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.