Acquisition of Real-time 3D and Confocal Microscopy for Bone Microstructure and Life History Studies of Human and Non-Human Primates

亨特学院人类学分析显微镜和成像中心（AMICA）是一个独特的环境，用于与人类学相关的图像的数字采集和处理。自1996年在美国国家科学基金会学术研究基础设施倡议下开始运作以来，AMICA一直致力于研究、知识传播和多个层面的教育机会。我们现在建议实施显微镜技术，以提高AMICA的三维成像和重建能力。我们有三个主要的研究目标，涉及实时3D和共聚焦显微镜在人类和非人类灵长类动物骨骼显微解剖和生活史中的应用。这些研究活动将在成像科学培训计划的背景下进行，包括本科生和研究生在他们学术生涯的各个阶段。这将包括在美国建立一个完善的同伴指导计划。第一个目的是研究人类中轴股骨继发性（哈弗氏）重构事件的三维分布（即分布和方向）之间的关系。我们的研究样本包括从1-95岁的已知年龄个体的尸检收集，其中保存了有关体重，身高和死因的信息。由于这种变异性可能受到骨骼生长过程和力学史的影响，因此了解哈弗斯系统三维构象中与年龄相关的变异性有望为人类生活史（例如，个体的年龄、健康状况和机械负荷史）提供见解。其次，我们的目标是测量灵长类动物骨骼中骨细胞腔隙的三维体积密度，假设其随个体发育年龄和体型的变化而变化。各种各样的灵长类类群将被研究，包括一些已知的个体生活史，代表不同的年龄和大范围的体型。这项工作的主要意义在于，骨细胞密度可能会随着这些灵长类分类群的生活史而变化，并且与其他技术相结合，对于重建已灭绝分类群的体型和/或年龄是有用的。第三，我们的目的是比较提供相对生长速度信息的骨组织类型的比例，以及量化相邻骨片种群的宽度，以便为确定Macaca和Ernthrocebus的骨骼生长速率变异性和生活史提供一些相对客观的标准。我们的样本来自已知生活史的幼年动物，其中一些已经获得了计算骨骼生长速率所必需的重要标签。这项工作具有重要的潜在意义，可以用于处理两种旧大陆猴的生长速率变化的高分辨率信息，并用于了解骨骼微观结构组织与灵长类动物生活史之间的关系。AMICA的传统二维（2D）显微镜一直是，并将永远是我们获取与骨组织组织和生长有关的骨微观结构信息的第一个点。尽管如此，可视化、数据捕获（即图像采集）和使用二维显微镜在第三维测量骨微结构特征是一项棘手的任务，原因是与来自组织学切片全厚度的焦点和焦点外信息的叠加有关。这使得三维解释和测量变得困难。我们建议购买三台显微镜来解决这些不足并满足我们的研究目标：Edge 8400和H160实时3D显微镜，以及Technical Instrument K2-S BIO共聚焦显微镜。除了它们的可视化潜力，从这些显微镜获得的图像将被重建，以提供精确测量的组织结构的三维数据集。8400依赖于多个倾斜照明光源，以获得非常高的分辨率（即高于传统的2D复合光显微镜）透射光3D可视化。该显微镜能够实时清晰地成像，在复合显微镜提供的放大倍数下，可以高分辨率地成像组织学薄片中存在的物体之间的3D关系，例如哈弗森系统的排列、骨细胞腔隙网络和骨片层。H160模拟了荧光显微镜，但其工作原理依赖于将反射光图像分解为左眼和右眼视图，这些图像被眼脑复合体实时正确地解释为3D图像。荧光标记的分布作为骨生长研究中生命标记的结果，在组织学薄切片的光学平面上被精确地可视化。这对于在我们的生长速率变异性研究中明确定位特定骨片的标签是至关重要的。K3共聚焦显微镜能够成像非常薄（例如0.35微米）的光学“切片”，深入到完整的大块骨的表面，以及在典型的组织学薄切片中包含的所有这些光学平面。这些非常高分辨率的二维图像允许对深层组织结构进行明确的解释，特别是在它们的三维空间被理解之后。

Bromage0079700The Analytical Microscopy and Imaging Center in Anthropology (AMICA), Hunter College, is a unique environment for the digital acquisition and processing of images of relevance to the anthropological sciences. Since it became operational in 1996 by virtue of an NSF Academic Research Infrastructure initiative, AMICA has remained true to research, the dissemination of knowledge, and educational opportunities at several levels. We propose now to implement microscopy technologies that advance a three-dimensional (3D) imaging and reconstruction capability in AMICA. We have three primary research objectives concerned with applications of real-time 3D and confocal microscopy to human and non-human primate bone microanatomy and life history. These research activities will take place in the context of an imaging sciences training program to include undergraduate and graduate students at all points in their academic careers. This will include a well established peer mentoring program in AMICA.The first objective is to examine the relationship between the 3D disposition (i.e. distribution and orientation) of secondary (Haversian) remodeling events in the human mid-shaft femur. Our study sample consists of an autopsy collection of known age individuals from 1-95 years for which information on weight, height, and cause of death are preserved. Knowledge of the age-related variability in the 3D conformation of Haversian systems, inasmuch as this variability may be affected by bone growth processes and mechanical history, is expected to provide insights into human life history (e.g., an individual's age, health status, and mechanical loading history).Second, we have the objective to the measure the 3D volumetric density of osteocyte lacunae in primate bone, as hypothesized to vary as a function of ontogenetic age and body size. A variety of primate taxa will be investigated, including some with known individual life history and representing