MRC National Institute for Medical Research, London

Science for Health

Main navigation

Research areas

Genetics and Development

Infections and Immunity

Neurosciences

Structural Biology

Tim Mohun group project:

High resolution imaging of embryo morphology

Click image to view at full-size

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

See the Wellcome Image Awards 2009 in HD on YouTube.

Studying the complex developmental changes in 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:

acquiring comprehensive section series 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 virtually impossible

Alternative methods for 3D imaging of morphology are therefore urgently needed. Modern 3D imaging techniques (eg. ultrasound, µMRI or optical projection tomography) do not yet provide anything like 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 as a means of combining high resolution with accurate 3D morphology. In this approach, images of the block surface are captured during sectioning of embedded samples, thereby obviating the need to align images from individual sections. Since successive episcopic images remain in perfect alignment, the data is ideally suited for analysis using a variety of video or 3D reconstruction software packages. With our collaborator, Wolfgang Weninger (Vienna University) we have developed two methods for analysing embryos based on episcopic imaging, EFIC and HREM.

Episcopic fluorescence imaging (EFIC)

Episcopic fluorescence imaging uses wax-embedded specimens, visualizing tissue by virtue of its auto-fluorescence using GFP illumination. The wax is rendered opaque using wax dyes in order to restrict visualization to the block surface and the method therefore yields 'negative' images of the tissue.

Autofluorescence in tissues of a mouse embryo

Whole embryo, viewed after clearing.

Episcopic image of section through heart and lungs of 16dpc embryo.

EFIC produces a series of images of the embedded specimen during the sectioning process itself, utilizing autofluorescence of the tissue to visualize its structure. The images are in perfect alignment and are readily analysed using video or 3D reconstruction software packages.

3D resolution is determined by the thickness of section, which can be as little as 2 microns. If thicker sections (5-7 microns) are cut, they can also be retained for conventional histological staining. The ability of EFIC to image tissue depends upon adequate and sufficiently varied auto-fluorescence to permit different tissue and cell types to be distinguished. This is not always the case and is especially limited in tissues from early stages of embryonic development. For this reason, we have sought alternative ways of visualising tissue that do not depend upon auto-fluorescence.

High Resolution Episcopic Microscopy (HREM)

HREM is an episcopic imaging procedure that uses plastic rather than wax-embedding medium. 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. With plastic embedding medium, a standard section thickness of 1-2 microns is feasible, with retained sections suitable for histology (eg. 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.

E10.5 mouse embryo imaged using HREM

The data set comprises 1,500 images captured during removal of 2 micron sections from the block.

Computer-derived, orthogonal sections were calculated using the 1,500 image data set originally obtained in the transverse plane.

An Embryo Imaging Pipeline

By utilising large format CCD cameras it is possible to retain considerable fine tissue detail whilst imaging the entire embryo. Used in this way, HREM imaging provides a very simple and effective way to document embryo morphology. Image data from normal embryos can complement to selected images obtained from standard anatomical reference sources and data from mutant embryos can be used for morphological phenotyping. The Wellcome Trust has recently provided funding for such an embryo imaging pipeline and comprehensive image data can be accessed at www.embryoimaging.org.

"Virtual dissection" with HREM data

HREM data can provide highly detailed 3D models of imaged samples. Because of the variation in greyscale range obtained from different tissues, useful 3D models can be obtained without any tracing of structures ('segmentation'), using the simplest of techniques, 3D volume rendering. This approach can provides remarkable detail, allowing 'virtual dissection' of an organ or tissue from any orientation.

E10.5 mouse embryo

HREM data visualised by 3D rendering using open source Osirix software

HH27 chick embryo

By combining the anatomical detail of HREM data sets with the flexible visualisation of 3D volume rendering, it is possible to analyse regions of the embryo that are structurally complex, such as the embryo head.

E14.5 mouse embryo head

The HREM data set comprises alternate images from a stack of 770, obtained by sectioning at 2 micron thickness.

3D model of the E14.5 mouse embryo head

Future Challenges

HREM imaging provides a simple and convenient way to analyse morphology of mammalian and avian embryos but it is also increasingly important to understand the expression of individual genes in a similar 3D context. With current protocols, HREM images morphology using GFP fluorescence filter sets and some success in simultaneous imaging of colorimetric signals has been achieved using longer wavelengths (e.g. RFP). Optimising this is an important challenge, enabling high resolution analysis of wholemount in situ hybridisation signals and lacZ reporter lines.

E14.5 mouse heart showing clones of lacZ-expressing cells

Current 3D modelling of data derived from specific signals is most easily achieved by using "isosurface rendering" of data to obtain an "object" to visualise. Whilst this has the advantage of convenience, it pays a considerable price in loss of morphological resolution, the resulting models representing only approximate 3D cartoons of the imaged organ or embryo. A second challenge lies in devising appropriate 3D modelling approaches that can render specific signals whilst retaining reasonable morphological verisimilitude. Since "volume rendering" maximises morphological accuracy, one fruitful direction might be the use of colour channels in such models to carry information of specific signal location and strength.

E11.5 mouse heart with endocardial cushions coloured

A third challenge is finding ways in which entire image data sets can be shared across the research community. HREM data sets are frequently several gigabytes in size, precluding easy electronic exchange. An effective alternative is to use modern web-based tools to allow viewing of entire HREM data sets online, as for example in www.embryoimaging.org.