Current trends in electron microscopy are focused on analysis of beam-sensitive samples. Cryo-electron tomography (cryo-ET), a method for studying the ultrastructure of vitrified biological specimen has recently become a popular approach in structural biology. A requirement for obtaining good quality results is that the samples are thin enough to enable transparency to an electron beam. However, a typical biological specimen is highly topographic with sizes far exceeding the required thickness, typically <500 nm [1].
Cryo-sectioning is a standard method used for thinning and slicing such samples. However, the integrity of the specimens can be easily degraded by common artifacts that are caused by knife marks, compression or crevasses. [2]. On the other hand, focused ion beam (FIB) nanofabrication is extensively used to reveal sub-surface information from bulk material, to shape samples for other analytical modalities, or to prepare an ultra-thin specimen for analysis in transmission electron microscopy (TEM).
We have successfully applied this technique to cryogenically frozen samples, thus demonstrating that it can be successfully used for preparing a TEM lamella from the vitrified specimen directly on the TEM grid. The quality of the prepared specimens was checked using cryo-TEM.
Moreover, a combination of the scanning electron microscope (SEM) with a nanomachining capabilities of FIB opens up a wider range of possibilities. FIB-SEM systems are widely used not only for routine preparation of ultra-thin TEM specimens but also for their capability of precise cross-sectioning and 3D volume imaging, applications which are possible in both ambient temperatures as well as in cryogenic conditions.
Appropriate applications with recent results from the structural analysis will be presented.
[1] J.Arnold et al, Biophysical Journal, Vol. 110 (2016), p. 860-869
[2] A.Al-Amoudi, D. Studer and J. Dubochet, Journal of Structural Biology, Vol. 150 (2005), p. 109-121