Observation of tissues in open aqueous solution by atmospheric scanning electron microscopy: Applicability to intraoperative cancer diagnosis

Abstract

In the atmospheric scanning electron microscope (ASEM), a 2- to 3-μm layer of the sample resting on a silicon nitride-film window in the base of an open sample dish is imaged, in liquid, at atmospheric pressure, from below by an inverted SEM. Thus, the time-consuming pretreatments generally required for biological samples to withstand the vacuum of a standard electron microscope are avoided. In the present study, various mouse tissues (brain, spinal cord, muscle, heart, lung, liver, kidney, spleen and stomach) were fixed, stained with heavy metals, and visualized in radical scavenger D-glucose solution using the ASEM. While some stains made the nuclei of cells very prominent (platinum-blue, phosphotungstic acid), others also emphasized cell organelles and membranous structures (uranium acetate or the NCMIR method). Notably, symbiotic bacteria were sometimes observed on stomach mucosa. Furthermore, kidney tissue could be stained and successfully imaged in <30 min. Lung and spinal cord tissue from normal mice and mice metastasized with breast cancer cells was also examined. Cancer cells present in lung alveoli and in parts of the spine tissue clearly had larger nuclei than normal cells. The results indicate that the ASEM has the potential to accelerate intraoperative cancer diagnosis, the diagnosis of kidney diseases and pathogen detection. Importantly, in the course of the present study it was possible to increase the observable tissue area by using a new multi-windowed ASEM sample dish and sliding the tissue across its eight windows.

Secretory glands and microvascular systems imaged in aqueous solution by atmospheric scanning electron microscopy (ASEM)

“…Serous vesicles, which contain a large number of protein, were strongly stained (Figure ), while vesicles containing mucin (Figure ) or lipid (Figure ) were weakly stained. This is in good agreement with previous results that show that PTA stains proteins (Maruyama et al, ) and chromatin (Memtily et al, ; Nishiyama et al, ). The results indicate that PTA staining in combination with ASEM might be an effective way to study various vesicle related phenomena; not only exocytosis but also endocytosis, the generation of microvesicles, membrane‐protein integration, trafficking of microvesicles in cells, as well as artificially synthesized vesicles for material science.…”

“…Thus, immunolabeling is as easy as for OM (Hirano et al, ; Kinoshita et al, ; Maruyama et al, ; Sato et al, ), making it possible to combine the technique with labeling procedures already used to study diabetes. ASEM might also be applicable to the study of other vasculature‐related diseases including cerebral infarction in which white blood cells and platelet accumulations are known to play critical roles; platelets (Hirano et al, ), white blood cells, e.g., dendritic cells and megakaryocites (Hirano et al, ), and blood cells in spleen (Memtily et al, ), have all been clearly imaged by ASEM.…”

“…ASEM images were recorded using the ClairScope ASEM system (JASM‐6200; JEOL, Tokyo, Japan) as described (Memtily et al, ). Briefly, the standard 35 mm bio‐ASEM specimen stage with eight 100‐nm thick (250 × 250 μm 2 ) SiN film windows was employed to observe tissue samples.…”

“…A PTA‐stained tissue segment immersed in 10 mg ml −1 (w/v) d ‐glucose (Dextrose; MP Biomedicals LLC), was laid cut face down on the SiN film of the ASEM specimen stage, and observed using the inverted SEM. While being monitored from above using the dissecting microscope, the tissue was pushed slightly to the side with tweezers to obtain the desired view (Memtily et al, ). The acceleration voltage of the SEM was 30 kV, backscattered electrons (BSE) from the tissue were recorded by a BSE imaging (BEI) detector (Figure a) for visualization.…”

“…The resolution immediately above the SiN film is 8 nm. By placing glutaraldehyde‐fixed tissue slabs on the SiN film of the ASEM specimen stage, mouse brain, smooth/skeletal muscle, lung, kidney, and stomach tissue (Memtily et al, ) and gold fish brain (Nishiyama et al, ), were successfully imaged. Comparison of the end plates of motor neurons formed on skeletal muscles of genetically modified and wild‐type Drosophila by ASEM, indicated involvement of T antigen in the localization of neuromuscular junction (NMJ) boutons and in the synaptogenesis of NMJs (Itoh et al, ).…”