I have done this using polarized light with Image pro
plus. Also here is a brief procedure of what I did, I was trying to
quantitate collagen in mouse disease lungs. You should remember if you are
trying to quantitate this let your scope warm up for 30 minutes prior to
capturing your images and keep all light settings (levels) / image size
the same. Your lamp temperature as it warms can give you different reading
(IOD) if not warmed up. I used a polarizer that screwed into to my scope
so I could rotate it to the right degree (I think 45) and it sits in place
so as not to move. I also did not use any NDF filters and I used the mouse
Kidney as positive control.
Here is a reference that I followed.
1. Gaoyun Yan, 2005 Therapeutic Dosing of anti-IL-13 Monoclonal Antibody
inhibits Asthma Progression in Mice. Journal of Pharmacology and
Experimental Therapeutics. 313: 8-15.
2. Juqueira LC, Picrosirius staining plus polarization microscopy, a
specific method for collagen detection in tissue sections. J.
Histochemistry 11(4) 447-55, 1979.
3. Histonet search:
picrosirius red (PSR) (EMS, Cat #26357) for Collagen deposition.
Picosirious Red Imaging
Slides where stained, imaged and saved as stated above. Images for PSR
collagen deposition where photographed under polarized light microscopy,
which yields a yellow, red and green birefringence of collagens type I,
II, III (Appendix 3). Background non-specific birefringence is removed as
stated above. Images are converted from RGB color images to HSI images and
the Intensity channel is used for analysis. The images are calibrated and
Threshholded on the red and yellow collagen fibers which represent mostly
the type I fibers. These areas are measured and counted using the
integrated optical density (IOD) feature, which represents the area
multiplied by the average intensity. This measurement is then exported to
excel and the two images for each sample are averaged to arrive at the
average intensity for the total sample.
Hope it helps.
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