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A simple setup for episcopic microtomy and a digital image processing workflow to acquire high-quality volume data and 3D surface models of small vertebrates

Abstract

The use of volume data and digital three-dimensional (3D) surface models in biology has increased quickly and steadily. Various methods are available to acquire 3D data, among them episcopic imaging techniques. Based on the episcopic microscopy with on-block staining protocol of Weninger et al. (Anat Embryol 197:341–348, 1998), we describe a simple and versatile setup for episcopic microtomy. It is composed of a consumer DSLR digital camera combined with standard histology equipment. The workflow of block surface staining and imaging, image processing, stack alignment, surface generation (including a custom Amira® macro), and 3D model editing is described in detail. For our sample specimen (Alytes obstetricans; Amphibia: Anura) we obtained images with a pixel size of 5.67 × 5.67 µm2. The generated image stacks allowed distinguishing different tissues and were well-suited for creating a 3D surface model. We analyzed the alignment quality achieved by various selections of specimen and fiducial marker spots. The fiducial spots had a significant positive effect on the alignment quality with the best alignment having a maximum mean alignment error of about 44.7 µm. We further tested the APS-C camera with combinations of macro lens, extension tube or teleconverter. The macro lens and extension tube yielded the smallest pixel size of 2.53 × 2.53 µm2. Considering data quality and resolution, and depending on object sizes and research goals, DSLR captured episcopic microtomy can be an alternative to other techniques, such as traditional histological sectioning or micro-computed tomography.

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Acknowledgements

We thank Anna-Maria Vogt and Lena Schwinger for skillful help with the lab-work. We also thank the students who have worked with episcopic microtomy data in our class or during their bachelor projects and helped gaining experiences with the workflow, in particular Stine Griep, Lisa Malina Hahne, Vivienne-Christine Ahrens, Juliana Stahl, and Mehria Sedik. We also thank the anonymous reviewers for their helpful comments on the manuscript.

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Correspondence to Karolin Engelkes.

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Electronic supplementary material

Below is the link to the electronic supplementary material.

Online Resource 1 Protocol for paraffin embedding of Alytes obstetricans (ZMH A12442). Supplementary material 1 (PDF 166 kb)

Online Resource 2 Scripts and documentation for Amira® macro MultiExport. Supplementary material 2 (ZIP 245 kb)

435_2017_386_MOESM3_ESM.pdf

Online Resource 3 Descriptive statistics of the sets of shortest distances between the observed midpoints and the, respectively, fitted lines. Supplementary material 3 (PDF 15 kb)

435_2017_386_MOESM4_ESM.pdf

Online Resource 4 Results of Kruskal–Wallis rank sum test and Dunn’s test on distance sets. Supplementary material 4 (PDF 321 kb)

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Engelkes, K., Friedrich, F., Hammel, J.U. et al. A simple setup for episcopic microtomy and a digital image processing workflow to acquire high-quality volume data and 3D surface models of small vertebrates. Zoomorphology 137, 213–228 (2018). https://doi.org/10.1007/s00435-017-0386-3

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Keywords

  • Block surface imaging
  • On-block staining
  • Alignment
  • Volume data
  • Surface model