Experiment 2

Measurement using Image Analysis

Introduction to ImageJ for the Life Sciences

Objectives:
At the end of the activity, the students should be able to:
• use the basic features of ImageJ for area measurement of complex object and for cell counting
• determine the photosynthetic portion of a variegated leaf
• determine the total number of cells in a photo using ImageJ
• recognize the role of basic image analysis as a tool in the life sciences

Introduction:
ImageJ is a freely available, public domain Java image processing program developed by National Institute
of Health (NIH) and the Laboratory for Optical and Computational Instrumentation. It can display, edit,
analyze, process, save and print 8-bit, 16-bit and 32-bit images. It can calculate area and pixel value
statistics of user-defined selections.
This has become a standard tool in many laboratories around the world because it is free, open source, and
very well supported. The software can be downloaded in this link:
https://imagej.nih.gov/ij/download.html.
Parts of ImageJ
Status Bar – gives information such as description of tools, grayscale & RGB pixel values and X, Y pixel
coordinates.

Tools – there are several drawing tools on the LHS of the toolbar. Red arrows shown in the figure indicate
more tools/options which are available whether by right or left hand mouse click or double-clicking.
Selections or Region of Interest (ROI): Drawing tools
Most commands in ImageJ will work on the image region which you need to select or segment. You can
select the whole image you are going to Edit-Selection-Select All.
You can define a specific region of interest (ROI) within the image using any one of the region selection
tools in the Menu toolbar (rectangular, oval, polygonal and freehand). To remove a selection, just click
the mouse outside the ROI.

Materials:
o ImageJ Software
o Sample Images

I. Determination of the photosynthetic portion of a variegated leaf

Physics 61.1 Physics and Geology Unit, DPSM, CAS, UP Manila

Variegated leaf – is a leaf which has both green and non-green parts. The green parts contain chlorophyll,
but the non-green parts do not contain chlorophyll, so they cannot photosynthesize.
Procedure
Open leaf image via Select File -> Open Samples -> Leaf
1. Convert scanned color image of leaf to grayscale: Image -> Type -> 8-bit
2. Set measurement scale. 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’
3. Draw a new line and confirm that the measurement scale is correct.
4. Threshold the leaf image using the automated routine: Process -> Binary -> Make Binary. Make
sure that the leaf and ruler is black and the background is white. Uncheck the ‘black background’
in the binary options if your background is black.
5. Calculate area of green portions:
Surround the leaf with the rectangular selection tool
Analyze -> Analyze Particles
Enter 50 as the minimum particle size, toggle ‘Show Outlines’, check ‘Display Results’ and click
‘OK’
6. Outline of analyzed area will be drawn. Data window gives an area of about 2000 mm2
depending on the calibration setting.

Output: Area of the photosynthetic region

II. Automated Cell Counting

Sometimes counting by hand is just not practical, such as when there are many cells per image or you
have many images to process. Counting can be automated, although depending on your image, this can
get to be a complicated process. The example below details a simple count from a single color
fluorescence image.
Procedure
1) Open the image to be counted (File-> Open Sample-> blobs(25k)). If it is a color image (RGB), as in
our example above, it will have to be converted to greyscale before proceeding. Check that you have set
Edit -> Options -> Conversions to “scale when converting.” Then use Image -> Type -> 16-bit to convert
to greyscale.
2) Once the image is in greyscale (8-bit or 16-bit) use Image -> Adjust -> Threshold (Cntl + Shift + T)
to highlight all of the structures you want to count. To highlight, either use the sliders or use the “set”

Physics 61.1 Physics and Geology Unit, DPSM, CAS, UP Manila

button to type in a known range of pixel intensities (if you want to threshold a whole set of images the
same way, for instance).
(Optional) Some particles may be touching already, shown here, or they may run together during the
threshold. This is somewhat fixable. Process -> Subtract background with rolling ball may help if you
find you are highlighting too many “noise” or background pixels. Once you have the area highlighted as
well as you can, click “apply.” This will create a binary version of the image with only two pixel
intensities: black = 0 and white = 255.

3) If you have particles that have merged together, Process -> Binary -> Watershed can often (but not
always) accurately cut them apart by adding a 1 pixel thick line where it feels the division should be.
The example at right has been thresholded, turned into a binary image with “apply” and then run through
the watershed program. For more information on other binary image tools, such as fill holes, see the
Menu Commands section of the ImageJ Documentation page at http://rsbweb.nih.gov/ij/docs/index.html.
4) Once you have a binary image of the particles you wish to count, go to Analyze -> Analyze Particles.
There are some choices here that can effect the counts from your images. Size will effect what size
particles to count. It will either be in pixels, or, if your image is calibrated, in a unit of measurement^2
(check under Image -> Properties (Ctrl + Shift + P). To count all particles, leave it at the default of 0 –
Infinity. If you are getting too many small “noise” pixels counted as pixels, or you want to exclude particles
based on size, adjust these numbers. Circularity excludes particles based on how close to perfectly round
they are. To include everything, keep at the default 0.00 – 1.00. To exclude things, adjust these numbers,
keeping in mind that 1.00 is a perfect circle and 0.00 is a straight line.
Data Sheets:
I. Determination of the photosynthetic portion of a variegated leaf
1. Attached a print screen of your final output. What is the area calculated by the software?
2. What are the other possible applications of this method of measurement?
II. Automated Cell Counting
1. What is the total cell count generated? Explain briefly how the software identified this count.
2. What is the average size of the cell measured?

References:
https://imagej.nih.gov/ij/docs/intro.html
Labno C. Two Ways to Count Cells with ImageJ. Retrieved from
unige.ch/medecine/bioimaging/files/3714/1208/5964/CellCounting.pdf

Physics 61.1 Physics and Geology Unit, DPSM, CAS, UP Manila