Comet Assay Analysis
this example as a Mathematica notebook. |
The single-cell gel electrophoresis (or comet) assay is a rapid and
sensitive fluorescence microscopic method for the detection of primary DNA
damage at the individual cell level. During the last 10 years, it has gained widespread
acceptance for genotoxicity testing. Compared to other genotoxicity tests, the
comet assay is an inexpensive and simple method that is well suited to analysis and
measurement by means of computer-assisted techniques [Christoph Helma and
Maria Uhl, Introduction in "A Public Domain Image Analysis Program for the Single
Cell Gel Electrophoresis (Comet) Assay," Mutation Research 466 (2000) 9-15].
for additional information and references.
This loads the package.
Here an example image is loaded (data the courtesy of Blake Whitaker, University of
This defines the regions of interest as user-selected rectangular areas in the
graphical coordinate system of the Mathematica front end. The coordinate
pairs represent the lower-left and upper-right vertices of the bounding rectangles
for each of six regions in the image.
In order to draw the regions of interest on the original image, define a utility function
that takes two vertices and returns a Line
graphics primitive depicting the bounding rectangle.
Here is a the original image data showing the regions of interest.
Proceed with extracting the comets from the original image.
This shows the individual comets.
The original images are in three-channel RGB format. Clearly the green channel signal
is strongly dominant, which is easily verified by displaying the planar format of an example comet.
A closer examination reveals the presence of weak signals in the red and blue channels,
especially in the tail section of the comet (interestingly, this effect is not discussed
in the scientific literature). Since all computer-aided quantification results are
traditionally presented in the context of gray-scale images, proceed with a color
This displays the histograms of the example comet images.
Next is a typical calculation. For example, the interest here is to obtain
the area of the head of the comet ("AH"), the total density of the head
("DNAH"), and the mean intensity of the head ("MH").
Usually, the same set of measurements is obtained for the tail, and the two results
are contrasted. In the literature on comet assays, the head is identified by the pixels
with the highest intensity. As is commonly used, the threshold value is 85 percent of
the maximum intensity in the image.
The images are binarized, adding a morphological filtering operation to smooth the
borders of the head regions.
This continues with identifying the regions and measuring the required properties,
additionally including calculations of the centroids and enclosing circles.
This displays the results in a tabular fashion.
The graphical elements used to annotate the comet images are calculated.
This is an alternative visualization of a comet image.
Finally, another property of a comet, the so-called comet profile, is calculated and displayed.
It is a plot of the cross-sectional density (density measured perpendicularly to the
lengthwise dimension of the comet).