Rapid imaging of mycoplasma in solution using Atmospheric Scanning Electron Microscopy (ASEM)

• Published in: Biochemical and biophysical research communications Vol. 417; no. 4; pp. 1213 - 1218
• Main Authors: Sato, Chikara, Manaka, Sachie, Nakane, Daisuke, Nishiyama, Hidetoshi, Suga, Mitsuo, Nishizaka, Takayuki, Miyata, Makoto, Maruyama, Yuusuke
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 27.01.2012
• Subjects: 60 APPLIED LIFE SCIENCES; BACTERIA; Bacterial Proteins - analysis; Bacterial Proteins - metabolism; BIOMEDICAL RADIOGRAPHY; CHEST; Correlative microscopy; DIAGNOSIS; ELECTRON MICROSCOPES; Electron microscopy; Environmental cell; Imaging; Immunohistochemistry; LABELLING; Microscopy, Electron, Scanning - methods; MYCOPLASMA; Mycoplasma - isolation & purification; Mycoplasma - metabolism; Mycoplasma - ultrastructure; Mycoplasma mobile; N-Acetylneuraminic Acid - chemistry; OPTICAL MICROSCOPES; OPTICAL MICROSCOPY; PATHOGENS; PNEUMONIA; RADIOLOGY AND NUCLEAR MEDICINE; SCANNING ELECTRON MICROSCOPY; SIALIC ACID; Silicon Compounds - chemistry; SILICON NITRIDES; Solutions; THIN FILMS; TRANSMISSION ELECTRON MICROSCOPY; VERTEBRATES; VIRUSES; PBS; RT; ASEM; Correlative microscopy; BEI; EM; Electron microscopy; Environmental cell; Imaging; Mycoplasma mobile; SiN; Bacteria; SEM; TEM; OM
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/j.bbrc.2011.12.111

► Mycoplasma mobile was observed in buffer with the Atmospheric Scanning Electron Microscope. ► Characteristic protein localizations were visualized using immuno-labeling. ► M. mobile attached to sialic acid on the SiN film surface within minutes. ► Cells were observed at low concentrations. ► ASEM should promote study and early-stage diagnosis of mycoplasma. Mycoplasma is a genus of bacterial pathogen that causes disease in vertebrates. In humans, the species Mycoplasma pneumoniae causes 15% or more of community-acquired pneumonia. Because this bacterium is tiny, corresponding in size to a large virus, diagnosis using optical microscopy is not easy. In current methods, chest X-rays are usually the first action, followed by serology, PCR amplification, and/or culture, but all of these are particularly difficult at an early stage of the disease. Using Mycoplasma mobile as a model species, we directly observed mycoplasma in buffer with the newly developed Atmospheric Scanning Electron Microscope (ASEM). This microscope features an open sample dish with a pressure-resistant thin film window in its base, through which the SEM beam scans samples in solution, from below. Because of its 2–3μm-deep scanning capability, it can observe the whole internal structure of mycoplasma cells stained with metal solutions. Characteristic protein localizations were visualized using immuno-labeling. Cells were observed at low concentrations, because suspended cells concentrate in the observable zone by attaching to sialic acid on the silicon nitride (SiN) film surface within minutes. These results suggest the applicability of the ASEM for the study of mycoplasmas as well as for early-stage mycoplasma infection diagnosis.