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: 8351202
• 负责人: Edward Mertz
• 金额: $ 9.8万
• 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/8351202
• 关键词: Adult; Age; Biochemical; Biochemical Process; Biocompatible Materials; Birth; Bone Diseases; Bone neoplasms; Cartilage; Cell Culture Techniques; Cells; Chemicals; Chloride Ion; Chlorides; Chondrocytes; Chondroitin; Collagen; Collagen Fibril; Computer Analysis; Connective Tissue; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cytoskeleton; Degenerative polyarthritis; Dehydration; Deposition; Diagnostics Research; Dyes; Dysplasia; Endocrine; Enzymes; Epiphysial cartilage; Exhibits; Financial compensation; Fingerprint; Fluorescence; Fluorescence Microscopy; Glycosaminoglycans; Goals; Growth; Heterogeneity; Image; Immature Bone; Inorganic Sulfates; Kinetics; Knock-in Mouse; Label; Libraries; Life; Light; Maps; Mature Bone; Measures; Mechanics; Microradiography; Microscope; Minerals; Modeling; Monitor; Morphologic artifacts; Mus; Mutation; Newborn Infant; Optics; Osteogenesis; Osteogenesis Imperfecta; Pathology; Pathway interactions; Polarization Microscopy; Property; Proteins; Proteoglycan; Regulation; Reproducibility; Resolution; Rest; Sampling; Severities; Signal Transduction; Skeletal Development; Solutions; Solvents; Specimen; Spectrum Analysis; Spottings; Stem cells; Structure; Surface; Techniques; Temperature; Time; Tissues; Unspecified or Sulfate Ion Sulfates; Water; Wild Type Mouse; absorption; adult stem cell; antiporter; articular cartilage; base; bone; cartilage development; chemical fingerprinting; chemical group; chondrodysplasia; density; design; design and construction; detector; femur head; improved; in vivo; interest; knock-down; mineralization; mouse model; novel strategies; polarized light; spectroscopic imaging; sulfation; tumor; uptake

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 16,384 micron-size spots in a tissue section. Chemical composition, orientation and interactions of chemical groups are determined within each spot 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 because water strongly absorbs infrared light, resulting in 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 collected a spectral library of well-purified and characterized components of connective tissues, which we measured with significantly improved spectro-chemical resolution. We also developed a new approach to quantitative mapping of collagen orientation in tissue sections by polarized infrared hyperspectral imaging. In addition, we designed a new thermo-mechanically stabilized, flow-through chamber for high-definition Raman microspectroscopy, allowing a simultaneous additional characterization of bone specimens with polarized and fluorescence microscopies. We also adapted analytical autoradiographic imaging with micrometer spatial resolution, bridging it with high-definition microspectroscopic imaging to correlate tissue composition with biochemical processes. During the last year, we adapted in vivo dynamic labeling of bone formation surfaces with fluorescence dyes which allows to demarcate bone regions formed at given time points and to perform high-definition Raman microspectroscopy on the same samples. We further extended high-definition spectral library of model connective tissue compounds and continued developing 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, bone tumors 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 progressive 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. We showed that in DTD mice, GAG sulfation was low compared to WT in the articular and proliferative zones but almost normal in the resting zone. 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 normally protects 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 growth plate. The disruption severity 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. We used quantitative microradiography to study 35S-sulfate incorporation into cartilage explants, to show that variability of undersulfation across different cartilage regions in DTD was associated with faster chondroitin synthesis rate in the articular and proliferative zones. This observation explained the undersulfation normalization with age, when the cartilage growth slows down, and provided basis for developing a kinetic model for the regulation of GAG sulfation and new potential targets for DTD treatment. We are also focusing on bone disorders. We previously studied bones in the Brittle mouse model of Osteogenesis Imperfecta treated 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. Using Raman microspectroscopy, we found that matrix mineralization heterogeneity near donor cells was lower within each material type, suggesting that better organization of matrix made by the donor cells may contribute to the amelioration of bone mechanical properties observed in the treated mice. We studied endocrine bone tumors caused by cyclic AMP signaling disruption in a mouse model with Prkar1a+/-/Prkaca+/- deletions in protein kinase A. Using polarized-light microscopy and Raman microspectroscopy, we found that tumor formation in adult mice causes periosteal deposition of immature cortical bone, in which collagen and mineral organizations are intermediate between those of woven and lamellar bone. These observations supported the hypothesis of the existence of periosteal adult stem cells capable of growing cortical bone. During the last year, we studied effects Prkar2a+/- and Prkar2b+/- deletions on new bone formation in tumors caused by Prkar1a+/- knock down in mouse using polarized-light microscopy, Raman microspectroscopy and dynamic bone labeling. We found unexpected partial compensation by such deletions of maturation of bone material at the levels of matrix mineralization and local collagen organization. Surprisingly, only Prkar2b+/-, but not Prkar2a+/-, was able to rescue global collagen organization, suggesting that regulatory subunit Prkar2b is involved in pathways responsible for global collagen organization in bone.

我们正在开发新的方法，定量，无标记的组织组织的红外显微光谱检查。在这种技术中，一个带有二维探测器阵列的红外光谱仪被连接到显微镜上。它同时测量组织切片中16384个微米大小斑点的红外吸收光谱。化学成分、取向和化学基团的相互作用在每个点内由化合物的独特光谱指纹确定，并绘制为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，因为水强烈吸收红外光，导致光学干涉伪影。然而，脱水会扭曲生物分子和组织结构，弄脏光谱指纹，降低化学和光谱分辨率。