Geometry-Dependent Spectroscopic Contrast in Deep Tissues

Abstract

Nano-structures of biological systems can produce diverse spectroscopic effects through interactions with broadband light. Although structured coloration at the surface has been extensively studied, natural spectroscopic contrasts in deep tissues are poorly understood, which may carry valuable information for evaluating the anatomy and function of biological systems. Here we investigated the spectroscopic characteristics of an important geometry in deep tissues at the nanometer scale: packed nano-cylinders, in the near-infrared window, numerically predicted and experimentally proved that transversely oriented and regularly arranged nano-cylinders could selectively backscatter light of the long wavelengths. Notably, we found that the spectroscopic contrast of nanoscale fibrous structures was sensitive to the pressure load, possibly owing to the changes in the orientation, the degree of alignment, and the spacing. To explore the underlying physical basis, we further developed an analytical model based on the radial distribution function in terms of their radius, refractive index, and spatial distribution.

Keywords: Infrared Optics; Medical Imaging; Optical Imaging; Spectroscopy.

Graphical abstract

Figure 1

FDTD Simulation Reveals Characteristic Spectroscopic Contrast of Motile Cilia (A) Schematic of light-tissue interactions in the respiratory mucosa. Color bar: light red and dark red color stand for short and long wavelengths in 700–950 nm, respectively. (B) Backscattered spectra of transversely oriented and regularly arranged nano-cylinders simulating the apical portions of motile cilia at the recovery stroke. These transversely oriented and regularly arranged nano-cylinders were generated by the same strategy but the different seeds. (C) Backscattered spectra of nano-spheres of the same diameter and refractive index as the cylinders in (B). Each curve in (B) and (C) was normalized to its maximum intensity. Curves with the spectral centroid shift toward the long wavelengths and short wavelengths were denoted with green and red color, respectively. Those without significant shift were denoted with orange color. λ_c is the center of gravity of the backscattered spectrum, and 825 nm is the center of gravity of the input light spectrum.

Figure 2

Geometry-Dependent Spectroscopic Contrast Images of Motile Cilia In Vitro and In Vivo (A) A representative OCT intensity image of motile cilia in HBE culture. (B) The corresponding spectral centroid shift image. (C) The corresponding HSV-mapped SOCT image. PBS, phosphate-buffered saline; EP, human bronchial epithelial monolayer; M, membrane of the Transwell insert; eff, effective stroke; re, recovery stroke. (D) A representative OCT intensity image of cultured sheep tracheal mucosa with functional mucociliary clearance in situ. Red arrow stands for the direction of the effective stroke. Note that the epithelium folded in the left side, so that the epithelial surface was parallel to the image plane, which “cut” through motile cilia perpendicularly. The 3D spatial relation between the image plane and the folded epithelium is explained in Figures S3B and S3C. (E) and (F) The corresponding spectral centroid shift image and HSV-mapped SOCT image, respectively. EP, epithelium; LP, lamina propria; M, mucus flow. The mark at 825 nm in the gray scale bars refers to the centroid of the input spectrum. The green hue indicates the long-wavelength shift in NIR, whereas the red hue represents shifts toward the short wavelength for all HSV-mapped images in this paper. Scale bars: 10 μm.

Figure 3

Geometry-Dependent Spectroscopic Scattering of Collagen Fibrils (A) FDTD predictions of backscattered spectra from densely packed well-aligned nano-cylinders simulating collagen fibrils in lamina cribrosa and sclera. (B) Backscattered spectra from mis-aligned nano-cylinders with the same diameter and refractive index as those in (A). Each spectrum was from a distinct spatial arrangement of cylinders generated using the same strategy. (C) A representative intensity cross-sectional image of swine optic nerve head. (D–F) Corresponding spectral centroid shift image (D), HSV-mapped SOCT image (E), and Masson's trichrome stained histology image (F), respectively. The spectroscopic artifacts at the top of (D) and (E) were the ghost image due to the autocorrelation artifacts of OCT signal. The cyan dashed boxes in (E) and (F) refer to the same region. LCR, lamina cribrosa; S, sclera; R, retina; PLR, prelaminar region; R, retina. Scale bars: 100 μm.

Figure 4

Geometry-Dependent Spectroscopic Contrast Is Negated by Thermal Denaturing of Collagen Fibrils Ex Vivo (A–F) (A–C) are the representative intensity image, spectral centroid shift image, and HSV-mapped SOCT image from the normal control mouse ear, respectively, whereas (D–F) are those of the burned mouse ear, respectively. EP, epidermis; D, dermis; AC, auricular cartilage. Scale bars: 100 μm.

Figure 5

Changes of the Spectroscopic Contrast of the Swine Coronary Arterial Wall during Pressure Loading (A) Representative cross-sectional intensity image acquired through the pericardium at the intracoronary hydrostatic pressure of 44.1 mm Hg. (B) Intensity image acquired in the same spot at the intracoronary hydrostatic pressure of 117.7 mm Hg. The bright interface at the bottom in (A) was from the lower surface of an air bubble, which moved out of the recorded view in (B) at high pressure (Video S4). (C) Spectral centroid shift image corresponding to (A). (D) Spectral centroid shift image corresponding to (B). FP, fibrous pericardium; SP, serous pericardium; A, adventitia; M, media; I, intima. Scale bars: 100 μm. (E) Dynamic correlation between the relative spectral centroid shift of the tunica media (blue line) and the media thickness reflecting the intracoronary pressure (red line). (F) Spectral curves at the selected time points. Spectral centroid λ_c in (E and F) is extracted from the nonframe-averaged spectral centroid shift images.