Studying complex developmental changes in embryo morphology is difficult, especially for mammalian and avian embryos that are relatively large and opaque. The traditional approach of using histological sections is very effective in providing high-resolution 2D images, but is less helpful for analysing morphology in 3D for several reasons:
- acquiring a comprehensive series of sections is extremely labour-intensive
- data is restricted to the plane of section chosen, which can be difficult to reproduce
- individual sections are variably distorted during the procedure, making accurate alignment of the sections virtually impossible.
Modern 3D imaging techniques (eg. ultrasound, µMRI or optical projection tomography) do not yet provide anything like the resolution achieved by histology and are therefore of limited use for studies of morphological detail.
A simple and effective alternative is to use block surface (episcopic) imaging of histological samples. This combines high resolution with accurate 3D morphology. Imaging the block surface of embedded samples during sectioning means that successive episcopic images are in perfect alignment, removing the need to align images from individual sections. The resulting data is extremely high resolution (1-3 μm3) making it ideally suited for analysis using a variety of video or 3D reconstruction software packages.
High Resolution Episcopic Microscopy (HREM) imaging is a method for analysing embryos based on episcopic imaging, developed together with Wolfgang Weninger of the Medical University of Vienna.
Samples are embedded in a methacrylate resin (JB4) that has been made highly fluorescent by the addition of dyes such as eosin and acridine orange. Tissue is visualized by virtue of its ability to suppress the fluorescence of the embedding plastic. This results in extremely high-resolution 'positive' images of the block surface, irrespective of the tissue type or developmental stage.
A standard section thickness of 1-2 microns can be achieved, and the sections retained are suitable for histology (e.g. H&E staining). HREM is suitable for use over a broad range of optical magnification (ranging from cellular to macroscopic) and different tissue types are readily distinguished.