1. Gel Diffusion Image Analysis Neil Forbes Nanotechnology Institute 2007

2. Software Download ImageJ –Install files is available at: http://rsb.info.nih.gov/ij/http://rsb.info.nih.gov/ij/ –Follow the Downloads link –Downloads are available for Windows, Macintosh and Linux Download Radial profiler –http://rsb.info.nih.gov/ij/plugins/radial-profile.htmlhttp://rsb.info.nih.gov/ij/plugins/radial-profile.html –Or in ImageJ: Help ImageJ Website Plugins Radial Profile Plot Radial Profile Plot –Download the plugin to the plugins folder in Image Program File –Restart ImageJ

3. Image Analysis Three methods from simplest to more complicated Method 1: –Measure the dye penetration distance each day using a ruler –Use graph paper to plot distance vs. time –The rate is the slope of the line

4. Image Analysis Method 2: –Display all of the images on the computer –Distances in the images need to be calibrated –Use the line function to measure the dye penetration distance (D) in pixels –Use a ruler to measure the width of the gel (W) in pixels, which we know is 60mm –The penetration distance is: –Plot the distance vs. time

5. Measurement by Hand

6. Image Analysis Method 3: –Open all of the images –Split into (R)ed, (G)reen and (B)lue images (RGB Split) –Pick the image with the most information –Draw a square box around the gel –Apply Radial profile from the plugins menu –Save the profile

7. Image Analysis Method 3 (cont): –Open all of the profiles in a spreadsheet (Excel) –Normalize the intensity: –Find the pixel that crosses 0.5 –Divide by the total number of pixels and multiply by 30 mm (the gel radius) –Plot distance vs. time

8. Software Analysis

9. Comparison – Pretty good! The dye penetrates at approximately 200 μm/hr

10. Image Analysis Finally, if the distance is normalized, complete distance vs. time plots can be generated. These compare nicely to theoretical predictions.