High-definition infrared micro-spectroscopic imaging of biomaterials

我们正在开发新的方法，定量，无标记的组织学检查的组织红外显微光谱。在这种技术中，具有2D检测器阵列的红外光谱仪连接到显微镜。它同时测量组织切片中16，000微米大小点的红外吸收光谱。每个点内化学基团的化学组成、方向和相互作用由化合物的独特光谱指纹确定，并绘制成2D图像。迄今为止，该技术在研究和诊断中的应用仅限于脱水组织。水强烈吸收红外光，导致光学干涉伪影。然而，脱水会扭曲生物分子和组织结构，涂抹光谱指纹，并降低化学和光谱分辨率。 为了克服这一限制，我们设计并构建了一个具有热机械稳定性的红外室，其允许将组织保持在所需温度的溶液中。通过减少干扰伪影，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。多功能的溶剂控制和增加的光谱精度的新室允许定性新的实验方法。例如，通过这项技术，我们可以区分胶原蛋白和其他蛋白质，解析不同的糖胺聚糖（GAG），甚至量化软骨中GAG硫酸化的程度。最近，我们通过结合红外和HPLC表征模型软骨素和透明质酸盐，提高了不同类型硫酸化GAG链的光谱化学分辨率。我们还开发了一种新的方法，通过偏振红外高光谱成像定量映射软骨切片中的胶原蛋白方向。 在过去的一年中，我们将模型化合物的高清晰度光谱库扩展到聚集蛋白聚糖，双糖蛋白聚糖，核心蛋白聚糖和II型胶原蛋白。我们还开发了一种新的光谱计算机分析，提高了化学分辨率和准确性。我们目前正在利用这种方法来表征成骨不全、软骨发育不良和其他结缔组织病理中的胶原基质组织。 具体来说，我们专注于研究由SLC 26 A2硫酸盐/氯化物反向转运蛋白突变引起的畸形性发育不良（DTD）的基因敲入小鼠模型。这些突变导致软骨细胞的硫酸盐摄取不足，导致对软骨发育和完整性至关重要的蛋白聚糖GAG链硫酸化不足。与其他先天性软骨发育不良一样，DTD延迟了骨骼发育，但表现出不寻常的进展。随着年龄的增长，硫酸化不足的情况正常化。然而，关节软骨随着年龄的增长而退化。为了了解软骨降解的机制，我们收集了新生DTD和野生型（WT）小鼠股骨头软骨和生长板中主要细胞外基质成分分布的5微米分辨率定量图像。在DTD小鼠中，与WT相比，关节和增殖区的GAG硫酸化较低，但静息区几乎正常。DTD中的硫酸化不足似乎与关节和增殖区的更快生长速率相关，这与软骨生长随年龄增长而减慢时的硫酸化不足正常化一致。在DTD小鼠中，偏振红外高光谱成像显示关节表面切向胶原纤维致密层破裂。切线胶原层通常存在以保护软骨免受摩擦损伤和滑液酶。它的破坏可能导致关节蛋白多糖耗竭，这是我们在出生时观察到的早期骨关节炎的标志，尽管GAG硫酸化正常化，但随着年龄的增长进一步进展。在DTD小鼠中，整个股骨头和生长板的胶原蛋白取向也被破坏。破坏的严重程度与GAG硫酸化不足的程度相关，但与胶原蛋白、非胶原蛋白和GAG链的密度无关，这表明GAG硫酸化可能对细胞合成定向基质至关重要。基于这些观察结果，我们提出了一个调节GAG硫酸化的动力学模型和DTD治疗的新的潜在靶点。 在过去的一年里，我们将红外技术的进步应用于拉曼显微光谱学，并开发了一种新的热机械稳定的流通室，可以同时对骨标本进行三重表征。该室允许记录成骨不全的脆性小鼠模型中的骨的高清晰度拉曼光谱，特别是在用绿色荧光蛋白标记的正常干细胞治疗后。使用荧光和偏振光观察，我们区分基质产生的非荧光宿主和荧光供体细胞和股骨皮质内区分不同类型的骨材料（编织，层状和细骨）。我们发现，在每种材料类型中，供体细胞附近基质矿化的异质性降低，这表明供体细胞基质的更好组织可能有助于改善治疗小鼠中观察到的骨机械性能。

We are developing new approaches to quantitative, label-free histological examination of tissues by infrared micro-spectroscopy. In this technique, an infrared spectrometer with a 2D detector array is attached to a microscope. It simultaneously measures infrared absorption spectra at 16,000 micron-size spots in a tissue section. Chemical composition, orientation and interactions of chemical groups within each spot are determined from unique spectral fingerprints of chemical compounds and plotted as 2D-images. To date, applications of this technique to research and diagnostics have been limited to dehydrated tissues. Water strongly absorbs infrared light, causing optical interference artifacts. However, dehydration distorts biomolecular and tissue structure, smears out spectroscopic fingerprints, and degrades chemical and spectral resolution. To overcome this limitation, we designed and constructed an infrared chamber with thermo-mechanical stabilization which allows keeping tissues in solution at desired temperature. By reducing the interference artifacts, we increased spectral reproducibility and chemical resolution by two orders of magnitude compared to commercial designs. Versatile solvent control and increased spectral accuracy of the new chamber allow qualitatively new experimental approaches. For example, with this technique we distinguish collagen from other proteins, resolve different glycosaminoglycans (GAG) and even quantify the extent of GAG sulfation in cartilage. Recently, we improved spectro-chemical resolution of different types of sulfated GAG chains by combing infrared and HPLC characterization of model chondroitins and hyaluronate. We also developed a new approach to quantitative mapping of collagen orientation across cartilage sections by polarized infrared hyperspectral imaging. During the last year, we extended