High-definition infrared micro-spectroscopic imaging of biomaterials

红外微光谱学是新一代的组织学允许定量，无标记成像的多个组件同时。在该技术中，将光谱仪连接到显微镜上，聚焦红外光，并在组织切片中微米大小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独特的峰指纹。使用一组探测器，可以同时确定样品中16000个点的特征并绘制为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，弄脏光谱指纹，降低化学和光谱分辨率。

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.