Fall 2013
Image Analysis with ImageJ
NOTE: There is an ‘ImageJ’ tutorial video available to step you through
image processing with ImageJ.
Video tutorial
available on D2L
Quantifying changes in the pattern of gene expression is a challenging task, even with the most
sophisticated microscope analysis software available. While it may be easy to qualitatively describe the
change in location of a GFP signal, we will be quantifying this change in lab today through
determining a ratio of nuclear to cytoplasmic GFP expression. To accomplish this task, we will use
ImageJ, a free, public domain Java image-processing program that is commonly used by research
scientists. The image processing protocol is provided below.
1. Open ImageJ through the Departmental Applications folder at the bottom of the computer
screen.
2. The ImageJ toolbar should be visible, but you will have to open each of your images
individually through the ImageJ File Menu bar (File 
Open…).
3. Begin by opening BOTH the light microscopy and epifluorescence microscopy images from
your initial experimental time point.
4. Place the images side-by-side so that you can easily see both images.
Lab 3 – Changes in Gene Expression Patterns with Environmental Stimuli - Protocols 1
Fall 2013
5. Select the epifluorescence image by clicking on the image window once. In the ImageJ file
menu bar, go to Image 
Type, and change the image type from RGB color to 8-bit. Your
image will change from color to black and white.
6. Next, go to Edit 
Invert. This will invert the black and white tones in your image.
7. Before you quantify the GFP expression, you need to select the type of measurement you
would like ImageJ to perform. Go to Analyze 
Set Measurements. Make sure that “Integrated
Density” is the only box checked.
8. To determine a nuclear to cytoplasmic ratio, you will begin the analysis by using the ImageJ
freehand tracing tool to trace a segment of the worm’s body and measure the integrated density
associated with this whole body segment. This measurement will be referred to as the “whole
body segment index.”
Lab 3 – Changes in Gene Expression Patterns with Environmental Stimuli - Protocols 2
Fall 2013
9. Next, click on any area on the image to get rid of the whole body trace that you made in the last
step. Then, draw small circles around the nuclear areas where there is GFP expression. Make
sure to hold down the “shift” key each time you trace an additional nuclear area to measure so
that all nuclei are quantified with a single value. Go to Analyze 
Measure, to measure the
integrated density. In addition to tracing the individual nuclear regions, count the number of
nuclear regions that you circled and add this number to your raw data table. You will subtract
the nuclear integrated density from the whole body segment index to determine a cytoplasmic
index. We will ask you to report the ratio of the nuclear to cytoplasmic GFP expression indices
for each time point in this lab (nuclear index/cytoplasmic index). Remember to average the
ratios obtained from 3 worms at each time point. Note: if you do not see any nuclei “lighting
up” with GFP for a particular time point, then you can subtract zero from the whole body
index to determine a cytoplasmic GFP index. For this scenario, use zero as the nuclear index.
A.
B.
Figure 2. Original color image of a daf-16::GFP worm prior to heat shock (a). An example of a whole
body index in a worm that does not appear to be expressing any nuclear GFP (b).
A.
B.
C.
Figure 3. Original color image of a heat-shocked daf-16::GFP worm after 45 minutes of heat shock (a). Example of a
cytoplasmic measurement (b) and a nuclear measurement (c) in a worm that does appear to be expressing nuclear GFP.
10. The final step in the quantification process is to transfer your whole body segment and nuclear
indices to a Microsoft Excel spreadsheet. We have created a template for you to use for your
raw data, but MAKE SURE TO SAVE YOUR GROUP’S COPY OF THE EXCEL
SPREADSHEET! In Excel, you can use a formula to perform the subtraction of the nuclear
index from the whole body segment index to calculate the cytoplasmic index only. For
Experiment 1, report the nuclear to cytoplasmic ratio for each time point (integrated density of
nuclear GFP/integrated density of cytoplasmic GFP). There are no units for this ratio. You will
need to pool the class data to create a data set for Experiment 2.
Lab 3 – Changes in Gene Expression Patterns with Environmental Stimuli - Protocols 3