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Cell size analysis using ImageJ

Shows the location of the filter box in the upper right corner of the Konqueror the file browser
Finding the right files
Use Konqueror to find the files you want to analyze.

You can use the filter to list only the relevant files. The filter box is highlighted in green in the picture to the left.

"Results" ".zip" and the name of the samples you want to analyze (ab meso in the picture) are useful filters.


LOCI Bio-Formats Import Window with the "Display associated metadata" option selected
Importing to ImageJ
Drag the file from Konqueror to the main ImageJ window to open it. It will display a dialog like the one to the left. Select "Display associated metadata" and "View stack with: Image5D". Nothing else should be selected.
Shows the "Scale factor" for .zvi files

Setting the image scale
When you first open ImageJ no scale is set. You only need to set it once as long as the scale is constant between images. Check the metadata for each image to make sure the scale is the same.

Scroll to the bottom of the metadata window. The attribute called "Scale factor for X" is what you want.

Go to Analyze->Set scale in the ImageJ main window and enter the following values.

Distance in Pixels: 1
Known Distance: The value from the metadata
Pixel Aspect Ratio: 1
Unit of length: micrometer
Global: Checked

A sample image loaded into Image%D. Shows how to open the color picker by selecting "Color" in the upper right of the window. Also shows the "color" drop down box and selecting the "ovl" option.
Image5D
Select "Color" to open a color chart. Use the slider to select the channels and choose an appropriate color. Then select "ovl" rom the drop down box to display the channels in an overlay.
An of spongy mesophyll cells from a leaf. The nuclei are stained, and the channel is blue. The cells are imaged with differenctial interference contrast filters and are grayscale. Show how to select an area to measure

Select an area of measurment
Use F6 for September 1 sample, F7 for August 5 samples.

A rectangle is randomly drawn. If all cells in the rectangle are mesophyll cells use it. If it contains other cells (i.e. veins) then move it or use the macro until it is in a suitable spot.

Press cnt+D to draw the rectangle.

A close up of the previous image. Shows several of the cells outlined in blue.

Measuring cells

Outline cells which
  • are completely enclosed by the rectangle
  • touch the top edge
  • touch the right edge unless it also touches the bottom edge (i.e don't measure cells in the bottom right corner).

Don't outline cells which

  • Touch the bottom edge
  • Touch the left edge unless it also touches the top edge (i.e. measure cells in the top left corner).

Use F12 to add the outline to the ROI Manager. Use "Show All" in the ROI Manager to show the selections.

This is identicle to the previous image and was included for consitant layout.
Save files
When finished, use F1 to save the file. It will ask for a name. Use the sample name. If the original file was named "10-9-1 ab meso.zvi" the sample name is "10-9-1 ab meso" without quotes. If you haven't saved before, it will ask for a directory. Use the same directory the original image was in.

Two files will be created. One with Results.csv added to the sample name, and one with .zip added to sample name.


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