Reviving formalin-fixed, paraffin embedded (FFPE) tissues for on-slide and multiscale correlative microscopy.
Authors
Affiliations (14)
Affiliations (14)
- School of Medical Sciences (Molecular Biomedicine Research Theme), The University of Sydney, Camperdown, NSW, 2006, Australia.
- Sydney Microscopy and Microanalysis, The University of Sydney, Camperdown, NSW, 2006, Australia.
- Charles Perkins Centre Histology Facility, The University of Sydney, Camperdown, NSW, 2006, Australia.
- Department of Pathology & Laboratory Medicine, University of Vermont, Burlington, 05405, VT, USA.
- Microscopy Imaging Center, Center for Biomedical Shared Resources, Larner College of Medicine, University of Vermont, Burlington, 05405, VT, USA.
- Division of Cardiovascular Medicine, University of Kentucky, Lexington, KY, 40536, USA.
- The Health Hub, Torrens University Australia, Surry Hills, NSW, Australia, 2010.
- Department of Rural Clinical Sciences, La Trobe Rural Health School, La Trobe University, Bendigo, VIC, Australia, 3550.
- Australian Centre for Microscopy and Microanalysis, The University of Sydney, Camperdown, NSW, 2006, Australia.
- Microscopy Australia (Sydney Node), The University of Sydney, Camperdown, NSW, 2006, Australia.
- School of Medical Sciences (Molecular Biomedicine Research Theme), The University of Sydney, Camperdown, NSW, 2006, Australia. [email protected].
- Sydney Microscopy and Microanalysis, The University of Sydney, Camperdown, NSW, 2006, Australia. [email protected].
- Australian Centre for Microscopy and Microanalysis, The University of Sydney, Camperdown, NSW, 2006, Australia. [email protected].
- Microscopy Australia (Sydney Node), The University of Sydney, Camperdown, NSW, 2006, Australia. [email protected].
Abstract
Correlative microscopy integrates complementary imaging modalities to enable structural analysis of biological samples across multiple length scales. Here, we present two correlative workflows for formalin-fixed, paraffin-embedded (FFPE) human tissues that extend the utility of archived clinical specimens for high-resolution imaging. The first, an on-slide correlative light and electron microscopy (CLEM) workflow, enables direct integration of brightfield light microscopy with scanning electron microscopy to resolve ultrastructural features from standard histological sections. The second, a multiscale three-dimensional workflow, combines X-ray micro-computed tomography with serial block-face scanning electron microscopy to enable targeted, volumetric ultrastructural analysis within a defined tissue context. Using these approaches, we demonstrate that FFPE tissues retain sufficient structural integrity to resolve cellular and subcellular architecture across liver, lung, brain, and heart specimens. Finally, we apply artificial intelligence-assisted image processing and segmentation to enable scalable, quantitative analysis of large-volume datasets. Together, these workflows establish FFPE specimens as a versatile and accessible resource for multiscale correlative imaging in both research and clinical settings.