gel diffusion image analysis neil forbes nanotechnology institute 2007
TRANSCRIPT
Gel Diffusion Image Analysis
Neil Forbes
Nanotechnology Institute 2007
Software
• Download ImageJ– Install files is available at: 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.html – Or in ImageJ: Help ImageJ Website Plugins
Radial Profile Plot– Download the plugin to the plugins folder in Image
Program File– Restart ImageJ
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
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
60Distance
mmD
W
Measurement by Hand
y = 0.2057x + 1.0715
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Time
Dis
tan
ce
(m
m)
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
Image Analysis
• Method 3 (con’t): – 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
minnorm
max min
I - II =
I - I
Software Analysis
y = 0.2106x + 1.7912
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Time (Hours)
Dis
tan
ce fr
om
Ed
ge
(m
m)
Comparison – Pretty good!
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Time (hours)
Dis
tan
ce (
mm
)
By Hand
Image Analysis
The dye penetrates at approximately 200 μm/hr
Image Analysis• Finally, if the distance is normalized, complete
distance vs. time plots can be generated. These compare nicely to theoretical predictions.
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Distance
Co
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Start
3 Hours
8 Hours
24 Hours
36 Hours
48 Hours
60 Hours
72 Hours