Tim Mohun: Projects

High Resolution Episcopic Microscopy (HREM) 

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. 

Episcopic Imaging 

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.   

mouse embryo head

'Virtual dissection' of a E14.5 mouse embryo head.

 

 

 

 

 

HREM 

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. 

Watch a video of Tim  Mohun explaining the HREM technique as part of the  Wellcome Image Awards 2009.   

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.