Observation of tissues in open aqueous solution by atmospheric scanning electron microscopy (ASEM)

[Speaker] Toshiko Yamazawa:1
[Co-author] Naotoshi Nakamura:2, Mari Sato:3, Chikara Sato:3
1:Department of Molecular Physiology, The Jikei University School of Medicine, Japan, 2:Department of Statistical Genetics, Graduate School of Medicine, Kyoto University, Japan, 3:Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Japan

Ca2+ signaling controls many physiological functions. In particular, key steps in transmitter release, muscle contraction, and secretion are controlled by Ca2+. Salivary glands secrete fluid and proteins in response to specific neurotransmitter-generated intracellular signals in exocrine /endocrine cells. There are three pairs of major salivary glands in mammals, the parotid glans, submandibular glands and sublingual glands. These glands secrete serous, mucous or mixed saliva via the proper main excretory ducts connecting the glandular bodies with the oral cavity. In Sjögren's syndrome, secretory production is diminished for the primary exocrine gland and the lacrimal or salivary glands, leading to dry eye and mouth. Here, we develop atmospheric scanning electron microscopy (ASEM) to tissues immersed in aqueous solution. Salivary glands and lacrimal glands were glutaraldehyde fixed, sectioned into slabs, stained with phosphotungstic acid (PTA), and observed in radical scavenger D-glucose solution from below by an inverted scanning electron microscopy (SEM), guided by optical microscopy from above to target the tissue substructures. A 2 to 3-µm specimen thickness was visualized by the SEM. In salivary and lacrimal glands, secretory vesicles and other organelles were clearly imaged at high resolution, and the former could be classified according to the degree of PTA staining. Using the same method on islets of Langerhans, the microvascular system used as an outlet by the secretory cells was also clearly observed. Microvascular system is also critically involved in the onset of diabetic complications and was clearly visible in subcutaneous tissue imaged by ASEM. The results suggest the usefulness of in-solution ASEM for histology, and the high-throughput ASEM is promising for the diagnosis of exocrine/endocrine-related disease.

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