Background to the invention

- the need for 3D data

The ability to visualise biological tissues in their correct 3D organisation is of great help to our understanding of biology, and is especially useful in the field of embryo development, in which tissues undergo an intricate array of movements relative to each other. The ability to map the 3D expression patterns of genes and proteins within these complex shapes is also useful, as it gives us insight into their functions, and gives us clues about which genes and proteins interact with each other. With the advent of complete genome sequences, microarray techniques and biological databases, it is likely that the growing need for a comprehensive database of gene expression will become a reality soon. Already a number of laboratories are initiating projects to maps gene expression patterns by the thousand.

The traditional method for creating 3D reconstructions of embryonic tissue is a laborious, time-consuming job, as it involves embedding the tissue, cutting it into hundreds of serial sections, mounting the sections, staining them, digitising them under a microscope, feeding them into an appropriate computer program and finally manually compensating for the distortions which have inevitably been introduced during the whole process.

Much effort has recently been put into the development of techniques which can image the structure of biological tissue in 3D, and in particular the mapping of gene expression patterns. By far the most commonly used is confocal microscopy. Typically, this is used to scan up to a depth of a few hundred microns, although the advent of dual-photon and multi-photon techniques means that good resolution images can be captured at greater depth. For some specimen which are larger than this depth, gene expression patterns can be built-up by scanning serial thick sections.

3-D reconstruction Shh expression in the ZPA of an E9 mouse limb bud. (A) A schematic to illustrate the orientations of sections (section colours relate to the borders around C and D). (B) One of the original optical sections scanned by the confocal microscope. (C) Virtual sections cutting though the 3-D reconstruction, starting at the posterior end of the limb bud and progressing anteriorly. (D) Virtual sections perpendicular to the previous two orientations, starting distally and progressing proximally. The thickened ectoderm of the AER (apical ectodermal ridge) can be seen in the first of these sections. ec - ectoderm, m - mesenchyme.

These images were generated at Caltech in the BioImaging Center. Click on the images to go to the relevant web-site. Sections through 3D images created by mMRI. Left, a sagital section through a reconstruction of an E11.5 mouse embryo (ex-vivo). Right, a section through a living tadpole, in which expression of the LacZ reporter gene is detected using the high-contrast agent whose molecular structure is shown above. This molecule is a substrate for the b-galactosidase enzyme (expressed from the LacZ reporter gene) which displays high MRI contrast after cleavage of its galactose side-chain.

Although mMRI holds much promise for imaging experiments in the future, especially with its ability to image living specimen, it also displays some limitations. We have developed a new imaging technique which has the following advantages over mMRI:

  1. It can image coloured stains. This allows us to image data from:
    • Normal in-situ hybridisation experiments (using the BCIP and NBT substrates)
    • Reporter gene experiments (such as LacZ and alkaline phosphatase)
  2. It can image fluorescent dyes. This allows us to image:
    • Multiple signals from the same tissue
    • Standard fluorescent antibody reagents
  3. Only takes about 15 minutes to image each channel. This speed allows us to image many specimen rapidly, and therefore to perform studies of natural variation in shape, gene expression, mutant phenotypes etc.
  4. It is much cheaper than mMRI, and works in conjunction with a normal "dissecting" microscope, making it accessible to many more biologists.