Cryo-electron microscopy
«Water is a beautiful substance. Its action is everywhere around us and in us. It has moulded the earth during geological times and it was the cradle of life. Even now, it is by far the most abundant constituent of living matter. Our body contains 65 % water and the most 'intelligent' part of it, our brain, is all water, except for a fifth of its mass. At the molecular level also, life works in, and with water. The liquid medium participates directly in many reactions and it provides the necessary mobility for the dynamics which is the essence of life. However, water has long been neglected in molecular biology.» (Dubochet J, et al. (1988). Q. Rev. Biophys. 21:129-228
). This is partly due to the fact that electron microscopes work under high vacuum and that at room temperature water evaporates under high vacuum. A biological specimen inserted without special care in the microscope dramatically collapses and is of little use for the study of fine structures, which the subnanometer resolution of transmission electron microscopes would in principle enable. Since the early days of electron microscopy in 1940s, biologists have developed tricks to alleviate this problem. We can distinguish two fundamental approaches. The first and earlier one consists of chemically fixing the specimen before staining it with heavy metals, gently dehydrating it, and embedding it in a resin. This processing preserves relatively well the original structure of the specimen and since the sample is now totally dry it can be introduced in the microscope without it collapsing. Yet this solution is not perfect: not every molecule are efficiently fixed, and when their natural solvent, water, is removed they either aggregate or get washed away. Furthermore, the necessary use of heavy-metal stains limits achievable resolution since the microscope does not image biological molecules but stain molecules bound to them.
In response to these problems, an alternative approach termed cryo-electron microscopy, or electron cryomicroscopy (cryo-EM), was developed in the early 1980s with the aim of keeping the specimen fully hydrated. Here the specimen is frozen very rapidly in order for the water not crystallize, a phenomenon termed vitrification. The specimen is then transferred to the microscope and observed at -180°C, a temperature where water does not evaporate even under high vacuum. No staining is necessary. The specimen is in principle preserved in its native state, and resolution is only limited by the relatively low signal-to-noise ratio inherent to cryo-EM images. For well-ordered structures, a single image can provide a resolution of about 1nm. If multiple copies of the same structures can be averaged, near-atomic (<5Å), or even atomic resolution (<3Å) can be achieved. 3-dimensional information can be obtained by single particle analysis if the specimen consists of purified biological molecules, or by electron tomography for non-pure samples, such as tissues, cells or organelles.
CEMOVIS
The majority of eukaryotic cells and several bacteria are too thick to be imaged by cryo-EM directly after vitrification. They must be thinned down. One approach to do that consists of cutting series of ultrathin sections (~60nm thick) with a diamond knife, at -150°C, in order for water to remain vitreous. CEMOVIS (cryo-electron microscopy of vitreous sections) has had the reputation of being an art restricted to a handful of highly skilled and trained scientists. We have introduced a set of tools that greatly facilitate the use of CEMOVIS (Studer et al. (2014) J. Struct. Biol. 185:125-128). This makes it possible to generate in a given amount of time volumes of observable specimen that are serveral orders of magnitude larger than what can be obtained with the alternative method, cryo-focused ion beam milling. Specimens prepared by CEMOVIS however currently suffer from compression along the cutting direction. It is to be expected that knives with better gliding properties could significantly reduce this effect.
Serial block face scanning electron microscopy (SBFSEM)
SBFSEM was pioneered in the mid 2000’s by Winfried Denk and colleagues. This method makes it possible to obtain the 3-dimensional ultrastructure of “very large” samples (very large, at the electron microscopic scale). Resin-embedded samples are mounted on an ultramicrotome, which is installed within the specimen chamber of a scanning electron microscope. The microtome cuts sections as thin as 15 nm; usually though section thickness is set to 50 nm. After each cut, the section is removed and the surface of the block is imaged at low voltage. The resulting images resemble transmission electron microscopy images, although their resolution is limited to around 20-30 nm. We apply SBFSEM in a number of collaborative projects in various fields such as neurobiology, vascular biology, and parasitology.
With this approach we study several aspects of synapse biology such as postsynaptic receptors organisation and synaptic transmission. People interested to join our group are welcome to contact Benoît Zuber (benoit.zuber@unibe.ch).
