Siadat Seyed Mohammad, Silverman Alexandra A, DiMarzio Charles A, Ruberti Jeffrey W
Department of Bioengineering, Northeastern University, Boston, MA 02115, USA.
Department of Electrical and Computer Engineering, Northeastern University, Boston, MA 02115, USA.
J Struct Biol. 2021 Mar;213(1):107697. doi: 10.1016/j.jsb.2021.107697. Epub 2021 Feb 2.
Collagen fibrils, linear arrangements of collagen monomers, 20-500 nm in diameter, comprising hundreds of molecules in their cross-section, are the fundamental structural unit in a variety of load-bearing tissues such as tendons, ligaments, skin, cornea, and bone. These fibrils often assemble into more complex structures, providing mechanical stability, strength, or toughness to the host tissue. Unfortunately, there is little information available on individual fibril dynamics, mechanics, growth, aggregation and remodeling because they are difficult to image using visible light as a probe. The principle quantity of interest is the fibril diameter, which is difficult to extract accurately, dynamically, in situ and non-destructively. An optical method, differential interference contrast (DIC) microscopy has been used to visualize dynamic structures that are as small as microtubules (25 nm diameter) and has been shown to be sensitive to the size of objects smaller than the wavelength of light. In this investigation, we take advantage of DIC microscopy's ability to report dimensions of nanometer scale objects to generate a curve that relates collagen diameter to DIC edge intensity shift (DIC-EIS). We further calibrate the curve using electron microscopy and demonstrate a linear correlation between fibril diameter and the DIC-EIS. Using a non-oil immersion, 40x objective (NA 0.6), collagen fibril diameters between ~100 nm to ~ 300 nm could be obtained with ±11 and ±4 nm accuracy for dehydrated and hydrated fibrils, respectively. This simple, nondestructive, label free method should advance our ability to directly examine fibril dynamics under experimental conditions that are physiologically relevant.
胶原纤维是胶原单体的线性排列，直径为20 - 500纳米，其横截面上包含数百个分子，是肌腱、韧带、皮肤、角膜和骨骼等多种承重组织中的基本结构单元。这些纤维通常组装成更复杂的结构，为宿主组织提供机械稳定性、强度或韧性。不幸的是，关于单个纤维的动力学、力学、生长、聚集和重塑的信息很少，因为使用可见光作为探针很难对它们进行成像。主要关注的量是纤维直径，很难在原位、动态、准确且无损的情况下提取该直径。一种光学方法，即微分干涉对比（DIC）显微镜已被用于观察小至微管（直径25纳米）的动态结构，并且已证明对小于光波长的物体尺寸敏感。在本研究中，我们利用DIC显微镜报告纳米级物体尺寸的能力来生成一条将胶原直径与DIC边缘强度偏移（DIC - EIS）相关联的曲线。我们进一步使用电子显微镜对该曲线进行校准，并证明纤维直径与DIC - EIS之间存在线性相关性。使用非油浸40倍物镜（数值孔径0.6），对于脱水和水合纤维，分别可以获得精度为±11纳米和±4纳米的100纳米至300纳米之间的胶原纤维直径。这种简单、无损、无需标记的方法应能提高我们在生理相关实验条件下直接检查纤维动力学的能力。
