Nanoscale Imaging of Collagen Gels with Focused Ion Beam Milling and Scanning Electron Microscopy

Abstract

In vitro polymerized type I collagen hydrogels have been used extensively as a model system for three-dimensional (3D) cell and tissue culture, studies of fibrillogenesis, and investigation of multiscale force transmission within connective tissues. The nanoscale organization of collagen fibrils plays an essential role in the mechanics of these gels and emergent cellular behavior in culture, yet quantifying 3D structure with nanoscale resolution to fully characterize fibril organization remains a significant technical challenge. In this study, we demonstrate that a new imaging modality, focused ion beam scanning electron microscopy (FIB-SEM), can be used to generate 3D image datasets for visualizing and quantifying complex nanoscale organization and morphometry in collagen gels. We polymerized gels at a number of concentrations and conditions commonly used for in vitro models, stained and embedded the samples, and performed FIB-SEM imaging. The resulting image data had a voxel size of 25 nm, which is the highest resolution 3D data of a collagen fibril network ever obtained for collagen gels. This resolution was essential for discerning individual fibrils, fibril paths, and their branching and grouping. The resulting volumetric images revealed that polymerization conditions have a significant impact on the complex fibril morphology of the gels. We segmented the fibril network and demonstrated that individual collagen fibrils can be tracked in 3D space, providing quantitative analysis of network descriptors such as fibril diameter distribution, length, branch points, and fibril aggregations. FIB-SEM 3D reconstructions showed considerably less lateral grouping and overlap of fibrils than standard 2D SEM images, likely due to artifacts in SEM introduced by dehydration. This study demonstrates the utility of FIB-SEM for 3D imaging of collagen gels and quantitative analysis of 3D fibril networks. We anticipate that the method will see application in future studies of structure-function relationships in collagen gels as well as native collagenous tissues.

Extracellular matrix compression temporally regulates microvascular angiogenesis

“…Islands smaller than 30 voxels (e.g., single cells, debris, or noise) were also removed using Amira. Thresholded images were exported for skeletonization and quantification of length and branching using the 4-D open snake method of the Farsight Toolkit (39).…”

An interactive ImageJ plugin for semi-automated image denoising in electron microscopy

“…Over the past years, there has been a substantial increase in the use of these techniques in life science research [7][8][9][10][11][12] . The advantage of generating high-resolution 3D information, and also a comprehensive view of a complete cell or tissue, has invited the scientific community to apply these techniques for many different research questions.…”

Mechanical Regulation of Microvascular Angiogenesis

“…single cells, debris, or noise) were also removed using Amira. Thresholded images were exported for skeletonization and quantification of length and branching using the 4-D open snake method (59) of the Farsight Toolkit (60,61).…”