Digitally Counting Colonies

2014-11-23

Digitally Counting Colonies
digitally counting cells
count cells
count colonies
count objects ImageJ

Basic process that seems to work on ImageJ

* Adjust brightness and contrast
* set image to an 8 bit image type
* threshold so that only the colonies are left
* Analyze particles and get a cell count

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Counting Colonies in One Image Using ImageJ

* Create image (take a picture of the plate with a phone, scan it with
  a scanner, or use a gel imaging instrument to take a picture of the
  plate (I do it this way))
* Open the image file
* Adjust the brightness and contrast as desired
  (Image->Adjust->Brightness/Contrast, adjust as necessary and then click
  apply)
* Convert the image from a red, green blue image to a grayscale 8 bit
  image (Image->Type->8 bit)
* Crop away unnecessary parts of the image by copying the part you want
  and creating a new image.
  o Select the oval tool. Highlight the region you are interested in.
    Copy this.
  o Make a new image (File->New->Internal Clipboard)

* Apply a threshold to the image so that only the colony spots are
  remaining.
  o Image->Adjust->Threshold. Adjust the minimum and maximum
    threshold levels so that only the colonies of interest and possibly
    some of the cropped background remain. Apply this threshold

* Count the particles
  o Analyze->Analyze Particles (may want to have the following
    settings applied: Size (pixel^2): 0-Infinity, Circularity: 0.00-1.00,
    Show: Outlines, display results, clear results, and summarize). Click
    OK

Automatically Counting Colonies in Many Images Using ImageJ

* First create a macro
  o Plugins->Macro->Record
  o perform all of the steps above for "Counting Colonies in One
    Image Using ImageJ" (note that adjusting the brightness and contrast
    does not seem to work so you may want to do that separately)
  o When all of the steps have been performed, click Create then
    File->Save As and save the macro with the name you want in the place
    you want. Note that the macro can be edited in any standard text
    editor

* Run the macro
  o Plugins->Macro->Run
  o Select your macro

* Set things up to process many images at once
  o I like to create one macro that does all of the image processing,
    and one macro to open all of the images and call the image processing
    macro
  o Example Image Processing Macro
    - run("Brightness/Contrast...");
      setMinAndMax(0, 157);
      call("ij.ImagePlus.setDefault16bitRange", 0);
      run("8-bit");
      makeOval(123, 45, 332, 330);
      run("Copy");
      run("Internal Clipboard");
      setAutoThreshold("Default");
      //run("Threshold...");
      setThreshold(134, 255);
      run("Convert to Mask");
      run("Analyze Particles...", "size=0-Infinity circularity=0.00-1.00
      show=Outlines display clear summarize");
      close();
      close();
      close();
    - Note that in order to obtain the values for the
      brightness/contrast command that you want for the setMinAndMax
      command you can play with an example image first. Adjust the
      brightness and contrast the way you want. Make note of the min and
      max values, and then click reset. To generate the commands in the
      Macro you can then click set, and then set the min and max values
      to the numbers you noted previously.

o Example file handling macro
    - open("L:\\Colonies on plate 2-16-12\\1 B 10-2.jpg");
      runMacro("L:\\Colonies on plate 2-16-12\\Macro.ijm");
      open("L:\\Colonies on plate 2-16-12\\2 B 10-2.jpg");
      runMacro("L:\\Colonies on plate 2-16-12\\Macro.ijm");
      etc. . . .

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Example Images from ImageJ Processing
Example of ImageJ Processing 3-25-12.zip
"C:\Users\kurtw_000\Documents\kurt\storage\CIM Research Folder\DR\2014\01-18-2014d1351\wikispaces download 01-18-2014d1351\01-18-2014d1513\Example of ImageJ Processing 3-25-12.zip"

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Here's an article about counting colonies
Optimized digital counting colonies of clonogenic assays using ImageJ
software and customized macros: Comparison with manual counting
Read More:
http://informahealthcare.com/doi/abs/10.3109/09553002.2011.622033

azim58wiki: