High-resolution episcopic microscopy: a rapid technique for high detailed 3D analysis of gene activity in the context of tissue architecture and morphology

Abstract

We describe a new methodology for rapid 2D and 3D computer analysis and visualisation of gene expression and gene product pattern in the context of anatomy and tissue architecture. It is based on episcopic imaging of embryos and tissue samples, as they are physically sectioned, thereby producing inherently aligned digital image series and volume data sets, which immediately permit the generation of 3D computer representations. The technique uses resin as embedding medium, eosin for unspecific tissue staining, and colour reactions (beta-galactosidase/Xgal or BCIP/NBT) for specific labelling of gene activity and mRNA pattern. We tested the potential of the method for producing high-resolution volume data sets of adult human and porcine tissue samples and of specifically and unspecifically stained mouse, chick, quail, frog, and zebrafish embryos. The quality of the episcopic images resembles the quality of digital images of true histological sections with respect to resolution and contrast. Specifically labelled structures can be extracted using simple thresholding algorithms. Thus, the method is capable of quickly and precisely detecting molecular signals simultaneously with anatomical details and tissue architecture. It has no tissue restrictions and can be applied for analysis of human tissue samples as well as for analysis of all developmental stages of embryos of a wide variety of biomedically relevant species.

Episcopic 3D Imaging Methods: Tools for Researching Gene Function

“…This includes methods such as "fast 3D serial reconstruction" [39], "Epi-3D" [40], "episcopic fluorescence image capturing" [41], "surface imaging microscopy" [42,43], "high resolution episcopic microscopy (HREM)" [44], and serial blockface scanning electron microscopy (SBF-SEM) [45]. The term "surface imaging microscopy" was used twice.…”

Three-Dimensional (3D) Visualisation of the Cardiovascular System of Mouse Embryos and Fetus

“…Since the images are captured at reproducible photo-positions, the digital images are aligned and can be immediately stacked together and converted to a volume data set. Several methods exist for generating volume data sets on the basis of the episcopic imaging technique [108][109][110][111][112]. Each has its advantages and disadvantages.…”

“…The morphology and tissue architecture of all the components of the cardiovascular system of all developmental stages of mouse embryos can be 3D visualised in high resolution [50,111]. Taking advantage of whole mount in situ hybridisation and whole mount staining, a few episcopic methods permit the 3D visualisation of gene expression and gene product patterns in respect to the cardiovascular system of mouse embryos/fetus [114].…”

“…It is however sufficient for 3D analysis of the expression patterns of multiple, selectively labelled (trans-)genes in small parts of embryos [106,107], or the morphology of cells in the walls of the heart and blood vessels. (not all) [108][109][110][111][112]114] Although the table lists the most promising "in vivo techniques", none of them currently enables the generation of useful volume data of the heart and great blood vessels of unborn mice. Note that each of the listed post mortem techniques has its specific advantages and disadvantages.…”

Multimodal imaging of mouse development: Tools for the postgenomic era

“…Two different approaches have been taken to remedy this problem. First, episcopic fluorescence image capture (EFIC), also referred to as surface imaging microscopy (SIM) or block-face imaging, has emerged as a relatively simple way to collect images from sequential physical sections through an embryo (Weninger et al, 1998(Weninger et al, , 2006Ewald et al, 2002;Weninger and Mohun, 2002; Table 1). The specimen is embedded in a block for sectioning, but before each section is cut, an image of the block face is taken, recording expression or histological data directly from the block, eliminating distortion caused by the mounting process.…”