Optical projection tomography as a new tool for studying embryo anatomy

Abstract

Optical projection tomography (OPT) is a new technique for three-dimensional (3D) imaging of small biological tissues. It is particularly useful for reconstructing vertebrate embryos and for examining the 3D anatomy of developing organs. The advantages of this technique over previous methods will be explained: in particular, its ability to image at a higher resolution than magnetic resonance imaging (MRI), while at the same time being able to image specimens much larger than those possible using confocal laser-scanning microscopy. Being an optical technique, OPT is also able to take advantage of the many coloured and fluorescent dyes which have been developed for tissue-specific or gene-specific staining. This becomes particularly important for the visualization of the 3D shapes of specific organs and tissues as it allows the computer to automatically determine the outline of the desired structure.

Fig. 1

OPT scan of a wildtype 10-day-old mouse embryo. The embryo was whole-mount stained for the expression patterns of two proteins using fluorescently labelled antibodies. In blue is the signal detected for antibodies against the HNF3b protein, and in green is the signal for neurofilament-a protein that is expressed in nerve cells. The red signal represents non-specific autofluorescence from the tissue, which highlights the overall anatomy of the embryo. This same data are displayed in two forms: on the left is a ‘virtual section’ through the reconstruction, and on the right is a 3D surface model of the same embryo.

Fig. 2

Four different views of an OPT scan of LacZ-expressing cells within the brain of a 13-day-old mouse embryo. The blue shapes show where the labelled cells were located. The white surface represents the lowest part of the brain tissue, i.e. the surface of the ventricles. If the tissue itself has been shown as a solid surface, then the LacZ cells would not be visible, as they are embedded within this tissue. Note that although the LacZ cells are shown in blue in this computer representation, the original imaging of this brain was performed in black-and-white. As such, the positive cells were distinguished simply by being darker than the surrounding tissue. This means that some other dark tissue in the brain (for example where blood has developed) is also visible as blue shapes in the images shown here (for example the blue shapes at the bottom of the fourth panel).

Fig. 3

False-colour computer-generated images of a TS21 mouse embryo which was stained with alcian blue and then scanned by OPT. The images illustrate another advantage of 3D data – that the tissue can be displayed with varying degrees of opacity, thereby allowing one to see more or fewer of the internal structures. (Staining and volume rendering of this embryo by Seth Ruffins at Caltech.)