Three-dimensional observation of foraminiferal cytoplasmic morphology and internal structures using uranium–osmium staining and micro-X-ray computed tomography

•Benthic foraminiferal cytoplasm that is stained with osmium and uranium is clearly visible with micro-X-ray CT.•Large vacuoles, plugs, and perhaps nuclei can be identified in X-ray CT images.•X-ray CT is able to quantify the cytoplasmic and vacuole volume in each chamber.•The method provides basic but crucial knowledge regarding foraminiferal ecology and biology.

Benthic foraminifera are common protists that inhabit a broad environmental range of the seafloor. Because of their large biomass, these organisms play crucial roles in biogeochemical cycles, but our knowledge of their ecology and cell biology are still limited. One reason is that the calcareous or agglutinated tests (shells) of foraminifera hamper observing or measuring the cytoplasm from outside the organism. Here, we report the three-dimensional (3-D) observation of the cytoplasm and several ultrastructures of deep-sea benthic foraminifera using the combined techniques of micro-X-ray computed tomography (CT) and the osmium- and uranium-based cytoplasm-staining methods that are standard protocols for transmission electron microscope (TEM) observation. Osmium and uranium bind to organic membranes, proteins, and nucleic acids, rendering them visible by X-rays due to their high X-ray attenuation and enabling their reconstruction as 3-D images. This methodology revealed not only the external cellular morphology but also vacuoles and plugs inside the cell. Furthermore, volumetric calculation of cytoplasm and vacuoles is possible using the obtained CT data to evaluate their biomass and roles of vacuoles. Our method offers rapid (less than an hour in this study) evaluation of whole single or multiple foraminiferal cells and can be applied to investigate the biology and ecology of the foraminifera and other testate eukaryotes whose internal features are obscured by their external test structures.