Wet STEM: a new development in environmental SEM for imaging nano-objects included in a liquid phase

Environmental scanning electron microscopy (ESEM) enables wet samples to be observed without potentially damaging sample preparation through the use of partial water vapour pressure in the microscope specimen chamber. However, in the case of latices in colloidal state or microorganisms, samples are not only wet, but made of objects totally submerged in a liquid phase. In this case, under classical ESEM imaging conditions only the top surface of the liquid is imaged, with poor contrast, and possible drifting of objects. The present paper describes experiments using a powerful new Scanning Transmission Electron Microscopy (STEM) imaging system, that allows transmission observations of wet samples in an ESEM. A special device, designed to observe all sorts of objects submerged in a liquid under annular dark-field imaging conditions, is described. Specific features of the device enable to avoid drifting of floating objects which occurs in the case of a large amount of water, thus allowing slow-scan high-definition imaging of particles with a diameter down to few tens of nm. The large potential applications of this new technique are then illustrated, including the imaging of different nano-objects in water. The particular case of grafted latex particles is discussed, showing that it is possible to observe details on their surface when submerged in water. All the examples demonstrate that images acquired in wet STEM mode show particularly good resolution and contrast, without adding enhancing contrast objects, and without staining.