Electron Microscopy and Diffraction of Wet Unstained and Unfixed Biological Objects

Ever since the invention of electron microscopy, there has been the desire to image biological samples and other samples, such as colloids, in their native liquid environment (as one can do with light microscopy), and various approaches have been developed throughout the years. The usage of microchip technology to produce micrometer-sized liquid enclosures with electron transparent silicon nitride (SiN) windows has spurred the research area of transmission electron microscopy (TEM) in liquid over the past decade. Solid material can be studied in situ in liquid layers of up to several hundreds of nanometers using liquid-cell TEM. Much thicker samples of up to 10 micrometers (μm) are available for the imaging of materials with a high atomic number (Z) in low-Z liquids using scanning transmission electron microscopy (STEM). In this chapter, a detailed discussion is presented of the practical aspects of the three most frequently used technical approaches for electron microscopy of liquid specimens: (1) environmental SEM (ESEM), (2) TEM and STEM of closed liquid cells, and (3) TEM and STEM of liquid flow devices. Details about the required equipment are also included. Liquid electron microscopy experiments need to be carried out carefully, and various factors need to be optimized. Nevertheless, user-friendly systems are now available, and exciting, novel scientific breakthroughs can be expected to result from the new capabilities to view images in liquid at a (sub-)nanoscale resolution.