Dual and multi-fluorescence Optical HREM allows for imaging a single dye within the standard HREM morphology image, for example imaging LacZ in the context of the overall sample.

Specific target structures within the sample are labelled with a fluorescent tag, as would be done in immunohistochemistry. The resin is then made opaque to provide a better signal-to-noise ratio, and imaging is carried out at the appropriate wavelength(s).

Up to 4 other fluorescence channels can be used in a similar way - preparation techniques are already published. (Walsh et al, 2020)

Dual and multiple fluorescence channel HREM supports:

  • Gene expression in context of the anatomy and tissue structure.
  • Microvascular mapping - fluorescent vascular stains can be injected into mouse or other animal models. The staining creates a 3D microvascular map of the sample on imaging.
  • 3D cell distribution - fluorescent staining of cell nuclei in a sample allows visualisation of the 3D cell density.
  • In vitro samples - 3D in vitro samples such as spheroids or 3D scaffolds can be stained and imaged using HREM to quantify 3D morphology of cells or other features of interest.
  • Autofluorescence - many organs e.g. lung and kidneys exhibit strong autofluorescence. This can be used to visualise larger scale phenotypic variations in these organs such as airway branching in lungs or glomeruli in kidneys.
  • Antibody staining – antibody stains may be compatible with HREM depending on the particular antibody of use and the solvent used for dehydration. A simple pre-test of the antibody on a cryosection with the intended solvent can be used to determine antibody compatibility. If the pre-test is successful, 3D whole mount staining times can be optimised for the sample size and antibody concentrations.

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.

References

  • Claire Walsh, Natalie Holroyd, Eoin Finnerty, Sean G. Ryan, Paul W. Sweeney, Rebecca J. Shipley, Simon Walker-Samuel
    Multi-fluorescence high-resolution episcopic microscopy (MF-HREM) for three dimensional imaging of adult murine organs.
    bioRxiv, 2020 https://doi.org/10.1101/2020.04.03.023978
  • 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

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