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Measuring Regeneration in ImageJ

This document provides instructions for using ImageJ to quantify regeneration in planarians by measuring the area of regenerated tissue (blastema) relative to total animal area. To control for animal size variability, ratios of blastema to total area will be calculated for individual animals, averaged within experimental groups, and compared statistically. The practice assignment involves setting the scale in ImageJ, taking triplicate measurements of total and blastema areas for provided images, and adding scale bars for presentation. Comparing the average area ratios across experimental groups will allow evaluation of whether regeneration is significantly different.

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131 views5 pages

Measuring Regeneration in ImageJ

This document provides instructions for using ImageJ to quantify regeneration in planarians by measuring the area of regenerated tissue (blastema) relative to total animal area. To control for animal size variability, ratios of blastema to total area will be calculated for individual animals, averaged within experimental groups, and compared statistically. The practice assignment involves setting the scale in ImageJ, taking triplicate measurements of total and blastema areas for provided images, and adding scale bars for presentation. Comparing the average area ratios across experimental groups will allow evaluation of whether regeneration is significantly different.

Uploaded by

Empowering Youth
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© © All Rights Reserved
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Taking measurements in ImageJ for regeneration:

Once you have delivered RNAi and amputated the animals, you will begin collecting
quantitative data to test your hypothesis on regeneration. Our goal is to measure the tissue
that is new regeneration. However, each animal is a different size and we need to control for
this variability. You can assume that bigger animals will have bigger blastemas (newly generated
tissue) because they have more mass with which to generate new cells. Therefore, we will
calculate a ratio of the blastema area to the total animal area. This will be discussed further in
class with the practice images.

Overall steps:
1) Measure the total area of the first animal 3X in the negative control group.
2) Measure the area of the blastema (non-pigmented tissue where you amputated) 3X for
that same animal.
3) Calculate the average for the three total area of the animal values.
4) Calculate the average for the three blastema area values.
5) Use the averages to create the ratio of new growth area:total area.
Blastema Area Average
Total Area Average
6) Repeat steps 1-5 for each animal in the negative control group. (You should have ~12
animals)
7) Calculate the overall average for the ratio values in the negative control group.
8) Repeat steps 1-8 for the positive control group and your gene of interest group.
9) We will compare the average ratios for each group and generate statistical values to
determine if the groups are significantly different for regeneration.

Notes to work efficiently: It will be best to complete steps 1-2 in ImageJ for your first animal
then copy the area measurements into your Excel spreadsheet then save after each animal. This
way if your program accidentally closes or you get distracted, you will have all your work
transferred and saved. Once steps 1 and 2 are complete on all the animals, you will use the
values to complete the remaining steps.

An excerpt from the master spreadsheet is shown below for the Negative Control Group. You
will be copying your measurements from ImageJ into the pre-formatted Excel sheet.

1
Practice measurement assignment
In Blackboard, there are two planarian images (a negative control and gene of interest) and a
ruler to use for practice. This is obviously not a full sample set. You will have a total of 36
animals to measure (or less dependent on the positive control). We will use the images on
Blackboard to practice taking measurements in triplicate, add scale bars to images and become
familiar with the ImageJ software. There are 5 places where you will complete a task during this
practice exercise and enter a screenshot or measurement into this document.

A. Setting the scale and measuring areas

1) Follow the steps in the ImageJ instructions (Posted on blackboard). Due to the size of
planarians, it is best to have the scale set in mm rather than cm.

Take a screen shot of the window for setting the scale and paste it here. This should
show how many pixels/mm.

2) Complete steps 1 (measure total area) and 2 (measure blastema) from page 1 for the
negative control image (NC1). Copy your measurements here. You should have 6 total
measurements (3X total area and 3X blastema).
Total area

2
Blastomere area

3) Complete steps 1 (measure total area) and 2 (measure blastema) from page 1 for the
gene image (G1). Copy your measurements here. You should have 6 total
measurements.

Total area

3
Blastomere area

Note: We will learn how to calculate the averages and ratios in Excel during a future lecture,
which will result in a completed chart similar to the image on page 1.

B. Adding a scale bar to images for presentation or publication:

4) Insert a cropped image of the Negative Control with a scale bar set to 0.5 mm. Position the scale
bar in the lower right corner and use black text.

4
5) Insert a cropped image of the Positive Control with a scale bar set to 1 mm. Position the scale
bar in the lower left corner and use white text.

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