Low-temperature low-voltage scanning electron microscopy (LTL VSEM) of uncoated frozen biological materials: A simple alternative
Introduction.Scanning Electron Microscopy (SEM) of biological materials, e.g. plants, fungi, insects, bacteria, host-pathogen interactions, and food systems, requires sample stabilization. Conventional strategies of chemical fixation (e.g. buffered glutaraldehyde, paraformaldehyde, osmium tetroxide), dehydration (ethanol or acetone series), critical point drying (CPD) and metal coating (Au/Pd) in a sputter coater or vacuum evaporator are often by applied. However, delicate biological materials may not withstand the stress of conventional preparation and acquire artifact such as collapse or shrinkage of cells or loss of soluble components (Fig la). Sophisticated devices and techniques are available which quick-freeze (<1.0 s), metal coat and transfer specimens under vacuum into the SEM. Herein I describe a simple, inexpensive alternative in which fresh biologicals are frozen slowly (< 1.5 min.) on an SEM cold stage, imaged directly without metal coating (Fig lb) at low SEM acceleration voltage (kV), and defrosted if needed with nitrogen gas (N2).