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Using transmission electron microscopy and 3View to determine collagen fibril size and three-dimensional organization.

ABSTRACT: Collagen fibrils are the major tensile element in vertebrate tissues, in which they occur as ordered bundles in the extracellular matrix. Abnormal fibril assembly and organization results in scarring, fibrosis, poor wound healing and connective tissue diseases. Transmission electron microscopy (TEM) is used to assess the formation of the fibrils, predominantly by measuring fibril diameter. Here we describe a protocol for measuring fibril diameter as well as fibril volume fraction, mean fibril length, fibril cross-sectional shape and fibril 3D organization, all of which are major determinants of tissue function. Serial-section TEM (ssTEM) has been used to visualize fibril 3D organization in vivo. However, serial block face-scanning electron microscopy (SBF-SEM) has emerged as a time-efficient alternative to ssTEM. The protocol described below is suitable for preparing tissues for TEM and SBF-SEM (by 3View). We describe how to use 3View for studying collagen fibril organization in vivo and show how to find and track individual fibrils. The overall time scale is ~8 d from isolating the tissue to having a 3D image stack.

SUBMITTER: Starborg T

PROVIDER: S-EPMC5642902 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Using transmission electron microscopy and 3View to determine collagen fibril size and three-dimensional organization.

Starborg Tobias T Kalson Nicholas S NS Lu Yinhui Y Mironov Aleksandr A Cootes Timothy F TF Holmes David F DF Kadler Karl E KE

Nature protocols 20130627 7

Collagen fibrils are the major tensile element in vertebrate tissues, in which they occur as ordered bundles in the extracellular matrix. Abnormal fibril assembly and organization results in scarring, fibrosis, poor wound healing and connective tissue diseases. Transmission electron microscopy (TEM) is used to assess the formation of the fibrils, predominantly by measuring fibril diameter. Here we describe a protocol for measuring fibril diameter as well as fibril volume fraction, mean fibril le ...[more]

PMID: 23807286

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Project description:Unlike white and brown adipose tissues, the bone marrow adipocyte (BMA) exists in a microenvironment containing unique populations of hematopoietic and skeletal cells. To study this microenvironment at the sub-cellular level, we performed a three-dimensional analysis of the ultrastructure of the BMA niche with focused ion beam scanning electron microscopy (FIB-SEM). This revealed that BMAs display hallmarks of metabolically active cells including polarized lipid deposits, a dense mitochondrial network, and areas of endoplasmic reticulum. The distinct orientations of the triacylglycerol droplets suggest that fatty acids are taken up and/or released in three key areas - at the endothelial interface, into the hematopoietic milieu, and at the bone surface. Near the sinusoidal vasculature, endothelial cells send finger-like projections into the surface of the BMA which terminate near regions of lipid within the BMA cytoplasm. In some regions, perivascular cells encase the BMA with their flattened cellular projections, limiting contacts with other cells in the niche. In the hematopoietic milieu, BMAT adipocytes of the proximal tibia interact extensively with maturing cells of the myeloid/granulocyte lineage. Associations with erythroblast islands are also prominent. At the bone surface, the BMA extends organelle and lipid-rich cytoplasmic regions toward areas of active osteoblasts. This suggests that the BMA may serve to partition nutrient utilization between diverse cellular compartments, serving as an energy-rich hub of the stromal-reticular network. Lastly, though immuno-EM, we've identified a subset of bone marrow adipocytes that are innervated by the sympathetic nervous system, providing an additional mechanism for regulation of the BMA. In summary, this work reveals that the bone marrow adipocyte is a dynamic cell with substantial capacity for interactions with the diverse components of its surrounding microenvironment. These local interactions likely contribute to its unique regulation relative to peripheral adipose tissues.

Dataset Information

Using transmission electron microscopy and 3View to determine collagen fibril size and three-dimensional organization.

Publications

Using transmission electron microscopy and 3View to determine collagen fibril size and three-dimensional organization.

Similar Datasets

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