high-definition spectral library of model compounds to aggrecan, biglycan, decorin and type II collagen. We also developed a new computer analysis of the spectra, increasing chemical resolution and accuracy. We are currently utilizing this approach for characterization of collagen matrix organization in Osteogenesis Imperfecta, chondrodysplasias and other connective tissues pathologies. Specifically, we are focusing on studies of a knock-in mouse model of Diastrophic Dysplasia (DTD) caused by mutations in SLC26A2 sulfate/chloride antiporter. These mutations result in deficient sulfate uptake by chondrocytes, leading to undersulfation of proteoglycan GAG chains crucial for cartilage development and integrity. Like other inborn chondrodysplasias, DTD has delayed skeletal development, but exhibits an unusual progression. The undersulfation is normalized with age. Nonetheless, the articular cartilage degrades with age. To understand the mechanism of the cartilage degradation, we collected 5-micron-resolution, quantitative images of distributions of major extra-cellular matrix components across femur head cartilage and growth plate in newborn DTD and wild type (WT) mice. In DTD mice, GAG sulfation was low compared to WT in the articular and proliferative zones but almost normal in the resting zone. The undersulfation in DTD appeared to be associated with faster growth rate in the articular and proliferative zones, consistent with the undersulfation normalization when the cartilage growth slows down with age. In DTD mice, polarized infrared hyperspectral imaging revealed disruption of a dense layer of tangentially oriented collagen fibrils at the articular surface. The tangential collagen layer is normally present to protect cartilage from frictional damage and synovial enzymes. Its disruption may cause articular proteoglycan depletion, a hallmark of early osteoarthritis which we observed at birth and which further progresses with age despite the normalization of GAG sulfation. Collagen orientation in DTD mice was also disrupted throughout the femur head and the growth plate. The severity of the disruption correlated with the extent of GAG undersulfation but not with densities of collagen, noncollagenous proteins and GAG chains, suggesting that GAG sulfation might be crucial for synthesis of the oriented matrix by cells. Based on these observations, we proposed a kinetic model for the regulation of GAG sulfation and new potential targets for DTD treatment. During the last year, we carried over infrared advances into Raman microspectrsocopy and developed a new thermo-mechanically stabilized, flow-through chamber which allowed a simultaneous triple characterization of bone specimens. The chamber allowed recording high-definition Raman spectra of bones in the Brittle mouse model of Osteogenesis Imperfecta, in particular, after treatment with normal stem cells labeled with green fluorescence protein. Using fluorescence and polarized-light observations, we distinguished matrix produced by non-fluorescent host and fluorescent donor cells and distinguished different types of bone material (woven, lamellar and fine-fibred) within femoral cortex. We found that heterogeneity of matrix mineralization near donor cells was decreased within each material type, suggesting that better organization of donor cells matrix may contribute to amelioration of bone mechanical properties observed in the treated mice.