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.

Figure 1

Schematic diagram of the ASEM and observation of heart tissues. (A) In the ASEM correlative microscope, tissue can be observed in aqueous liquid by the SEM from below through the SiN film of the ASEM dish and by OM from above, allowing various manipulations. Since the optical axes of the two microscopes are aligned, the central region of the sample overview obtained by OM can be imaged immediately afterwards at higher resolution by SEM. (B) Slab of heart tissue stained with PTA, immersed in radical scavenger D-glucose and observed by ASEM at low magnification. Longitudinal cardiomyocytes are evident and sometimes branched. (C and D) Higher magnification image of the white rectangle in the preceding panel. Myofibrils can be brightly distinguished above the background. (E) ASEM image of another cardiac muscle area. Nuclei (open arrowhead) and intercalated discs (black arrowhead) are visible; the nuclei between myocytes (white arrowheads) might belong to attached fibroblasts. Sarcomere striations are visible in myocytes: A-bands (white arrow) are broad bright zones, while I-bands are dark zones (black arrow). The Z-line can be faintly recognized as an indistinct thin white line at the center of I-bands. Inset, diagram of the sarcomeres.

Figure 2

ASEM images of neural tissues stained with PTA. In these images, bright nuclei are distinguished above the background, while cytoplasm and nerve fibers are darker, as they were only weakly stained. (A) Vertical section of the cerebrum showing a layered structure. (B and C) Higher magnification image of the white rectangle in the preceding panel. Different kinds of neurons with nuclei have various dendrites and are surrounded by glia cells. (D) Horizontal section of the cerebellum. The three clearly different layers visible are probably the molecular layer, the granular layer and cerebellar white matter. (E and F) Higher magnification images. Nuclei have brightly stained patches that are probably chromatin and nucleoli. (G) Vertical section of thoracic spinal cord. Neural networks can be distinguished, mainly running from top to bottom. (H and I) Higher magnification images. Minor connections in various directions are apparent.

Figure 3

ASEM images of liver. (A) Low magnification image of a tissue slab stained with UA. Hepatocytes, and probably sinusoids are visible. (B and C) Higher magnification image of the white rectangle in the preceding panel. Hepatocytes, sometimes with their nucleus (n) near the SiN support, can be clearly distinguished from erythrocytes (e). The brightly stained patches evident within some nuclei are probably chromatin and nucleoli. (D) Hepatocytes at the surface of a different tissue slab stained with PTA. Slightly more heavily stained collagen-like fibers (c) are visible as brighter strands. A nucleus (n) and fat droplet-like structures (f) can also be observed.

Figure 4

ASEM image of kidney cortex tissue stained with PTA. The features evident in the characteristic view are putatively assigned as: Bowman’s capsule (BC), podocytes (visceral epithelial cells; white arrowheads), a glomerulus (G), proximal convoluted tubules (PCT) and distal convoluted tubules (DCT).

Figure 5

ASEM images of gastrocnemius skeletal muscle stained with PTA. (A) Low magnification image. (B) Higher magnification image of the white rectangle in the preceding panel. Filament networks are evident as bright strands on striped striated muscle fibers. (C) Another specimen area. The muscle fiber has A-bands, evident as broad bright zones (white arrows), and I-bands, evident as dark zones (black arrows). Z-lines look like a faint thin white line in the middle of the I-bands (black arrowheads). Nuclei are bright (white arrowheads). Inset, diagram of the sarcomere.

Figure 6

ASEM images of digestive tract. (A) Low magnification images of the mucosal side of stomach stained with PTA. (B) Higher magnification image. Commensalism of bacteria (arrow) is revealed. (C) Another area of stomach stained by the modified NCMIR method. (D) Higher magnification image; bright symbiotic bacteria (arrow) are observed.

Figure 7

Images of spleen tissue. (A) OM. The spleen was cross-sectioned to obtain 5- to 6-μm, thin-sections, H&E stained and observed by OM. (B) ASEM. The spleen was independently cross-sectioned to obtain 200-μm-thick tissue slabs, stained with PTA and observed at low magnification by ASEM. (C–F) Higher magnification ASEM images of the white rectangle in the preceding panel. Differently shaped blood cells, presumably including lymphocytes, were observed.

Figure 8

Comparative ASEM observation of normal lung and lung metastasized by breast cancer cells. (A) OM of an H&E-stained thin-section of normal lung. Nuclei are stained blue and the cytoplasm is stained red. (B) Low magnification ASEM image of an independently-prepared tissue slab stained with both Pt-blue and PTA. Alveoli with alveolar ducts, a vein system and trachea are visible. (C and D) Higher magnification images of the white rectangle in the upper panel. Normal size nuclei are observed (arrowhead). (E–H) Comparative observation of tissue excised from a lung metastasized with breast cancer cells. (E) OM of an H&E-stained thin section. (F–H) ASEM of an independently-prepared Pt-blue- and PTA-stained thick slab at the same magnification as the corresponding left panels. Regular alveoli, alveolar ducts and alveolar cells are only faintly discernable. Most of the space is occupied by cells of different shapes with larger nuclei (arrowhead), i.e., cancer cells.

Figure 9

Comparative ASEM observation of normal spinal cord and spinal cord metastasized by breast cancer cells. Tissues are stained with both Pt-blue and PTA. (A and B) ASEM image of a slab of normal spinal cord. Normal size nuclei are observed at the end of fibers (arrowhead). (C and D) Comparative observation of tissue excised from a spinal cord metastasized with breast cancer cells. Unusually large nuclei similar to those seen in lung metastasized by breast cancer cells (Fig. 8F–H), were observed (arrowhead).

Figure 10

Wide area imaging by shifting a tissue on the ASEM dish. (A) Schematic diagram of the procedure. The shift caused by pushing with the tweezers is precisely controlled by low magnification monitoring using the upper OM (see Fig. 1A). (B) Schematic diagrams of a one-window and an 8-window ASEM dish. All windows are 250×250 μm. (C) Images of spinal cord tissue initially recorded from windows a, b, c and d. (D) Images recorded in the same windows after the tissue has been pushed in one direction causing it to slip across the SiN-film window. (E) Higher magnification image of window b in panel C and of window b′ in panel D. (F) Merged image of windows b and b′.