Fourier transform-second-harmonic generation imaging of biological tissues

Abstract

Fourier transform-second-harmonic generation imaging is employed to obtain quantitative metrics of collagen fibers in biological tissues. In particular, the preferred orientation and maximum spatial frequency of collagen fibers for selected regions of interest in porcine trachea, ear, and cornea are determined. These metrics remain consistent when applied to collagen fibers in the ear, which can be expected from observation. Collagen fibers in the trachea are more random with large standard deviations in orientation, and large variations in maximum spatial frequency. In addition, these metrics are used to investigate structural changes through a 3D stack of the cornea. This technique can be used as a quantitative marker to assess the structure of collagen fibers that may change due to damage from disease or physical injury.

Application of quantitative second-harmonic generation microscopy to dynamic conditions

“…Here we use 10 mW of average power, 1.2 s acquisition time, and 512 x 512 pixel area comprising an 8 x 8 grid (and thus each grid contains 64 pixels). Details on 2D quantitative-SHG analysis to calculate localized fiber orientation can be found elsewhere [15]. The processing times varied from 300 to 800 ms depending on the density of fibers.…”

“…An FFT spectrum of the ROI is also displayed [ Fig. 1(b), bottom right corner], which can be used to directly determine preferred fiber orientation [15].…”

“…Further, the push to identify useful quantitative metrics for this imaging modality has resulted in several approaches [9][10][11][12][13][14]. For example, in recent years, Fourier transform-second harmonic generation imaging (FT-SHG) has been developed, whereby either 2D or 3D spatial-frequency analysis is used to quantify the spatial organization of structures at cellular scales (300 nm-100 µm), particularly type I collagen fibers [15][16][17][18][19][20][21]. Indeed, using measures such as preferred orientation (of collagen fibers) and orientation anisotropy, FT-SHG has been utilized to differentiate between healthy and injured tendon [17], assess age-related changes in cortical bone [18], quantify dissimilarities in stromal collagen fiber organization in human breast biopsies at different pathological stages [19], and to quantify the 3D spatial arrangement of collagen fibers, in general, and especially in the cervix [20,21].…”

Effect of recombinant interleukin-12 on murine skin regeneration and cell dynamics using in vivo multimodal microscopy

“…The weaker SHG signals observed in the wound bed suggested that there was a lower type I collagen density since type I collagen gives stronger SHG signals [12]. To evaluate the change in collagen structure during the process of wound healing, five small areas around the wound bed on each SHG image of each animal were manually selected, and an ellipse-fit was performed on the magnitude of the 2D Fourier transform of each small image area as described in Section 2.5 [16]. The eccentricity value of the ellipse was used to evaluate the preferred orientation of the collagen fibers immediately around the wound bed, which can be an implication of collagen contraction since the collagen fibers are stretched toward the center of the wound during the contraction process to achieve wound closure [5,7].…”

Local assessment of myelin health in a multiple sclerosis mouse model using a 2D Fourier transform approach

“…Recently, researchers have characterized the local tissue architecture of collagen imaged with second harmonic microscopy by using a two-dimensional Fourier transform (2D-FT) method [19][20][21]. More specifically, the average fiber orientation as well as the directional anisotropy within the image were extracted from the 2D-FT and then used to calculate the correlation between the orientations of neighboring domains [22].…”