Subdiffraction Fluorescence imaging of Bone Biomechanics

描述(申请人提供)：假设可以使用次衍射量子点成像以5 nm或更高的分辨率测量小鼠皮质骨组织成分的机械诱导扭曲，这一假设将得到测试。最终目的是研究骨组织负荷的动力学，特别是骨细胞结构和细胞周围基质成分内的变形。结果将与有限元分析模型相结合，以估计应变分布。在最初的实验中，将使用不同发射波长的量子点标记对小鼠皮质骨矿物质和基质胶原进行化学或免疫化学染色。标记试剂的浓度将被调整，以确保组织被稀疏地标记(大约每2-5微米一个标记)，从而使荧光产生于单个点。使用高NA物镜和超灵敏的电子倍增CCD成像器，荧光显微镜将观察到一个点的衍射点重心的变化，从而观察到纳米运动。在法向加载和塑性变形区，矿物和基质的相对运动将相互关联。标签的发射波长将被选择，以优化骨组织自发荧光最小化、相机量子效率和发射光谱带宽之间的权衡。为了共同定位不同发射波长的量子点，它们将被连接到可光裂解的试剂上，带到组织上的同一点，然后用短暂的紫外光脉冲分离。我们的候选光解连接物是双(对氨基苯基)二硫化物，它将允许两个不同的量子点共局域化。或者，将随机应用标签，并通过对观察到的运动矩阵进行统计分析来获得相关性。随着该项目这一阶段的成功，将扩大对细胞骨架元素的标记。

DESCRIPTION (provided by applicant): The hypothesis that mechanically induced distortions of components of murine cortical bone tissue can be measured with 5 nm resolution or better using subdiffraction quantum dot imaging will be tested. The ultimate aim is to study the dynamics of loading of bone tissue with a particular focus on deformation within osteocyte structures and pericellular matrix components. The results will be coupled with finite element analysis models to estimate strain distributions. In the initial experiments murine cortical bone mineral and matrix collagen will be stained chemically or immunochemically using quantum dot labels with different emission wavelengths. The concentrations of the labeling reagents will be adjusted to guarantee that the tissue is sparsely (about 1 label every 2-5 microns) labeled, so that fluorescence results from single dots. Nanometer motion will be observed by fluorescence microscopy as changes in center of gravity of the diffraction spot of a dot, using a high NA objective and an ultrasensitive electron-multiplied CCD imager. Mineral and matrix relative motions will be correlated under normal loading and in the plastic deformation region. The emission wavelengths of the labels will be chosen to optimize the trade-offs between minimization of bone tissue autofluorescence, camera quantum efficiency and emission spectrum band width. To colocalize quantum dots of different emission wavelengths they will be attached to photocleavable reagents, brought to the same point on the tissue and then separated with a brief pulse of UV light. Our candidate photocleavable linker is bis(p-aminophenyl) disulfide, which will allow colocalization of two different quantum dots. Alternatively, labels will be applied randomly and the correlations obtained by statistical analysis of the matrix of observed movements. With success of this phase of the project, extension to labeling cytoskeletal elements will be undertaken.