Table 1.
Scoring parameters for the collagen assessment in histological sections.
Figure 1.
Schematic layout of the experimental setup for recording in vivo autofluorescence and microscopic studies. Typical overlaid ex vivo and in vivo autofluorescence spectra of granulation tissue at 325 nm excitation with peaks for collagen (A) & NADH (B) shown in the inset.
Figure 2.
Autofluorescence patterns with varying age and gender.
Influence of age on autofluorescence pattern of collagen in un-wounded skin (A). Average collagen intensity was plotted against different age group (in weeks). Collagen intensity values with respect to normalized NADH levels for male and female animals of un-wounded control (B), un-illuminated control (C), and optimum laser dose treated group (D) on different post-wounding days.
Figure 3.
Typical in vivo autofluorescence spectra.
Typical normalized in vivo autofluorescence spectra of un-illuminated control, unwounded control and 2 J/cm2 treated animals on day 0 (A), day 5 (B), day 10 (C), day 30 (D), day 45(E), and day 60 (F) post-wounding. Inset- Comparision of the spectral range 390–405 nm indicating the collagen peak of the all the experimental group animals.
Figure 4.
Collagen intensity and the corresponding area under the curve (AUC) values from in vivo granulation tissue/skin autofluorescence.
Collagen intensity with respect to normalized NADH values on days 0, 5, 10, 30, 45 and 60 post-wounding for un-wounded control, un-illuminated control and optimum laser treated animals indicating the increased levels of collagen in the laser treated group (A). The AUC values for un-wounded control, un-illuminated control and optimum laser treated animals were also indicating the elevated levels of collagen in the test group (B). Level of significance bP<0.01, aP<0.001 and no symbol = insignificant compared to un-illuminated control.
Figure 5.
Collagen birefringence under polarized light microscopy.
Histological sections of the un-wounded control, un-illuminated control, and 2 J/cm2 treatment group on day 5, 10, 30 post-wounding stained with a direct red visualized by the polarized microscope (10X). Collagen birefringence pattern of un-wounded skin (A, B, C,), Un-illuminated control (D, E, F) and optimal laser treated group (G, H, I) on days 5, 10, 30 post-wounding respectively. A skin section with hair follicles (A, B, C), Loosely formed early collagen (D), moderate collagen (E), birefringent yellow/red fibers (F), Moderately birefringent red/yellow fibers (G), thick strongly birefringent yellow/red fibers (H) and strongly birefringent thick red fibers (I) as indicated by arrows. Dotted arrow indicates epidemis/epidermal layer and solid arrows indicate the occurrence of collagen.
Table 2.
Qualitative scoring for the collagen assessment in histological sections of Un-wounded control.
Table 3.
Qualitative scoring for the collagen assessment in histological sections of Un-illuminated control.
Table 4.
Qualitative scoring for the collagen assessment in histological sections of optimum laser treated group.
Figure 6.
Comparison of the change in collagen levels measured by autofluorescence and polarized microscopy (image analysis).
Bar graph indicates the total collagen (% scores) derived from image analysis of Picro-Sirius red stained histological sections. Total amount of collagen for each group was computed by adding the type I (yellow and red fibers) and type III (green fibers) together. Scatter plot indicates collagen intensity with respect to normalized NADH levels. Results were represented as mean ±SEM. Level of significance bP<0.05, aP<0.001 and no symbol = nonsignificant compared to un-illuminated control.