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Introduction to Biological Image Analysis



ImageJ is a free public domain image analyzing software used for image processing and analysis techniques. The basic steps for image analysis using the software are discussed in the following sections.



 One can freely download and install ImageJ from the website (Refer Simulator to learn the installation process).The ImageJ window is shown below.



ImageJ window


ImageJ Toolbars





  • Among this, rectangular selection tool, oval selection tool, Polygon selection tool and free hand selection tool are generally used for area selection of the imported image. The status bar reports the coordinates, dimensions, and aspect ratio of the selection’s in the box. Location, width, height, and aspect ratio are displayed in the status bar during the selections.
  • Tools important to impact cratering activities are the Straight Line Selection tool and the Angle Tool.
  • Line selection tool is useful for measuring distances between two points. Angle Tool requires selecting three points in an image.  The angle is displayed in the status bar while the selection is being created or adjusted while image processing.
  • Magnifying Glass tool may be used to zoom in out of particular areas of an image.
  • Point selection tool is useful in pointing specific areas of the image. Point selection is mainly to, count objects in the image.
  • Creates a selection by tracing objects of uniform color or threshold objects. Widely using in the gel analysis studies to plot the band intensity.
  • Text tool is useful in adding text to images.
  • Application of Scrolling tool in an image makes the image larger than its window.
  • Color picker tool helps to picking up’ colors from any open image.


Menu Bar



File --->  Open:  Opens and displays the selected image in a separate window.

File ---> Close:  Closes the currently active image in the window.

File---> Save: Saves the active image in a separate folder.


Edit ---> Undo: Reverses the most recent image editing operation. Only one “undo” step is possible.

Edit ---> Copy: Copy the contents of the selected region of interest (ROI) of an image or any other window to the clipboard. If ROI-selection is not included, the entire active image is then copied.

Edit ---> Paste: Inserts the contents of the clipboard into the active image. The pasted image is selected automatically, allowing it to be dragged with the mouse.  One can click outside the selection to terminate the paste.

Selection of Region of Interest (ROI): The selection of a specified area of an image is a basic operation for image analysis.

Edit --->Selection ---> Select All:  create a rectangular ROI that has the same size as the image. 

Edit ---> Selection --->Select None: Deactivate the selection created in the active image.

Edit ---> Selection ---> Specify:  This help to select a precise region. The specific values can be provided in a specific window.

Grey Value Histogram: ImageJ calculates and displays a grey level histogram of the imported image.


Image ---> Adjust ---> Threshold is useful to adjust the lower and upper values of the threshold.

 The threshold values segments the image into features of interest and background. Pixels with grey values greater than or equal to the lower threshold and less than or equal to the upper threshold are displayed in red.

Image ---> Colour ---> Split Channels converts the image into three channels (Red, Green and Blue). Among this one image can be used for the image analysis.

Image ---> Adjust ---> colour balance determines the basic brightness and contrast of the image. One can provide Minimum and maximum level adjustments in this section.

Image ---> Look up table ---> Invert LUT, helps to adjust the brightness and contrast of the image so that the specific region in image is displayed as black in a white background.


Process ---> Subtract background reduces the back ground intensity of the image.

Process ---> Binary ---> Watershed, this separates the adjacent cells.


Analyze ---> Set Measurements select specific measurements needed for analyzing the imported image

Analyze ---> Measure, provide the selected measurements of the imported image.


Cite this Simulator:

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