Sample preparation for Electron Microscopy
Imaging a biological specimen with a transmission electron microscope (TEM) is not a trivial task, mostly because of the intrinsic nature of the electron/matter interactions that are responsible for the image formation. In order to be imaged, the specimen has to be placed inside a high vacuum, and should be very thin. For these reasons, biological specimens can hardly be imaged in their native state and need to be processed. Therefore, sample preparation is a crucial step, which represents the essential, fundamental activity in an electron microscopy facility such as the EMCF.
The EMCF offers a range of sample immobilization techniques. These cover conventional techniques (chemical fixation) as well as cryo-methods (plunge-freezing, high pressure freezing (HPF)) which are routinely deployed and are used in experiments on single particles, cell cultures and multicellular organisms or tissues. Over the past years, the EMCF has acquired a strong expertise and reputation on cultured cells (bacteria, yeast cells, mammalian cells) built on successful experiments in many projects.
High pressure freezing and Freeze substitution
EMCF staff and autonomous users process a variety of samples by HPF and freeze substitution (FS). In particular the method is well established in house for yeast cells, cell suspensions, adherent culture cells, drosophila and mouse embryos, starfish oocytes and C. elegans.
While sample preparation is crucial before introducing the samples in the high vacuum of the EM, and to bring sufficient contrast for the creation of an EM image, it is important that the specimens are thin enough to transmit electrons. Therefore, creating ultra-thin sections of resin embedded or of frozen samples is an obligatory step when working on most specimens in cell biology. For this, the facility is equipped with a set of 5 ultramicrotomes, 3 of which having the ability to be switched from room temperature to cryo-sectioning modes. The machines are routinely used by the staff of the facility, and by trained users. They can be booked via the online booking system. Cryo-sectioning is part of a routine protocol for preparing the sections used for immuno-gold labeling (the Tokuyasu technique) that are offered as a service in the facility. This technique can also be used on vitrified samples, when one plans to image their sample by cryo-electron microscopy. CEMOVIS is not offered as a full service, but the facility has the expertise and the equipment for it and is often consulted for advice and for training in this field.