Quantitative differentiation of normal and scarred tissues using second-harmonic generation microscopy.
The aim of this study was to differentiate normal and scarred hamster cheek pouch samples by applying a quantitative image analysis technique for determining collagen fiber direction and density in second-harmonic generation microscopy images. This paper presents a collagen tissue analysis of scarred cheek pouches of four adult male Golden Syrian hamsters as an animal model for vocal fold scarring. One cheek pouch was scarred using an electrocautery unit and the other cheek was used as a control for each hamster. A home-built upright microscope and a compact ultrafast fiber laser were used to acquire depth resolved epi-collected second-harmonic generation images of collagen fibers. To quantify the average fiber direction and fiber density in each image, we applied two-dimensional Fourier analysis and intensity thresholding at five different locations for each control and scarred tissue sample, respectively. The resultant depth-resolved average fiber direction variance for scarred hamster cheek pouches (0.61 ± 0.03) was significantly lower (p < 0.05) than control tissue (0.73 ± 0.04), indicating increased fiber alignment within the scar. Depth-resolved average voxel density measurements indicated scarred tissues contained greater (p < 0.005) fiber density (0.72 ± 0.09) compared to controls (0.18 ± 0.03). In the present study, image analysis of both fiber alignment and density from depth-resolved second-harmonic generation images in epi-detection mode enabled the quantification of the increased collagen fiber deposition and alignment typically observed in fibrosis. The epi-detection geometry is the only viable method for in vivo imaging as well as imaging thick turbid tissues. These quantitative endpoints, clearly differentiating between control and scarred hamster cheek pouches, provide an objective means to characterize the extent of vocal fold scarring in vivo in preclinical and clinical research. In particular, this non-invasive method offers advantages for monitoring scar treatments in live animals and following the effects of scarring-related treatments such as application of steroids or drugs targeting pathways involved in fibrosis. SCANNING 38:684-693, 2016. © 2016 Wiley Periodicals, Inc.
Department of Mechanical Engineering, The University of Texas at Austin, Austin, Texas.
Murat
M
Yildirim
Department of Biomedical Engineering, Tufts University, Medford, Massachusetts.|Department of Biomedical Engineering, University of Arkansas, Fayetteville, Arkansas.
Kyle P
KP
Quinn
Department of Surgery, Harvard Medical School, Center for Laryngeal Surgery and Voice Rehabilitation, Massachusetts General Hospital, Boston, Massachusetts.
James B
JB
Kobler
Department of Surgery, Harvard Medical School, Center for Laryngeal Surgery and Voice Rehabilitation, Massachusetts General Hospital, Boston, Massachusetts.
Steven M
SM
Zeitels
Department of Biomedical Engineering, Tufts University, Medford, Massachusetts.
Irene
I
Georgakoudi
Department of Mechanical Engineering, The University of Texas at Austin, Austin, Texas.|Department of Biomedical Engineering, The University of Texas at Austin, Austin, Texas.
Adela
A
Ben-Yakar
D000818:Animals; D002610:Cheek; D002921:Cicatrix / Q000473:pathology*; D003094:Collagen / Q000032:analysis; D006224:Cricetinae; D008297:Male; D008647:Mesocricetus; D008853:Microscopy / Q000379:methods*
D003094:Collagen
Fourier transform; collagen fiber alignment; collagen fiber density; hamster cheek pouch; image analysis; scar remodeling; second-harmonic generation microscopy; ultrafast fiber lasers; vocal fold scarring