Standard HREM is single-channel.
Single channel Eosin - standard HREM
The resin is mixed with Eosin B which cause it to exhibit broad fluorescence in the green portion of the visible spectrum. Samples are stained with Eosin B and where the stain binds to the sample the fluorescence is inhibited, so more eosinophilic proteins appear darker against the bright fluorescence of the resin. When imaged with a GFP filter set (~495nm/~520nm), this provides good contrast for viewing structural or phenotypic variation.
Single channel HREM is ideal for:
- Whole embryo phenotyping – HREM is widely used to identify phenotypic variations in mouse and other embryos; this is particularly useful in mouse gene knockout imaging.
- Isolated organ - imaging of isolated organs such as the heart, to look for organ specific anomalies and quantify phenotypic features e.g. heart chamber volume.
- Vascular imaging – in certain organs, for example the liver and skin, vasculature can be imaged with negative contrast; the fluorescent resin fills the vasculature making it bright whilst the stained tissue appears dark. With appropriate post processing to remove any bright structures that are not vasculature, 3D vasculature maps can be rendered.
HREM is successful with all developmental stages of all the principal biomedically relevant model organisms, including specimens in which the autofluorescence is too low to allow proper analysis with EFIC - episcopic fluorescence image capturing (e.g. quail embryos or early embryonic stages). In addition, HREM overcomes the limited ability of EFIC for visualisation of specifically contrasted tissues. [EFIC is reduced to analyses of transgene expression pattern inside solid organs because it uses the extinction of tissue autofluorescence by LacZ (i.e. a ‘‘negative’’ contrast method).] HREM directly detects the product of colour reactions (i.e. a ‘‘positive’’ contrast method) and preserves the morphological information of stained structures since the staining reaction does not obscure tissue morphology. Furthermore, HREM enables distinction of strong and weak signals (Fig. 5) and thus, in principle, permits visualisation of gene product quantities.Weninger et al, 2006, https://doi.org/10.1007/s00429-005-0073-x
Figure 3 in the above publication shows block surface images of whole mount stained specimens obtained with HREM where images have been taken with a GFP filter set, contrasted with those captured with a Leica TX2 filter set. The GFP image shows detailed anatomical structures in the head region of a wild-type zebrafish embryo, including the lens and otic vesicle.
An additional GFP image of a mouse embryo shows detail such as the formation of cushions at the atrioventricular junction.
- Geyer, S.H., T.J. Mohun and W.J. Weninger
Visualizing vertebrate embryos with episcopic 3D imaging techniques.
Scientific World Journal, 2009. 9: p. 1423-37. PubMed abstract
- Weninger, W.J., S.H. Geyer, T.J. Mohun, D. Rasskin-Gutman, T. Matsui, I. Ribeiro,
F. Costa Lda, J.C. Izpisua-Belmonte, and G.B. Muller
High-resolution episcopic microscopy: a rapid technique for high detailed 3D analysis of gene activity in the context of tissue architecture and morphology.
Anat Embryol (Berl), 2006. 211(3): p. 213-21. PubMed abstract
Dual and multiple fluorescence
Optical HREM is also suited to dual and multiple fluorescence.