The main constituent of all living organisms is water. In fact, at least 70% can be water and it is involved in many of the processes of life. Within even the simplest of living organisms there are a myriad of dynamic processes going on at any one time. These make biological samples incompatible with observation in an electron microscope. The vacuum inside the microscope and the damage from the electron beam mean it’s not possible to look at live organisms.

In order to preserve a living organism for electron microscopy the specimen is commonly fixed and dehydrated. Unfortunately, these chemical processes cause structural changes to the organism. The cross-linking of a fixative will lead to some physical modifications, and the removal of the water through dehydration will cause substantial shrinkage.

However, frozen water, ice, can be observed in the electron microscope. If the freezing process is done in such a way as to prevent or limit ice crystal formation, the preservation of a hydrated specimen should be of sufficiently high quality to be observed in this frozen state in the electron microscope.

Because of this ideal preservation, cryo-EM is compatible with both structural and analytical studies on biological samples from cells to viruses, food products to polymers. It offers a way to see biological structures in high resolution, even at the molecular level.

A reconstructed and segmented tomogram of a mitochondrion in a mouse embryonic fibroblast (MEF) cell. Green is the inner membrane, red the outer membrane, blue the cristae membranes and yellow the ATP synthases. Movie courtesy of Georg Ramm, Monash University.

A. Escherichia coli processed at room temperature by chemical fixation, dehydration and embedding in resin before being stained with heavy metals. B. Legionella pneumophila frozen by plunge freezing and observed in a cryo-TEM still in the vitrified state. The structure in A. shows many artefacts that are caused by this processing. The outer and plasma membranes are difficult to discern and very wrinkled in appearance. The peptidoglycan material of the periplasm is largely dissolved during the processing. The DNA of the nucleoid has precipitated into coarse strands. In B. the membranes clearly exhibit the true straight bilayer appearance and the periplasm is still fully intact. The DNA of the nucleoid is difficult to see as it consists of fine stands that fill the ribosome-free regions. Due to the lack of heavy metals in the sample in B. the contrast is very low. Images courtesy of Rick Webb, University of Queensland and Debnath Ghosal, University of Melbourne. Bar 100nm.