Automated high‐resolution three‐dimensional fluorescence imaging of large biological specimens

Kazakia, Galateia J.; Lee, J. J.; Singh, Mandeep; Bigley, R. F.; Martin, R. Bruce; Keaveny, Tony M.

doi:10.1111/j.1365-2818.2007.01721.x

Cited by 11 publications

(22 citation statements)

References 30 publications

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“…Marrow was removed with a low pressure water jet. Specimens were fixed in 10% neutral buffered formalin, dehydrated in increasing concentrations of alcohol and embedded undecalcified in methyl methacrylate made opaque through the addition of Sudan black dye [15]. …”

Section: Methodsmentioning

confidence: 99%

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Voxel size and measures of individual resorption cavities in three-dimensional images of cancellous bone

Tkachenko

Slyfield

Tomlinson

et al. 2009

Bone

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Cavities formed by osteoclasts on the surface of cancellous bone during bone remodeling (resorption cavities) are believed to act as stress risers and impair cancellous bone strength and stiffness. Although resorption cavities are readily detected as eroded surfaces in histology sections, identification of resorption cavities in three-dimensional images of cancellous bone has been rare. Here we use sub-micron resolution images of rat lumbar vertebral cancellous bone obtained through serial milling (n=5) to determine how measures of the number and surface area of resorption cavities are influenced by image resolution. Three-dimensional images of a 1mm cube of cancellous bone were collected at 0.7 X 0.7 X 5.0 μm/voxel using fluorescence based serial milling and uniformly coarsened to four other resolutions ranging from 1.4 X 1.4 X 5.0 to 11.2 X 11.2 X 10 μm/voxel. Cavities were identified in the three-dimensional image as an indentation on the cancellous bone surface and were confirmed as eroded surfaces by viewing two-dimensional cross-sections (mimicking histology techniques). The number of cavities observed in the 0.7 X 0.7 X 5.0 μm/voxel images (22.0 ± 1.43, mean ± SD) was not significantly different from that in the 1.4 X 1.4 X 5.0 μm/voxel images (19.2 ± 2.59) and an average of 79% of the cavities observed at both of these resolutions were coincident. However, at lower resolutions, cavity detection was confounded by low sensitivity (<20%) and high false positive rates (>40%). Our results demonstrate that when image voxel size exceeds 1.4 X 1.4 X 5.0 μm/voxel identification of resorption cavities by bone surface morphology is highly inaccurate. Experimental and computational studies of resorption cavities in three-dimensional images of cancellous bone may therefore require images to be collected at resolutions of 1.4 μm/pixel in-plane or better to ensure consistent identification of resorption cavities.

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Section: Methodsmentioning

confidence: 99%

“…The serial milling approach has been described in detail previously [15]. Serial milling is a sectioning approach similar to serial grinding [16].…”

Section: Methodsmentioning

confidence: 99%

Voxel size and measures of individual resorption cavities in three-dimensional images of cancellous bone

Tkachenko

Slyfield

Tomlinson

et al. 2009

Bone

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“…Another technique that permits high resolution 3-D imaging of bone matrix is epifluorescence-based serial block face imaging [60, 61]. In this technique, multiple fluorochromes within a single specimen can be reconstructed and visualized in three dimensions (Fig.…”

Section: In Vivo Histomorphometry and Sequential Fluorochrome Labelingmentioning

confidence: 99%

“…5). Kazakia et al [60] used this method to study fluorochrome-labelled microdamage in trabecular bone (5mm diameter) with voxel size of 3 × 3 × 8 µm 3 . Local damage volume fraction (an indicator of matrix heterogeneity) and global damage volume fractions based on the 3-D reconstruction, were computed to be 0.1–0.28 and 0.15 respectively.…”

Section: In Vivo Histomorphometry and Sequential Fluorochrome Labelingmentioning

confidence: 99%

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Multiscale imaging of bone microdamage

Poundarik

Vashishth

2015

Connective Tissue Research

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Bone is a structural and hierarchical composite that exhibits remarkable ability to sustain complex mechanical loading and resist fracture. Bone quality encompasses various attributes of bone matrix from the quality of its material components (type-I collagen, mineral and non-collagenous matrix proteins) and cancellous microarchitecture, to the nature and extent of bone microdamage. Microdamage, produced during loading, manifests in multiple forms across the scales of hierarchy in bone and functions to dissipate energy and avert fracture. Microdamage formation is a key determinant of bone quality, and through a range of biological and physical mechanisms, accumulates with age and disease. Accumulated microdamage in bone decreases bone strength and increases bone’s propensity to fracture. Thus, a thorough assessment of microdamage, across the hierarchical levels of bone, is crucial to better understand bone quality and bone fracture. This review article details multiple imaging modalities that have been used to study and characterize microdamage; from bulk staining techniques originally developed by Harold Frost to assess linear microcracks, to atomic force microscopy, a modality that revealed mechanistic insights into the formation diffuse damage at the ultrastructural level in bone. New automated techniques using imaging modalities such as microcomputed tomography are also presented for a comprehensive overview.

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Three-dimensional dynamic bone histomorphometry

Slyfield

Tkachenko

Wilson

et al. 2011

Journal of Bone and Mineral Research

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Dynamic bone histomorphometry is the standard method for measuring bone remodeling at the level of individual events. While dynamic bone histomorphometry is an invaluable tool for understanding osteoporosis and other metabolic bone diseases, the technique’s two-dimensional nature requires the use of stereology and prevents measures of individual remodeling event number and size. Here, we use a novel three-dimensional fluorescence imaging technique to achieve measures of individual resorption cavities and formation events. We perform this three-dimensional histomorphometry approach using a common model of postmenopausal osteoporosis, the ovariectomized rat. The three-dimensional images demonstrate the spatial relationship between resorption cavities and formation events consistent with the hemi-osteonal model of cancellous bone remodeling. Established ovariectomy was associated with significant increases in the number of resorption cavities per unit bone surface (2.38 ± 0.24 mm−2 SHAM v. 3.86 ± 0.35 mm−2 OVX, mean ± SD, p < 0.05) and total volume occupied by cavities per unit bone volume (0.38 ± 0.06% SHAM v. 1.12 ± 0.18% OVX, p < 0.001), but no difference in surface area per resorption cavity, maximum cavity depth, or cavity volume. Additionally, we find that established ovariectomy is associated with increased size of bone formation events due to merging of formation events (23,700 ± 6,890 μm2 SHAM v. 33,300 ± 7,950 μm2 OVX). No differences in mineral apposition rate (determined in 3D) were associated with established ovariectomy. That established estrogen depletion is associated with increased number of remodeling events with only subtle changes in remodeling event size suggests that circulating estrogens may have their primary effect on the origination of new basic multicellular units with relatively little effect on the progression and termination of active remodeling events.

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Automated high‐resolution three‐dimensional fluorescence imaging of large biological specimens

Cited by 11 publications

References 30 publications

Voxel size and measures of individual resorption cavities in three-dimensional images of cancellous bone

Voxel size and measures of individual resorption cavities in three-dimensional images of cancellous bone

Multiscale imaging of bone microdamage

Three-dimensional dynamic bone histomorphometry

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