Figure 1.
Birefringent images of skin CBs.
In the papillary layer (P), CBs are distributed parallel to the epithelial surface (arrow) and are thinner than the CBs of the reticular region (R), which are orthogonally distributed. The birefringence brightness originally exhibited by the CBs in the papillary layer in A appears compensated for in black after using Sénarmont’s method in B, provided that these structures are positioned at 45° with respect to the crossed polarizer-analyzer planes. The non-compensated birefringence in B is evident for CBs positioned perpendicularly to the CBs exhibiting birefringence compensation. CBs with helically intertwined and chiral aspects forming circular structures in the reticular region can be identified due to their birefringence characteristics (C, arrow). Different brilliances and hues of gray are also observed in the birefringent images of the CBs depending on their patterns of macromolecular orientation in the dermis (D). Bars = 100 µm (A–C) and 50 µm (D).
Figure 2.
Schematic representation and images of birefringence brilliance meaning.
Representation of the oriented relationship of collagen fibers or CBs with respect to the plane of polarized light (PPL) of the microscope polarizer (X-axis) and analyzer (Y-axis). During examination of the samples, the microscope polarizer and the analyzer remain crossed, while the microscope stage is rotated to detect changes in the birefringence brilliance of the CBs relative to their orientation. The Z-axis represents birefringence brilliance variance in OR, which is represented as 3D information because the birefringence brilliance intensity depends on the CB orientation with respect to the PPL of the crossed polarizer-analyzer [15], [23]. High OR values are obtained in the Z-axis when the long axis of a fiber (for example, a 14.8-µm-thick nylon fiber with maximal internal molecular order) (a) is oriented at 45° (the direction of the white arrow on the black background) with respect to the crossed polarizer-analyzer (X and Y). When the long axis of this fiber is oriented parallel to one of the crossed polarizers (black arrows) by rotation of the microscope stage, birefringence extinction occurs. A CB fiber hypothetically composed of two helical chains is represented (b), such that when the long axis of the fiber is positioned parallel to the polarizers, only the portion of the chains parallel to the polarizer will appear black, whereas the portion of the chains positioned at 45° with respect to the crossed polarizer-analyzer will show birefringence brilliance. Differently oriented collagen birefringent images are also shown (c).
Figure 3.
A model of collagen organization in skin.
An interpretation of how a collagen fiber network is interwoven (A), based on work from Gibson et al. [1– Fig. 12]. The insert in A is a higher magnification of the intertwined fibers, also showing a 3D distribution of the fibers. When this network is stretched in any direction, the fibers become oriented parallel to the stretching direction (B).
Figure 4.
Birefringence of skin CBs observed after rotating the stage of the polarizing microscope.
Although birefringence as a phenomenon is evident in CBs in any given position of the field of observation while rotating the microscope stage from A to I, changes in the birefringent image characteristics are observed. A. Most CBs are positioned at 45° with respect to the polarizers; a circular arrangement of CBs is evident in the center of this image. Such an arrangement is maintained in B through I, despite changes in their birefringence. D. Most CBs appear to be oriented at 45° with respect to the polarizer-analyzer, but they are oriented in the opposite direction with respect to the preceding figures. Following the clockwise rotation of the microscope stage from D-I, changes in the birefringent images continue to be observed. I. Almost all fibers forming the CBs attain a relatively maximal extinction position. The large arrows in the X- and Y-axis directions represent the polarizer and analyzer PPL, respectively. Bar = 100 µm.
Figure 5.
Birefringent images with focal plane changes.
The microscope’s focal plane was sequentially altered from the upper to the lower surface (A to F) of a thick section of skin. The morphological changes observed depend on the track followed by the CBs from one focal position to the next. The large arrows in the X- and Y-axis directions represent the polarizer and analyzer PPL, respectively. Bar = 50 µm.
Figure 6.
Surface plots of birefringent images of skin CBs.
In A and B, false colors were correlated with the birefringence brilliance images shown in Figures 5A and 5F, respectively. The differences in image focal planes due to the paths followed by the CBs from one focal plane to the subsequent deeper one in the same section are represented by different colors. The scale inserted on the left in A and B corresponds with pixel values that are representative of the various false colors. Bar = 50 µm.
Figure 7.
Circular and helical arrangement aspects of skin CBs.
A 3D circular arrangement of CBs is envisioned in a thick skin section. Birefringence compensation is observed in the collagen fibers oriented at 45° with respect to the crossed polarizer-analyzer. The various hues of gray are a function of differences in fiber orientation, resulting in birefringent images that range from complete compensation (black) to non-compensation (brilliance). Bar = 50 µm.
Figure 8.
Birefringence image analysis of skin CBs.
A frequency histogram of birefringence gray average (GA) values expressed in pixels, obtained after the skin sections were immersed in water and the long axes of the skin CBs were positioned at 45° with respect to the crossed polarizer-analyzer. A polydispersed distribution of the GA values is observed.
Figure 9.
Skin CB birefringence varies as a function of the refractive index of the immersion fluids.
Plots of optical retardation (OR) values in nanometers against the refractive index of the different immersion fluids were obtained for the skin CBs in 170-day-old (red line) and 240-day-old (black line) rats. Each point on the curves is the arithmetic mean of 90 OR values from four rats. Error bars are indicated. Asterisks indicate differences significant at P<0.05, as determined by ANOVA, when comparing OR values obtained from the skin of rats of different ages. Bi is identified at the refractive index (1.46) corresponding to the smallest OR obtained. See text for details.