various ages and a large range of body sizes. The primary significance of this work is that bone cell density may be found to vary in respect to the life histories characterizing these primate taxa and to be useful, in combination with other techniques, for the reconstruction of body size and/or age in extinct taxa.Third, we aim both to compare the proportions of bone tissue types, that provide information about relative growth rate, as well as to quantify the widths of populations of contiguous bone lamellae in order to have some relatively objective criterion for the determination of bone growth rate variability and life history in Macaca and Ernthrocebus. Our sample derives from juvenile animals of known life history, some of whom have received vital labels necessary for the calculation of bone growth rate. This work is significant as a potential means of finessing high resolution information about growth rate variation in two Old World Monkey's specifically, and for appreciating relationships between aspects of bone microstructural organization and primate life history generally.AMICA's conventional two-dimensional (2D) microscopy has been, and always will be, our first point of access for information about bone microstructure relating to bone tissue organization and growth. Despite this, the visualization, data capturing (i.e. image acquisition), and measurement of bone microstructural features in the third dimension using 2D microscopy is an intractable task for reasons having to do with the superimposition of both in focus as well as out of focus information derived from the full thickness of histological sections. This makes 3D interpretations and measurements difficult. We propose to acquire three microscopes to address these deficiencies and to meet our research objectives: the Edge 8400 and H160 Real-Time 3D microscopes, and the Technical Instrument K2-S BIO Confocal microscope. In addition to their visualization potential, images acquired from these microscopes will be reconstructed to provide 3D data sets of histological structures for accurate measurement.The 8400 is dependent upon multiple oblique illuminating light sources to obtain very high (i.e. higher than conventional 2D compound light microscopy) resolution transmitted light 3D visualization. This microscope enables one to image with clarity in real-time, and at magnifications offered by a compound microscope, high resolution imagery of the 3D relationships existing between objects within histological thin sections, such as the arrangement of Haversian systems, networks of osteocyte lacunae, and bone lamellae.The H160 emulates a fluorescence microscope but its operating principle relies on splitting of the reflected light image into left- and right-eye views which are interpreted in real-time, correctly, as a 3D image by the eyebrain complex. The distribution of fluorescing label as a result of the administration of vital label in bone growth research, is precisely visualized at optical planes distinct from all others within a histological thin section. This is essential for the unambiguous localization of label to specific bone lamellae in our growth rate variability research.The K3 Confocal microscope is able to image very thin (e.g. 0.35 micron) optical "sections" deep to the surface of intact, bulk bone as well as in all such optical planes contained within typical histological thin sections. These very high resolution 2D images allow unambiguous interpretations of deep histological structures particularly once these have been understood in their third dimension.