Retinal angiogenesis
We are interested in the hypothesized effects of DEE on blood vessel formation (angiogenesis).
We consider post-natal day 3 (“P3”) retinal cells from mice; some have mutant genes while others are normal.
At this age, the blood vessels are on a single plane (superficial layer) which means we don’t need to worry about complicating 3D effects.
AngioTool
AngioTool, a self-contained program for analyzing blood vessels, will be used to avoid re-inventing the wheel. In this specific case, the NIH also gives away the wheels which have been used in over 100 papers.
AngioTool’s processing chain
We use AngioTool 0.6a, and we are also using a table and figure from their paper in this section.
- Multi-scale second order local structure (Hessian)
- Second order derivatives through convolutions with derivatives of Gaussians
- Computationally favorable
- Introduces a scale factor that matches vessel size
- Map the eigenvectors of the Hessian onto an ellipsoid where the axis’ semi-lengths are magnitudes of respective eigenvalues
- Magnitudes of eigenvalues describe structure
- Magnitudes of eigenvalues describe structure
- Second order derivatives through convolutions with derivatives of Gaussians
- Convex Hull
- Given a binary image, the convex hull is the set of pixels contained within the smallest convex polygon that will surround all of the foreground pixels.
- Used for calculating area
- Skeletonization
- Given a binary image, skeletonization yields a frame which is 1 pixel-wide.
- Used for calculating vessel lengths
AngioTool outputs values such as
I was handed unlabelled data (i.e. I was blinded) and told to tweak the knobs and dials of AngioTool.
- Vessel Thickness
- Values for Vessel Thickness varied between 8 and 15.
- The difference in wild type and mutant is most reflected here.
- Small Particle Size
- Setting this to a high value such as 3000 was occasionally necessary for removing “islands” in the background.
- Fill Holes
- Frequently set to 0 due to being unncessary.
We obtain images such as
where the features are
| Blue dots | Red lines | Yellow outlines |
|---|---|---|
| Vessel junction | Vessel skeleton | Vessel area |
and the AngioTool results are saved in an Excel file.
At this point, we can dive into our Jupyter Notebook titled RetinalCells-PaireTtest.ipynb
Misc. info
GSL I-B4 isolectin was used as a marker for endothelial cells.