Erlandsen Stanley, Lei Ming, Martin-Lacave Ines, Dunny Gary, Wells Carol
Department of Genetics, Cell Biology and Development, University of Minnesota Medical School, Minneapolis, MN 55455, USA.
Microsc Microanal. 2003 Aug;9(4):273-8. doi: 10.1017/S1431927603030502.
The outer surfaces of three microorganisms, Giardia lamblia, Enterococcus faecalis, and Proteus mirabilis, were investigated by cryo-immobilization followed by sublimation of extracellular ice and cryocoating with either Pt alone or Pt plus carbon. Cryocoated samples were examined at -125 degrees C in either an in-lens field emission SEM or a below-the-lens field emission SEM. Cryocoating with Pt alone was sufficient for low magnification observation, but attempts to do high-resolution imaging resulted in radiolysis and cracking of the specimen surface. Double coating with Pt and carbon, in combination with high resolution backscatter electron detectors, enabled high-resolution imaging of the glycocalyx of bacteria, revealing a sponge-like network over the surface. High resolution examination of bacterial flagella also revealed a periodic substructure. Common artifacts included radiolysis leading to "cracking" of the surface, and insufficient deposition of Pt resulting in the absence of detectable surface topography.
通过冷冻固定，随后升华细胞外冰，并单独用铂或铂加碳进行冷冻镀膜，对三种微生物——蓝氏贾第鞭毛虫、粪肠球菌和奇异变形杆菌的外表面进行了研究。在-125摄氏度下，使用镜内场发射扫描电子显微镜或镜下场发射扫描电子显微镜对冷冻镀膜样品进行检查。单独用铂进行冷冻镀膜足以进行低倍观察，但进行高分辨率成像时，标本表面会发生辐射分解和开裂。铂和碳的双重镀膜，结合高分辨率背散射电子探测器，能够对细菌的糖萼进行高分辨率成像，揭示其表面的海绵状网络。对细菌鞭毛的高分辨率检查也揭示了一种周期性的亚结构。常见的伪像包括导致表面“开裂”的辐射分解，以及铂沉积不足导致无法检测到表面形貌。
