Episcopic 3D Imaging Methods: Tools for Researching Gene Function

Abstract

This work aims at describing episcopic 3D imaging methods and at discussing how these methods can contribute to researching the genetic mechanisms driving embryogenesis and tissue remodelling, and the genesis of pathologies. Several episcopic 3D imaging methods exist. The most advanced are capable of generating high-resolution volume data (voxel sizes from 0.5x0.5x1 microm upwards) of small to large embryos of model organisms and tissue samples. Beside anatomy and tissue architecture, gene expression and gene product patterns can be three dimensionally analyzed in their precise anatomical and histological context with the aid of whole mount in situ hybridization or whole mount immunohistochemical staining techniques. Episcopic 3D imaging techniques were and are employed for analyzing the precise morphological phenotype of experimentally malformed, randomly produced, or genetically engineered embryos of biomedical model organisms. It has been shown that episcopic 3D imaging also fits for describing the spatial distribution of genes and gene products during embryogenesis, and that it can be used for analyzing tissue samples of adult model animals and humans. The latter offers the possibility to use episcopic 3D imaging techniques for researching the causality and treatment of pathologies or for staging cancer. Such applications, however, are not yet routine and currently only preliminary results are available. We conclude that, although episcopic 3D imaging is in its very beginnings, it represents an upcoming methodology, which in short terms will become an indispensable tool for researching the genetic regulation of embryo development as well as the genesis of malformations and diseases.

Keywords: 3D modelling; development; embryo; episcopic microscopy; gene expression.; imaging.

Fig. (1)

Set up used for episcopic 3D data generation. A camera (c), sitting on an optic (o) is aligned perpendicular to the surface of a block of embedding medium (b) containing a specimen. Note that the field of view (marked by grey lines) should be as narrow as possible. Its position cannot be changed during sectioning. k, knife.

Fig. (2)

Flow chart of the episcopic 3D data generation protocol. Note that this flow chart provides only the main steps. Depending on the method, additional steps might be necessary for analysing gene expression patterns.

Fig. (3)

From pixel to voxel. a. Schematic drawing of three subsequent two-dimensional (2D) images. Each image consists of 10 x 13 pixels. b. Volume data created from images of 1 µm thick sections. The voxels are cubic, because the distance between the images is the same as the length of the x and y coordinate of the pixels of the 2D images. c. Volume data created from images of 7 µm thick sections. While the 2D resolution (resolution of the 2D images) is relatively high, the 3D resolution (resolution of the volume data set) is low.