Safinya Group Research
Electron Microscopy

The resolution of optical microscopy and therefore the minimum size of structures that can be observed is limited by the wavelength of light. Special techniques, such as fluorescence microscopy and differential interference contrast (DIC) which are used in our laboratory allow detection of structures smaller than the wavelength of light, but for detailed structural investigation on the sub-micrometer scale, electron microscopy (EM) is a more appropriate tool. EM allows real-space observation of structures from micrometer down to a few nanometer in size. In this way, it is a technique that not only compements optical microscopy, but also (small angle) x-ray scattering, which gives information about structures on a similar length scale but as an ensemble average. Since obtaining statistical information from EM data is often very tedious and time consuming, the combination of SAXS and EM is a very powerful and efficient tool to investigate nanoscale structures, as e.g. our work on MT assembly by multivalent cations has shown [1].

Sample preparation is an important part of EM methodology, and we use a variety of techniques depending on the system under investigation. The best technique for investigating very fragile structures present in aqueous solution is cryogenic TEM (cryo-TEM), where samples are rapidly frozen in liquid ethane and then imaged, as opposed to the combination of staining and drying employed in conventional TEM (EM requires a high vacuum to be applied, thus the need for dried samples or very low temperatures). To image novel lipid phases and CL–DNA complexes, we perform cryo-TEM at the National Resource for Automated Molecular Microscopy (NRAMM) at the Scripps Institute in San Diego. To image microtubules by transmission EM (TEM), we use both whole-mount and plastic embedding techniques. The later allows cross-sectional imaging, which is important for assessing MT diameter and identifying assemlies such as the living necklace phase [1]. We perform transmission electron microscopy (TEM) at the TEM facility in the Biological Sciences Department at UCSB.

[1] Needleman, D. J.; Ojeda-Lopez, M. A.; Raviv, U.; Miller, H. P.; Wilson, L.; Safinya, C. R.: Higher-order assembly of microtubules by counterions: From hexagonal bundles to living necklaces. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 16099-16103.