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

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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.