Quantitative differentiation of normal and scarred tissues using second‐harmonic generation microscopy

Abstract

Summary: 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.

Quantification of collagen fiber structure using second harmonic generation imaging and two‐dimensional discrete Fourier transform analysis: Application to the human optic nerve head

“…The angular distribution of the integrated DFT power spectrum is then extracted and used to calculate and map the preferred orientation of the collagen fibers, the aligned fiber content and the degree of fiber recruitment around the main direction. The method also features an in‐built correction for tile edge artefact that adopts the PPSID algorithm, avoiding the significant loss of peripheral image information encountered with commonly used alternative windowing correction methods , and resulting in comparatively minimal distortion of the resulting calculated fiber distribution. The latter issue has been largely overlooked in previous studies that utilize DFT to quantify fiber organization from SHG images.…”

“…Because regular images are not periodic, the discontinuity across the frame edge introduces a centrally positioned cross‐shaped artefact in the frequency domain . To correct this, the use of an apodization windowing function to enforce a smooth transition at the image borders has been widely adopted . A Hanning window is a commonly utilized method, which is a cosine‐based function that smoothly reduces the signal to zero at the image edges, and is given by: …”

“…Notably, this approach has been found to be superior in detecting directionality of fibrillar structures when compared with other techniques such as mean intercept length and line fraction deviation methods . Several approaches have been adopted to calculate the dominant fiber orientation and anisotropy from DFT patterns, the most common being ellipse fitting to the power spectrum , and to extract the magnitude of the frequency components as an angular fiber distribution .…”

“…Since the early 2000s second harmonic generation (SHG) microscopy has evolved as a useful imaging tool for imaging the suprafibrillar assembly of the collagen network in various connective tissues, including skin , tendon , muscle , cornea , sclera and cardiovascular tissue . It has also been successful in detecting and quantifying the effects of various deleterious connective tissue conditions and insults, such as corneal photothermal damage , keratoconus , heat burns , matrix remodelling in tumor microenvironments , as well as tissue fibrosis and scarring .…”

Bioluminescence and second harmonic generation imaging reveal dynamic changes in the inflammatory and collagen landscape in early osteoarthritis

“…For SHG imaging, photons emitted at 400 ± 10 nm were detected using a non-descanned photomultiplier tube detector. Endogenous TPEF emitted by chondrocytes was detected at 525 ± 25 nm [48][49][50][51][52]. A water-immersion 63x objective (NA 1.2; 220 μm working distance) was used for articular cartilage imaging.…”