Subsurface examination of a foliar biofilm using scanning electron- and focused-ion-beam microscopy

摘要: The dual beam scanning electron microscope, equipped with both a focused ion- and electron-beam (FIB SEM) is novel tool for the exploration of subsurface structure biological tissues. FIB can remove predetermined amount material from selected site to allow when coupled SEM or ion-beam microscopy (SIM) could be suitable examine bacterial biofilms on leaf surface. suitability chemical cryofixation was examined use surfaces. control agent, Burkholderia pyroccinia FP62, that rapidly colonizes surface forms biofilms, inoculated onto geranium leaves incubated in greenhouse 7 14 days. Cryofixation not examination because it created frozen layer over cracked exposed protective cap required milling accurately deposited. With chemically fixed samples, possible precisely mill single cross section (5μm) sequential sections without any damage surrounding Biofilms, days post-inoculation (DPI), were composed 2-5 cell layers while DPI ranged 5 greater than 30 layers. Empty spaces between bacteria cells observed 7- 14-DPI. Sequential inferred empty often continuous FP62 possibly make up network channels throughout biofilm. useful observe composition foliar