Geometry-Dependent Spectroscopic Contrast in Deep Tissues

摘要

class="head no_bottom_margin" id="sec1title">IntroductionIn contrast to that produced by selective absorption, the spectral modulation of light reflected or scattered by a non-luminous object is mainly due to elastic interactions between the input light and micro- and nano-structures. This spectral modulation may bring about spectroscopic contrasts manifested as spectral centroid shifts in the back-scattered light fields. Since Robert Hooke and Isaac Newton revealed the basis of structural coloration (, ), fascinating colors created by natural structures have attracted considerable research interests (, ). In biology, structural colors are commonly observed under sunlight or white light on the surfaces of animals and plants (, , , ) (skin, feathers, flowers, and epicarp). However, as turbid tissues are opaque to visible light, the direct observation of structural colors originating from beneath biological tissues is extremely difficult.The spectroscopic properties of deep biological tissues are commonly investigated within the near-infrared (NIR) window (650–1,350 nm), where the light has its maximum penetration. For more than a decade, an NIR reflectance microscopic technique termed spectroscopic optical coherence tomography (SOCT) has been developed (, , , ). SOCT detects the light signals from a micrometer-scale sample volume while rejecting the light from the background, a unique capability known as optical sectioning, which makes it possible to probe microstructures through 1- to 2-mm opaque tissues. The effect of scatterer size on the back-scattered spectra has led to the use of SOCT to evaluate the cell nuclei (, , , href="#bib47" rid="bib47" class=" bibr popnode">Xu et al., 2005) and probe nanoscale information (href="#bib2" rid="bib2" class=" bibr popnode">Azarin et al., 2015, href="#bib53" rid="bib53" class=" bibr popnode">Yi et al., 2013). The spectra of random microspheres packing have been investigated extensively (href="#bib38" rid="bib38" class=" bibr popnode">Tseng et al., 2006); detailed understanding of nanoscale shape on spectroscopic contrast, however, remains elusive.At the nanometer level, the two fundamental geometries of biological tissues are spherical geometry, such as mitochondria and various intracellular granules, and cylindrical geometry, such as motile cilia, collagen fibrils, and elastic fibers. It is intriguing to know how different nanoscale geometries may alter the spectrum of scattered light and whether it is possible to extract biologically relevant information from their spectroscopic signatures. The answers to these questions are pertinent to a variety of clinical and scientific research fields where optical reflectance imaging techniques are widely used, such as noninvasive anatomical and functional imaging of microstructures in the respiratory mucosa, the blood vessel wall, the posterior segment of the eye, the skin, and the gastrointestinal mucosa (href="#bib10" rid="bib10" class=" bibr popnode">Fujimoto, 2003, href="#bib13" rid="bib13" class=" bibr popnode">Huang et al., 1991, href="#bib18" rid="bib18" class=" bibr popnode">Liu et al., 2011, href="#bib36" rid="bib36" class=" bibr popnode">Tearney et al., 1997, href="#bib51" rid="bib51" class=" bibr popnode">Yelin et al., 2006, href="#bib54" rid="bib54" class=" bibr popnode">Yun et al., 2006), nanoscale mapping of nuclear architecture (href="#bib40" rid="bib40" class=" bibr popnode">Uttam et al., 2015, href="#bib43" rid="bib43" class=" bibr popnode">Wang et al., 2010), or changing of partial wave spectroscopy signals by chromosome condensation (href="#bib14" rid="bib14" class=" bibr popnode">Kim et al., 2011). In this study, we established numerical models of nano-spheres and nano-cylinders and discovered that transversely oriented and regularly arranged nano-cylindrical scatterers are more likely to generate spectral centroid shifts toward the long wavelengths within the spectral window of 700–950 nm than nano-spheres, which tend to exhibit shifts toward the short end. Using a form of SOCT that operates in 700–950 nm, we verified the above-mentioned predictions on a variety of natural nano-cylindrical structures, such as motile cilia and extracellular matrix containing nanoscale fibrous structures, which exhibited a striking and consistent spectroscopic contrast against the surrounding background tissues. To our knowledge, this study is the first to demonstrate geometry-dependent spectroscopic contrasts from nanometer-scale features in deep mammalian tissues in situ and in vivo. Interestingly, we additionally found that this geometry-dependent spectroscopic contrast is sensitive to external stress, probably due to the changes in the orientation, degree of alignment, and spacing of the nano-cylinders.