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

Nature Protocols volume 8, pages 1433–1448 (2013)Cite this article

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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.

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Figure 1: Embryonic chick metatarsal tendon prepared using the reduced osmium protocol and imaged using 3View and TEM.
Figure 2: TEM of postnatal tendon.
Figure 3: Semiautomated measurements of fibril diameter from transverse sections.
Figure 4: 3View analysis of a conventionally en bloc–stained resin-embedded sample of a newborn mouse tendon.
Figure 5: Complete collagen fibril at the plasma membrane of a fibroblast in embryonic mouse tendon.
Figure 6: Screen shots of an IMOD model window from 'complete-fibril.mod'.
Figure 7: Alternative views of an image stack produced in IMOD.
Figure 8: Example output images from dm2mrc and clip showing the effects of smoothing on image appearance.

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Acknowledgements

We thank the Wellcome Trust for generous grant support (091840/Z/10/Z, 083898/Z/07/Z, 081406/Z/06/Z). We acknowledge the help of support staff in the Electron Microscopy Facility, Faculty of Life Sciences, University of Manchester. We thank D. Golijanin for help with IMOD segmentation.

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Author notes

  1. Tobias Starborg, Nicholas S Kalson and Yinhui Lu: These authors contributed equally to this work.

Authors and Affiliations

  1. Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, University of Manchester, Manchester, UK

    Tobias Starborg, Nicholas S Kalson, Yinhui Lu, David F Holmes & Karl E Kadler

  2. Faculty of Life Sciences, University of Manchester, Manchester, UK

    Tobias Starborg, Nicholas S Kalson, Yinhui Lu, Aleksandr Mironov, David F Holmes & Karl E Kadler

  3. Faculty of Medicine and Human Sciences, Imaging Sciences and Biomedical Imaging Institute, University of Manchester, Manchester, UK

    Timothy F Cootes

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Contributions

Y.L. and A.M. optimized the staining and embedding procedures for collagen fibril–containing tissues. T.S. and N.S.K. developed the 3View procedures. T.S. wrote the shell scripts. D.F.H. developed the single-fibril approaches (TEM and STEM) and provided expertise in quantitative analyses. Y.L. cut all the TEM sections. T.S. and N.S.K. prepared the 3D reconstructions. T.F.C. provided the solution for calculating mean fibril length. K.E.K. motivated the implementation of 3View for studies of collagen, oversaw data collection and interpretation and equipped the laboratory. Everyone contributed to writing the manuscript. K.E.K. wrote the final draft.

Corresponding authors

Correspondence to David F Holmes or Karl E Kadler.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Data 1

A Shell Script for producing an image stack from 3View images. Requires pre-installation of IMOD. The Shell Script produces the image stack, floats and smooths the images in the stack, and flips the images about the x-axis. (TXT 5 kb)

Supplementary Data 2

IMOD-generated model file of a cell (green, day 13 embryonic chick metatarsal tendon fibroblast), three extracellular matrix collagen fibrils (yellow) and a short entire fibril (red) within a recessed fibripositor (gray). Nucleus, blue. To view this file, install IMOD and use the following command: $ 3dmodv complete-fibril.mod (ZIP 209 kb)

Supplementary Data 3

An example dataset of 3View images. The dataset shows 100 images from 400 × 100 nm slices through embryonic mouse tail tendon. The images have been binned ×4 to reduce the file size from 4096 × 4096 to 1024 × 1024. The voxel dimensions are 45 × 45 × 100 nm. The DigitalMicrograph images were converted to TIF format using mrc2tif. N.B. the images in this data set are flipped about the x-axis. The flipping means that the scanning line order is inverted, i.e. the image is generated from the bottom of the screen. The reversed image line order affects the appearance of some of the section fragments that fall onto the surface during image acquisition. (ZIP 81663 kb)

Supplementary Data 4

A cropped volume (at original resolution) of the same image stack that was used to generate 'example_bin4'. The dataset shows 100 images from 400 × 100 nm slices through embryonic mouse tail tendon. The images have been cropped at full resolution. The voxel dimensions are 11 × 11 × 100 nm. The DigitalMicrograph images were converted to TIF format using mrc2tif. (ZIP 80521 kb)

Supplementary Video 1

Step-through movie of 250 images produced by 3View showing the range of size of elements that can be segmented. The sample was embryonic mouse-tail tendon cut at 100 nm intervals. IMOD was used to generate the 3D reconstruction of a cell (green) and individual collagen fibrils in the extracellular matrix (blue), a fibril within a fibripositor (yellow), and a fibril within a fibricarrier (purple). Other elements in the image stack that have not been segmented are nuclei, endoplasmic reticulum, mitochondria, and the Golgi apparatus. (MOV 28268 kb)

Supplementary Video 2

Step-through movie of 117 images produced from 'complete-fibril.mod'. The movie shows a short complete fibril (circled in red) in a plasma membrane recess (recessed fibripositor). The movie demonstrates the high quality imaging that is possible with reduced osmium staining and 3View. Scale bar, 500 nm. (MOV 17339 kb)

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Starborg, T., Kalson, N., Lu, Y. et al. Using transmission electron microscopy and 3View to determine collagen fibril size and three-dimensional organization. Nat Protoc 8, 1433–1448 (2013). https://doi.org/10.1038/nprot.2013.086

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