Examples of Image Analysis Using ImageJ

Area Measurements of a Complex Object

Problem: Determine the photosynthetic (i.e., green) portion of a
variegated leaf.

(Open leaf image via Select ***File → Open Samples → Leaf***)

+----------------+-----------------------------------------------------+
| ![](medi       |  Convert scanned color image of leaf to grayscale: |
| a/image1.png){ |                                                     |
| width="1.14027 | ***Image → Type → 8-bit***                          |
| 77777777777in" |                                                     |
| he             |  Set measurement scale:                            |
| ight="1.002777 |                                                     |
| 7777777778in"} | Draw a line over a 50 mm section of the ruler then  |
|                | ***Analyze → Set Scale***                           |
|                |                                                     |
|                | In *Set Scale* window enter 50 into the \'Known     |
|                | Distance\' box and change the \'Unit                |
|                |                                                     |
|                | of Measurement\' box to mm , check \'Global\'       |
|                |                                                     |
|                |  Draw a new line and confirm that the measurement  |
|                | scale is correct.                                   |
+----------------+-----------------------------------------------------+
| ![](medi       |  Threshold the leaf image using the automated      |
| a/image2.png){ | routine:                                            |
| width="1.14027 |                                                     |
| 77777777777in" | ***Process → Binary → Make Binary***                |
| he             |                                                     |
| ight="1.002777 | The automated threshold includes only the dark      |
| 7777777778in"} | green areas.                                        |
+----------------+-----------------------------------------------------+
| ![](medi       |  Calculate area of green portion:                  |
| a/image3.png){ |                                                     |
| width="1.14027 | Surround the leaf with the rectangular selection    |
| 77777777777in" | tool                                                |
| he             |                                                     |
| ight="1.002777 | ***Analyze → Analyze Particles***                   |
| 7777777778in"} |                                                     |
|                | Enter 50 as the minimum particle size, toggle       |
|                | \'Show Outlines\', check "Display                   |
|                |                                                     |
|                | Results' and click \'OK\'                           |
|                |                                                     |
|                | Outline of analyzed area will be drawn. Data window |
|                | gives an area of about 2000 mm^2^ depending on the  |
|                | calibration setting.                                |
+----------------+-----------------------------------------------------+
| *(See bottom   |                                                     |
| of page for an |                                                     |
| alternative    |                                                     |
| method for     |                                                     |
| measuring      |                                                     |
| areas.)*       |                                                     |
+----------------+-----------------------------------------------------+
| ![](medi       |  Threshold new image of leaf using manual          |
| a/image4.png){ | settings:                                           |
| width="1.14027 |                                                     |
| 77777777777in" | ***Image → Adjust →Threshold*** and play with       |
| he             | sliders to include all of leaf in red               |
| ight="1.002777 |                                                     |
| 7777777778in"} | and click 'Apply'                                   |
|                |                                                     |
|                | The manual threshold setting includes all of the    |
|                | leaf area.                                          |
+----------------+-----------------------------------------------------+
| ![](medi       |  Calculate area of entire leaf:                    |
| a/image5.png){ |                                                     |
| width="1.14027 | Enclose the leaf with the rectangular selection     |
| 77777777777in" | tool                                                |
| he             |                                                     |
| ight="1.002777 | ***Analyze → Analyze Particles***                   |
| 7777777778in"} |                                                     |
|                | Use previous window settings and click \'OK\'       |
|                |                                                     |
|                | Outline of entire leaf is automatically drawn.      |
|                |                                                     |
|                | Data window gives an area of about 2450 mm^2^.      |
+----------------+-----------------------------------------------------+

This analysis suggests that about 82% of leaf surface is dark green.
These values should be manually confirmed before beginning a
\'production run\' of measurements.

+-----------------------------------------------------------------------+
| An alternative procedure for measuring areas:                         |
|                                                                       |
| ***Analyze → Set Measurements***, check \'Limit to Threshold\'. After |
| converting to a binary image,                                         |
|                                                                       |
| *select **Analyze → Measure***                                        |
|                                                                       |
| This procedure is simpler but does not draw an outline of the         |
| measured area.                                                        |
+-----------------------------------------------------------------------+

**(more on reverse side)**

**Examples of Image Analysis Using ImageJ (continued)**

**[Particle Counting and Analysis.]{.underline}**

Problem: Count and determine the size distribution of a collection of
echinoderm embryos.

**(Open embryos image via Select *File → Open Samples → Embryos***)

+-------------------+--------------------------------------------------+
| ![]               |  Draw line over the scale bar and select        |
| (media/image6.jpe | ***Analyze → Set Scale***                        |
| g){width="1.75in" |                                                  |
| h                 | In *Set Scale* window enter 100 into the \'Known |
| eight="1.3125in"} | Distance\' box                                   |
|                   |                                                  |
|                   | and Change the \'Unit of Measurement\' box to um |
|                   | , check \'Global\'                               |
|                   |                                                  |
|                   |  Confirm that the measurement scale is correct. |
+-------------------+--------------------------------------------------+
| ![]               |  Convert the image to grayscale:                |
| (media/image7.jpe |                                                  |
| g){width="1.75in" | ***Image → Type → 8-bit***                       |
| h                 |                                                  |
| eight="1.3125in"} |                                                  |
+-------------------+--------------------------------------------------+
| ![]               |  Threshold the image using the automated        |
| (media/image8.jpe | routine:                                         |
| g){width="1.75in" |                                                  |
| h                 | ***Process → Binary → Make Binary***             |
| eight="1.3125in"} |                                                  |
|                   |  Surround the scale bar with the rectangular    |
|                   | selection tool and                               |
|                   |                                                  |
|                   | clear the contents (***Edit → Clear***)          |
+-------------------+--------------------------------------------------+
| ![](media/image9. |  Analyze Particles:                             |
| jpeg){width="1.63 |                                                  |
| 33333333333333in" | ***Analyze → Analyze Particles***                |
| height="1.225in"} |                                                  |
|                   | Enter 20 as the minimum particle size, toggle    |
|                   | \'Show Outlines\', check                         |
|                   |                                                  |
|                   | 'Display Results', 'Summarize' and 'Record       |
|                   | Stats' and click \'OK\'                          |
|                   |                                                  |
|                   | Twenty five embryos are counted, numbered and    |
|                   | outlined.                                        |
|                   |                                                  |
|                   | The data window lists the area (in um^2^) for    |
|                   | each embryo. These data could be copied to a     |
|                   | spreadsheet.                                     |
+-------------------+--------------------------------------------------+
| **Threshold:      | A summary of the particle count is also shown in |
| 0-0**             | another data window.                             |
|                   |                                                  |
| **Count: 25**     |                                                  |
|                   |                                                  |
| **Total Area:     |                                                  |
| 3177.6 µm\^2**    |                                                  |
|                   |                                                  |
| **Average Size:   |                                                  |
| 127.1 µm\^2**     |                                                  |
|                   |                                                  |
| **Area Fraction:  |                                                  |
| 3.72%**           |                                                  |
+-------------------+--------------------------------------------------+

As mentioned in the previous example, this technique should be manually
validated before collecting experimental data.