High-definition infrared micro-spectroscopic imaging of biomaterials

红外显微光谱学是新一代组织学，可以同时对多种成分进行定量、无标记成像。在这项技术中，光谱仪被连接到显微镜上，以聚焦红外光，并在组织切片中的微米尺寸的斑点内检测其吸收光谱。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都有一个独特的峰指纹。使用探测器阵列，可以同时确定样品中16,000,000个点的特征，并将其绘制为2D图像。这项技术在研究和诊断中的应用迅速增长，使用的是脱水组织，因为水强烈地吸收红外线并引起强烈的光学干扰伪影。但脱水会扭曲生物分子结构和组织结构，抹去光谱指纹，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细胞，可以使组织保持在所需的溶液和温度下。与商业设计相比，我们将光谱重复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实现的。在去年，我们进一步开发了这种电池，不仅可以处理水溶液，还可以处理生理温度下的有机溶剂和接近饱和的水蒸气。新电池的多功能溶剂控制和提高的光谱精确度将允许在化学、物理和生物医学研究中采用全新的实验方法。 在过去的一年里，我们测量了野生型(WT)和突变新生小鼠股骨头软骨中主要细胞外基质成分的定量、5微米分辨率分布。该突变体具有SLC26A2硫酸盐/氯逆向转运蛋白纯合子突变，模拟患有遗传性发育不良的人类软骨病理。我们的技术可以分辨不同类型的糖胺多聚糖(GAG)及其硫酸盐化程度，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硫酸盐化的程度都朝着股骨头中心增加。在突变体中，关节表面的GAG硫酸盐含量是正常的2.3倍，但头部中心的含量几乎正常。这种正常化可能是由于硫酸根不足的GAG降解较快，或由于GAG分解代谢导致细胞内硫酸盐增加或GAG合成速度较慢所致。该突变还影响了其他细胞外基质成分的浓度和空间分布。在突变体中，糖基分布接近均匀，但在关节表面附近被耗尽。突变体中非胶原蛋白的浓度降低了1.8倍。在两种基因型中，从关节表面向股骨头中心逐渐减少(1.5倍)。WT中的胶原浓度也向股骨头中心逐渐减少(2倍)，但在突变的股骨头中几乎保持一致，可能是由于发育迟缓。在关节表面，突变体中的胶原浓度约为WT的1.5倍。胶原、GAG和糖硫酸盐密度的降低可能是导致突变软骨弹性降低和滑膜酶通透性增加的原因之一，从而导致萎缩性发育不良软骨的退化。

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.