High-Resolution Cryoscanning Electron Microscopy of Biological Samples

  • Paul Walther


Cryoscanning electron microscopy of fast frozen samples is the most direct approach for imaging aqueous organicmaterial at nanometer scale resolution. It circumvents artifact formation caused by chemical fixation and drying. Cryofixation is preferentially done by high-pressure freezing because it allows for fixation of native samples up to 200 μm thick and 2mm wide with minimal or no ice crystal damage. Because in the SEM only the surface of the sample (natural or artificial) is visible, water-covered biological surfaces must be made accessible to the electron beam by either freeze substitution, critical-point drying or freeze drying. If one wishes to view the inner part of a frozen sample, it must be opened by fracturing or by cryosectioning and then be coated with an electrically-conductive layer both to prevent charging and to produce contrast at the sample surface. Here we describe a method developed specifically for frozen-hydrated bulk samples: doublelayer coating. In this approach, the frozen sample is coated in a manner similar to that used to produce a TEM-replica, e.g., with a shadow of platinum (at an angle of 45°) followed by an additional layer of carbon. The sample is then cryotransferred to an SEM equipped with a cold stage and imaged using the material-dependent backscattered electron signal (BSE), which shows the platinum distribution. With this method, charging artifacts and the effects of beam damage are significantly reduced comparedwith secondary electron imaging of conventionally-coated frozen samples. Although currently the resolution of the replica technique cannot be surpassed, double compared to layer coating greatly facilitates the processing of brittle rapidly frozen samples, because in contrast to TEM, no removal of the biological material from the replica is necessary. This makes cryo-SEM especially suitable for the analysis of high-pressure frozen samples. For small samples, such as macromolecular complexes or viral particles, the best resolution was achieved by a uniform coating with about 1.5 nm of tungsten and high-resolution imaging with secondary electrons. This method provides an extremely good signal-to-noise ratio and allows for macromolecular resolution without image averaging and has, therefore, a high potential for imaging of single events, such as actin-membraneinteractions.


Tobacco Mosaic Virus Freeze Sample Primary Beam Uncoated Sample Freeze Substitution 
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© Springer Science+Business Media, LLC 2008

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  • Paul Walther

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