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

Bromage 0079700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，用于数字采集和处理与人类学科学相关的图像。自1996年通过NSF学术研究基础设施倡议开始运作以来，AMICA一直致力于研究，知识传播和各级教育机会。我们现在建议实施显微技术，推进三维（3D）成像和重建能力的AMICA。我们有三个主要的研究目标与应用程序的实时三维和共聚焦显微镜人类和非人类灵长类动物的骨骼显微解剖和生活史。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其学术生涯的各个阶段。这将包括一个完善的同伴指导计划在AMICA。第一个目标是检查三维处置（即分布和方向）的二次（哈弗）重塑事件在人类股骨中段之间的关系。我们的研究样本由1-95岁已知年龄个体的尸检收集组成，其中保留了体重，身高和死因的信息。哈弗系统的3D构象中与年龄相关的变异性的知识，因为这种变异性可能受到骨生长过程和机械历史的影响，预计将提供对人类生活史的见解（例如，个体的年龄、健康状况和机械负荷史）。其次，我们的目标是测量灵长类动物骨骼中骨细胞陷窝的3D体积密度，假设其作为个体发育年龄和身体大小的函数而变化。将调查各种灵长类动物分类群，包括一些已知的个人生活史和代表各种年龄和大范围的身体大小。这项工作的主要意义是，骨细胞密度可能会发现不同的生活史特征，这些灵长类类群和有用的，结合其他技术，为重建身体大小和/或年龄在灭绝taxa.Third，我们的目标都是比较骨组织类型的比例，提供有关相对生长速度，并对相邻骨板的宽度进行量化，为确定猕猴和红猴的骨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，其中一些动物已经接受了计算骨生长速率所需的重要标签。这项工作是重要的，作为一种潜在的手段，精细化高分辨率的信息增长率的变化，在两个旧世界猴的具体，并欣赏之间的关系方面的骨微结构组织和灵长类动物的生活史一般。这是我们获取有关骨组织组织和生长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），并使用2D显微镜在第三个维度上的骨微结构特征的测量是一个棘手的任务，原因是必须与叠加的焦点以及从组织切片的全厚度导出的焦点外信息。这使得3D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缺陷并满足我们的研究目标：Edge 8400和H160实时3D显微镜以及技术仪器K2-S BIO共聚焦显微镜。除了其可视化潜力，从这些显微镜获取的图像将被重建，以提供组织结构的3D数据集，用于精确测量。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。这种显微镜使人们能够实时清晰地成像，并在复合显微镜提供的放大倍数下，对组织学薄切片内物体之间存在的3D关系进行高分辨率成像，例如哈弗系统的排列，骨细胞陷窝的网络，H160模拟荧光显微镜，但其工作原理依赖于将反射光图像分成左-右-左-左-右-右-左-右-左-右-右-以及右眼视图，其被眼脑复合体实时正确地解释为3D图像。在骨生长研究中给予生命标记导致的荧光标记的分布在组织学薄切片内与所有其他光学平面不同的光学平面上精确可视化。这对于在我们的生长速率变异性研究中将标记明确定位到特定的骨板是必不可少的。K3共聚焦显微镜能够对非常薄（例如0.35微米）的光学“切片”进行成像，深入到完整、大块骨的表面，以及典型组织学薄切片中包含的所有此类光学平面。这些非常高分辨率的2D图像允许对深层组织结构进行明确的解释，特别是一旦这些结构在其第三维中被理解。

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.