High-definition infrared micro-spectroscopic imaging of biomaterials

生物材料的高清红外显微光谱成像

• 批准号: 7594260
• 负责人: Edward Mertz
• 金额: $ 25.06万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7594260
• 关键词: Affect; Biochemical; Biocompatible Materials; Biomedical Research; Bone and Cartilage Funding; Cartilage; Catabolism; Cells; Characteristics; Chemicals; Chloride Ion; Chlorides; Collagen; Cultured Cells; Cytoskeleton; Dehydration; Development; Diagnostics Research; Dysplasia; Elasticity; Enzymes; Fingerprint; Generations; Genotype; Glycosaminoglycans; Goals; Head; Histology; Human; Image; Inorganic Sulfates; Knock-in Mouse; Label; Life; Light; Measures; Mechanics; Microscope; Modeling; Monitor; Morphologic artifacts; Mus; Mutation; Newborn Infant; Optics; Organic solvent product; Pathology; Permeability; Physiological; Proteins; Purpose; Rate; Reproducibility; Resolution; Sampling; Solutions; Solvents; Spatial Distribution; Spectrum Analysis; Spottings; Structure; Surface; Techniques; Temperature; Time; Tissues; Unspecified or Sulfate Ion Sulfates; Water; absorption; antiporter; aqueous; chemical group; density; design; desire; detector; femur head; interest; mutant; size; spectroscopic imaging; sugar; sulfation; vapor

Infrared micro-spectroscopy is a new generation of histology allowing quantitative, label-free imaging of multiple components simultaneously. In this technique a spectrometer is attached to a microscope to focus infrared light and detect its absorption spectrum within a micron-size spot in a tissue section. From these spectra chemical composition, orientation and interactions of chemical groups can be determined, because every chemical group has a unique fingerprint of peaks in the absorption spectra. Using an array of detectors, the characteristics from 16,000 thousand spots across the sample can be simultaneously determined and plotted as 2D-images. Rapidly growing applications of this technique to research and diagnostics use dehydrated tissues, because water strongly absorbs infrared light and cause strong optical interference artifacts. But dehydration distorts biomolecular and tissue structure, smears out spectroscopic fingerprints, and degrade chemical and spectral resolution. To overcome this limitation, over the last years we developed an infrared cell that allows to keep tissues in desired solution and temperature. We increased spectral reproducibility and chemical resolution by two orders of magnitude compared to commercial designs, which we achieved by thermo-mechanical stabilization reducing interference artifacts. During the last year we further developed the cell to handle not only aqueous solutions, but also organic solvents and nearly saturated water vapors at physiological temperatures. Versatile solvent control and increased spectral accuracy of the new cell will allow qualitatively new experimental approaches in chemical, physical and biomedical research. During the last year we measured quantitative, 5-micron resolution distributions of major extra-cellular matrix components across femur head cartilage of wild type (WT) and mutant newborn mice. The mutant has a knocked-in homozygous mutation in SLC26A2 sulfate/chloride antiporter modeling cartilage pathology in humans with diastrophic dysplasia. Our technique resolved different glycosaminoglycan (GAG) types and the extent of their sulfation and distinguished between collagen and other proteins. We found that the extent of GAG sulfation increased toward the femur head center both in the mutant and WT. In the mutant, the GAGs were 2.3-times undersulfated at the articular surface, but nearly normal in the head center. This normalization may be caused by faster degradation of undersulfated GAGs, increased intracellular sulfate due to GAG catabolism or slower rate of GAG synthesis. The mutation also affected the concentrations and spatial distributions of other extra-cellular matrix components. Sugar groups were nearly uniformly distributed in WT, but depleted near the articular surface in the mutant. Concentration of non-collagenous proteins was 1.8 times lower in the mutant. It gradually decreased (1.5 fold) from the articular surface toward the femur head center in both genotypes. Collagen concentration in WT also gradually decreased (2 fold) toward the femur head center, but remained nearly uniform across the mutants femur head probably due to delayed development. At the articular surface, collagen concentration in the mutant was about 1.5 times lower than in WT. The lower densities of collagen, GAGs and sugar sulfate may be responsible for lowering mutant cartilage elasticity and increasing its permeability to synovial enzymes, contributing to the observed cartilage degradation in diastrophic dysplasia.

红外显微光谱是新一代的组织学技术，可以同时对多种成分进行定量、无标记成像。 在这种技术中，光谱仪连接到显微镜上，以聚焦红外光并检测其在组织切片中微米大小的斑点内的吸收光谱。 从这些光谱中可以确定化学基团的化学组成、取向和相互作用，因为每个化学基团在吸收光谱中具有独特的峰指纹。 使用探测器阵列，可以同时确定样品上16，000千个点的特征，并绘制成2D图像。 这种技术在研究和诊断中的快速增长的应用使用脱水组织，因为水强烈吸收红外光并引起强烈的光学干涉伪影。 但是脱水会扭曲生物分子和组织结构，模糊光谱指纹，降低化学和光谱分辨率。 为了克服这一限制，在过去的几年里，我们开发了一种红外线电池，可以将组织保持在所需的溶液和温度下。 与商业设计相比，我们将光谱再现性和化学分辨率提高了两个数量级，这是通过热机械稳定减少干扰伪影来实现的。 在过去的一年中，我们进一步开发了细胞，不仅处理水溶液，而且有机溶剂和接近饱和的水蒸气在生理温度。 多功能的溶剂控制和增加的光谱精度的新细胞将允许在化学，物理和生物医学研究的定性新的实验方法。 在过去的一年中，我们测量了野生型（WT）和突变新生小鼠股骨头软骨的主要细胞外基质成分的定量，5微米分辨率分布。 该突变体在SLC 26 A2硫酸盐/氯化物反向转运蛋白中具有敲入纯合突变，从而模拟患有畸形性发育不良的人类的软骨病理学。 我们的技术解决了不同的糖胺聚糖（GAG）类型和它们的硫酸化程度，并区分胶原蛋白和其他蛋白质。 我们发现，在突变体和WT中，GAG硫酸化的程度向股骨头中心增加。 在突变体中，GAG在关节面硫酸盐不足2.3倍，但在头部中心几乎正常。 这种正常化可能是由于未充分硫酸化的GAG的更快降解、由于GAG催化剂引起的细胞内硫酸盐增加或GAG合成速率较慢引起的。 该突变还影响了其他细胞外基质成分的浓度和空间分布。 糖组几乎均匀分布在野生型，但耗尽附近的突变体的关节面。 非胶原蛋白的浓度在突变体中低1.8倍。 从关节面向股骨头中心逐渐减少（1.5倍），在两种基因型。 WT中的胶原蛋白浓度也朝向股骨头中心逐渐降低（2倍），但在整个突变体股骨头中保持几乎均匀，这可能是由于发育延迟。 在关节表面，突变体中的胶原蛋白浓度比WT低约1.5倍。 胶原蛋白、糖胺聚糖和硫酸糖的密度较低可能是降低突变软骨弹性和增加其对滑液酶的渗透性的原因，有助于观察到的畸形性发育不良中的软骨降解。