated intensity. The "total" column is the integrated intensity within the ROI without background correction.
?. [Another example: western blot]
Run the Band/Peak Quantification Tool macro, and set the "estimate background from" option to "top/bottom".
In this case, it would be more reasonable to estimate top and bottom sides of the ROI. Also, the sizes of ROIs can be different, depending on the intensity of the band (generally, high-intensity band requires larger ROI).
?. [Another example: western blot]
Select ROIs and add to the ROI Manager as described above.
?. [Another example: western blot]
Open the image.
The example data was extracted from a published article(DegasperiA, Birtwistle MR, Volinsky N, Rauch J, Kolch W, Kholodenko BN(2014)Evaluating Strategies to Normalise Biological Replicates of Western Blot Data. PLoS ONE 9(1): e87293.). The data was from western blot of serially diluted BSA(2-folddilution) detected with an enhanced chemiluminescence reagent and a CCD camera system.
?. [Quantification of a dot blot with traditional lane-profile method]
Select next lane and press Cmd + 2 (or Ctrl + 2 or Analyze > Gels > Select Next Lane). Repeat for the other lanes.
?. [Quantification of a dot blot with traditional lane-profile method]
Press Cmd + 3 (or Ctrl + 3 or Analyze > Gels > Plot Lanes) to plot lane profiles.
?. [Quantification of a dot blot with traditional lane-profile method]
Draw lines at the bottom of the peaks, so that uneven background is accounted for.
A limitation of this method is that baseline can not be drawn for small peaks, as the lane profile tool does not offer zooming function.
?. [Quantification of a dot blot with traditional lane-profile method]
From Image J tool bar, select Wand tool. Click the peaks and the calculated peak area is displayed in the Results window.
Note that this wand tool quantify area surrounded by lines (closed area). So you need to make the peak area closed by lines.
?. [Quantification of dot blot with this macro]
If needed, invert the image ("Edit > Invert").
By placing the cursole on the image, you can see intensity value on the Image J main window. Background should be low intensity and signals should show high intensity. Basically, ImageJ shows background as black and high-intensity region as white.
?. [Quantification of dot blot with this macro]
Start ROI Manager, which you can find at "Analyze > Tools > ROI Manager", and check “Show all” and “Labels” options.
?. [Another example: western blot]
The quantification results will be shown in the Results window. Save as CSV file or copy to Excel for further analysis.