We previously demonstrated that human osteosarcoma cells (SAOS‐2) induce contact‐dependent apoptosis in endothelium, and expected similar apoptosis in human gingival fibroblasts (h‐GF) using SAOS‐2 alkaline phosphatase (AP) to identify cells. However, h‐GF apoptosis did not occur, despite reduction in AP‐negative h‐GF number (p < 0.01) and enhancement of this by h‐GF TNFα pretreatment (p < 0.01). We suggest that TNFα‐enhanced transfer of membrane AP from SAOS‐2 to h‐GF would explain these data. This idea was investigated using fluorescence prelabelled cells and confocal laser scanning microscopy. Co‐cultures of membrane‐labelled h‐GF (marker‐DiO) and SAOS‐2 (marker‐DiD) generated dual‐labelled cells, primarily at the expense of single labelled h‐GF (p < 0.001), suggesting predominant membrane transfer from SAOS‐2 to h‐GF. However, opposite directional transfer predominated when membrane labels were reversed; SAOS‐2 further expressed green fluorescent protein (GFP) in cytoplasm and nuclei, and h‐GF additionally bore nuclear label (Syto59) (p < 0.001). Cytoplasmic exchange was investigated using h‐GF prelabelled with cytoplasmic DDAO‐SE and nuclear Syto59, co‐cultured with SAOS‐2 expressing GFP in cytoplasm and nuclei, and predominant cytoplasmic marker transferred from h‐GF to SAOS‐2 (p < 0.05). Pretreating h‐GF with TNFα increased exchange of membrane markers (p < 0.04) but did not affect either cell surface area profile or circularity. Dual‐labelled cells had a morphological phenotype differing from SAOS‐2 and h‐GF (p < 0.001). Time‐lapse microscopy revealed extensive migration of SAOS‐2 and cell process contact with h‐GF, with the appearance of SAOS‐2 indulging in ‘cellular sipping’ from h‐GF. Similar exchange of membrane was seen between h‐GF and with other cell lines (melanoma MeIRMu, NM39, WMM175, MM200‐B12; osteosarcoma U20S; ovarian carcinoma cells PE01, PE04 and COLO316), while cytoplasmic sharing was also seen in all cell lines other than U20S. We suggest that in some neoplasms, cellular sipping may contribute to phenotypic change and the generation of diverse tumour cell populations independent of genetic change, raising the possibility of a role in tumour progression. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Summary Leaf function is intimately related to the size, shape, abundance and position of cells and chloroplasts. Anatomy has long been assessed and quantified in two dimensions with 3D structure inferred from 2D micrographs. Serial block face scanning electron microscopy (SBF‐SEM) was used to reconstruct 95 cells and 1173 chloroplasts from three wheat and nine chickpea leaves (three samples each from three chickpea genotypes). Wheat chloroplast volume was underestimated by 61% in mesophyll cells and 45% in bundle sheath cells from 2D micrographs, whereas chickpea mesophyll chloroplast volume was underestimated by 60% using simple geometrical models. Models of chickpea spongy and palisade cells both under‐ and overestimated surface area and volume by varying degrees. These models did not adequately capture irregular shapes such as flattening of chloroplasts or lobed spongy mesophyll cells. It is concluded that simple geometrical models to estimate chloroplast and cell 3D volume and surface area from 2D micrographs are inadequate, and that SBF‐SEM has strong potential to contribute to improved understanding of leaf form and function.

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Following injury to the central nervous system, axons and myelin distinct from the initial injury site undergo changes associated with compromised function. Quantifying such changes is important to understanding the pathophysiology of neurotrauma; however, most studies to date used 2 dimensional (D) electron microscopy to analyse single sections, thereby failing to capture changes along individual axons. We used serial block face scanning electron microscopy (SBF SEM) to undertake 3D reconstruction of axons and myelin, analysing optic nerves from normal uninjured female rats and following partial optic nerve transection. Measures of axon and myelin dimensions were generated by examining 2D images at 5 µm intervals along the 100 µm segments. In both normal and injured animals, changes in axonal diameter, myelin thickness, fiber diameter, G-ratio and percentage myelin decompaction were apparent along the lengths of axons to varying degrees. The range of values for axon diameter along individual reconstructed axons in 3D was similar to the range from 2D datasets, encompassing reported variation in axonal diameter attributed to retinal ganglion cell diversity. 3D electron microscopy analyses have provided the means to demonstrate substantial variability in ultrastructure along the length of individual axons and to improve understanding of the pathophysiology of neurotrauma.

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To-date serial block-face scanning electron microscopy (SBF-SEM) dominates as the premier technique for generating three-dimensional (3-D) data of resin-embedded biological samples at an unprecedented depth volume. Given the infancy of the technique, limited literature is currently available regarding the applicability of SBF-SEM for the ultrastructural investigation of tissues. Herein, we provide a comprehensive and rigorous appraisal of five different SBF-SEM sample preparation protocols for the large-volume exploration of the hepatic microarchitecture at an unparalleled X, Y and Z resolution. In so doing, we qualitatively and quantitatively validate the use of a comprehensive SBF-SEM sample preparation protocol, based on the application of heavy metal fixatives, stains and mordanting agents. Employing the best-tested SBF-SEM approach, enabled us to assess large-volume morphometric data on murine parenchymal cells, sinusoids and bile canaliculi. Finally, we integrated the validated SBF-SEM protocol with a correlative light and electron microscopy (CLEM) approach. The combination of confocal scanning laser microscopy and SBF-SEM provided a novel way to picture subcellular detail. We appreciate that this multidimensional approach will aid the subsequent research of liver tissue under relevant experimental and disease conditions.

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