The process of preparing biological samples for transmission electron microscopy is typically a very protracted method that can take several days to complete. This means that the results for experiments are achieved slowly, hampering the progress of the research. Several “rapid” protocols have been described over the years [ 1, 2, 3] but even these take 1 to 2 days and have never been widely accepted. This presentation will show new protocols that allow for the entire process, from live specimen to fully polymerised resin embedded sample to be completed in under 1 hour, with results that are equivalent to those achieved by the standard protocols.
The standard processing method can be divided into several sections: fixation, dehydration, infiltration with resin and resin polymerisation. Typically, each step of the double fixation in glutaraldehyde followed by osmium tetroxide is 1 to 2 hours. However, it has long been known that the chemical reactions that occur to give good structural preservation are extremely fast and take less than 1 minute to complete [ 4]. This means that any extension of time beyond 1 minute for these steps is only to ensure that the fixative fully diffuses throughout the entire sample. So, if it were possible to enhance the diffusion, the process of fixation could be greatly shortened. Two methods for achieving an increased diffusion rate have been used successfully in this study, allowing a time as short as 5 minutes in each fixative step.
The first method involves the use of microwave energy. This significantly increases the diffusion of the fixatives as well as promoting the chemical reactions involved.