Standard HREM mode is single-channel. HREM is also suited to dual and multiple fluorescence.
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.
Dual and multiple fluorescence
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).
Dual or multiple fluorescence are suited for:
- 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.
- Cell tracking (short/mid term) – injecting cells pre-loaded with dyes designed for short/mid-term cell tracking, either into organs or systemically allows the locations of the cells to be identified at later time points.
- 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, for example the 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.