Biomedical Engineering Reference
In-Depth Information
picrosirius, bind differentially depending on the tissue properties (physical structures, amino acid com-
position) and the size of the dye molecules. Due to differential binding, trichromes poorly stain base-
ment membranes, reticulum fibers, and thin collagen fibers resulting in an underestimation of collagen
content or lack of structural information [40]. The shortcomings of trichromes should be noted, since
in clinical settings trichromes are primarily used. Polarized picrosirius red, however, is a direct dye that
is collagen specific, while standard H&E stains are not specific for collagen. Picric acid dye molecules
align their long axis (46 Å) parallel to the collagen fibers to enhance the birefringence of collagen in
polarized microscopy. The basic sulfonic groups of lysine, hydroxylysine, and arginine in collagen bind
well with acidic picrosirius red dye molecules. However, quantification studies of collagen must proceed
with caution since the picric acid dye molecule interaction is not simply stoichiometric but depends on
the configuration and substituents of dye molecule, ratio of ionic to nonionic sites and tendency to form
aggregates [41]. Picrosirius red staining methods have been used to study normal and abnormal intesti-
nal walls and vaginal mucosa in research settings [42,43].
Further investigation into picrosirius red and direct comparison with SHG in its ability to detect col-
lagen signatures is a vital component of the potential incorporation of collagen signatures in diagnostic
environments. If picrosirius red is validated, it can provide a cheap and simple method for pathologists
to incorporate collagen signature analysis into their workflow as the importance of collagen in disease
progression is validated and introduced into the clinical setting.
Picrosirius red and SHG both demonstrate the ability to distinguish similar patterns of collagen
deposition in ECM in various stromal components. In Figure 16.4, the imaging techniques are com-
pared inside the stroma of a rather developed mouse mammary tumor. Straightened collagen is visually
detectable with both techniques. Figure 16.5 further illustrates the techniques' ability to distinguish
collagen deposition patterns in early dysplasia. Both picrosirius red and SHG are able to distinguish
between the dense collagen in the tunica adventitia surrounding the blood vessels and collagen com-
posing the tumor stroma and boundary. The collagen signal from the tunica adventitia is equivalent in
FIgurE 16.4
(
See color insert.
) Collagen in stroma imaged by both polarized picrosirius and SHG images all
taken at 40× magnification from Col1a1
tm1Jae
/+ PyVT (+/+) palpable mammary mouse tissue. (a) H/E, (b) polarized
picrosirius red, (c) multiphoton and second harmonic composite, (d) second harmonic. Zoomed in (150%) regions
demonstrate differences in collagen fiber detection between techniques. Scale bars = 10 μm.
