Multi-fluorescence high-resolution episcopic microscopy (MF-HREM) for three dimensional imaging of adult murine organs

Claire Walsh, Natalie Holroyd, Eoin Finnerty, Sean Ryan, Paul Sweeney, Rebecca Shipley, Simon Walker-Samuel

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Three-dimensional microscopy of large biological samples (greater than 0.5 cm^3) is transforming biological research. Many existing techniques require trade-offs between image resolution, sample size and method complexity. A simple robust instrument with the potential to perform large volume 3D imaging currently exists in the form of the Optical HREM, however the development of the instrument to date is limited to single fluorescent wavelength imaging with non-specific eosin staining. This work presents developments to realize the potential of the HREM to become Multi-fluorescent High Resolution Episcopic Microscopy (MF-HREM). MF-HREM is a serial-sectioning and block-facing wide-field fluorescence imaging technique, which does not require tissue clearing or optical sectioning. Multiple developments are detailed in sample preparation and image post-processing to enable multiple specific stains in large samples, and show how these enable segmentation and quantification of the data. The application of MF-HREM is demonstrated in a variety of biological contexts: 3D imaging of whole tumor vascular networks and tumor cell invasion in xenograft tumors up to 7.5 mm^3 at resolutions of 2.75 mm, quantification of glomeruli volume in the adult mouse kidney, and quantification of vascular networks and white matter track orientation in adult mouse brain.
Original languageEnglish
Article number2100110
Number of pages13
JournalAdvanced Photonics Research
Early online date26 May 2021
Publication statusE-pub ahead of print - 26 May 2021


  • serial-sectioning
  • HREM
  • deconvolution
  • tumor
  • whole-mount


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