HREM IMAGING REVEALS DETAILS OF ABNORMAL EMBRYO DEVELOPMENT


High Resolution Episcopic Microscopy (HREM) is a technique that allows embryos to be imaged in unprecedented 3D detail. It’s the main imaging method used by the DMDD programme – we use it to identify a wide range of developmental abnormalities that result from mouse gene knockouts. The detail revealed by HREM models can be seen in this video of an embryo with a profound ventricular septal defect. At the time of imaging, the embryo was just over 1cm long.

 

 

But despite the many benefits of HREM it’s still a relatively uncommon technique to use, because a commercial system has only recently become available. In this post we’ll give a quick overview of how HREM works (and why it’s so useful).


MAXIMISING 3D IMAGE RESOLUTION

Many 3D imaging methods work by taking a series of images through the whole volume of a tissue sample, after cutting it into thin sections. The resulting ‘stack’ of section images can then be used to build a 3D model using software such as Osirix or Amira. The resolution of the final model depends on both the thickness of the sections and the accuracy with which the final images can be aligned. Sometimes this can involve using external markers to help align the images.

However, sections can often become distorted as they are cut and captured – even using external markers the final alignment can often be poor. HREM overcomes this problem by imaging the face of the block (so-called ‘episcopic imaging’) rather than the individual sections.

The samples (in our case, embryos) are embedded in a hard plastic resin, which contains fluorescent dyes. Tissue at the surface of the block can be visualised against the bright background of the fluorescent plastic, and this simple approach gives remarkable detail. The sections are not distorted, and the relative alignment of the images is well known. As a result the resolution of the 3D model is limited only by the thickness of the cut sections.

 

Click to view larger image.

HREM models at 3 µm resolution allow detailed 3D investigation of developing embryos.

 

Since the plastic resin is very hard, HREM sections can be cut as thin as 1 µm. In the DMDD programme we use sections between 1 and 3 µm (depending on the size of the embryo), and around 3000 images are recorded for an embryo at E14.5. At this resolution, features such as individual nerves and blood vessels can be identified, as shown in the image above.


WHY USE HREM?

HREM data has allowed the DMDD team to identify nearly 400 different phenotypes in knockout mouse embryos. These range from large-scale organ malformations to the abnormal development of nerves and blood vessels. In the image below, a mutant embryo was found to have an absent hypoglossal nerve. Since this nerve controls tongue movement, the phenotype typically results in neonatal death when the pup is unable to suckle.

 

Click to view larger image.

This view from the DMDD database shows a mutant embryo (right) from the knockout line D930028MRik, and its wild-type litter-mate (left). The mutant embryo has an absent hypoglossal nerve on both the right and left sides of the head. The same nerves are clearly visible in the wild-type embryo.

 

The DMDD programme has used HREM to image whole mouse embryos. But the technique has also been used to image embryos, organs and tissues from other animals, as well as samples from plants. It provides a way to resolve details that may not be visible in other 3D models.

If you would like more information or advice about how to image a tissue sample using HREM, please email contact@dmdd.org.uk.

DMDD HREM image and phenotype data can be found at dmdd.org.uk.


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