Whole-ExM: Nanoscale imaging of all anatomical features over whole vertebrate via covalent staining and expansion microscopy

Abstract

Systematic understanding of biological systems necessitates investigating the entire organism at subcellular levels, but this has not yet been achieved. Here, we demonstrate whole-ExM, which enables three-dimensional nanoscale imaging of anatomical features across the entire vertebrate by whole-body staining with NHS-ester fluorophores and physically expanding the specimens four-fold. To accomplish this, we first optimize the fluorophore selection and labeling procedure to visualize a broader range of anatomical structures. We then develop an expansion protocol suitable for whole-body vertebrates having calcified body parts. Through this process, we successfully revealed the nanoscale details of diverse organs, which have corresponding organ counterparts in humans, over the intact organismal-level of specimens. Finally, we also verified that whole-ExM is compatible with target-specific fluorescent labeling methods, and its resolution is high enough to exhibit a wide range of structures that can be imaged only with electron microscopy. We believe that whole-ExM will pave the way for new developments in biology and medicine with its outstanding performance.