black. Therefore, for CryoET, samples should be less than ~500 nm thick to achieve "macromolecular" resolution (~4 nm). For this reason, most ECT studies have focused on purified macromolecular complexes, viruses, or small cells such as those of many species of Bacteria and Archaea. Cryotomography was used to understand encapsulation of 12 nm size protein cage nanoparticles inside 60 nm sized virus-like nanoparticles. Larger cells, and even tissues, can be prepared for CryoET by thinning, either by cryo-sectioning or by focused ion beam (FIB) milling. In cryo-sectioning, frozen blocks of cells or tissue are sectioned into thin samples with a cryo-microtome. In FIB-milling, plunge-frozen samples are exposed to a focused beam of ions, typically gallium, that precisely whittle away material from the top and bottom of a sample, leaving a thin lamella suitable for ECT imaging. The strong interaction of electrons with matter also results in an anisotropic resolution effect. As the sample is tilted during imaging, the electron beam interacts with a relatively greater cross-sectional area at higher tilt angles. In practice, tilt angles greater than approximately 60–70° do not yield much information and are therefore not used. This results in a "missing wedge" of information in the final tomogram that decreases resolution parallel to the electron beam. For structures that are present in multiple copies in one or multiple tomograms, higher resolution (even ≤1 nm) can be obtained by subtomogram averaging. Similar to single particle analysis, subtomogram averaging computationally combines images of identical objects to increase the signal-to-noise ratio. A major obstacle in CryoET is identifying structures of interest within complicated cellular environments. One solution is to apply correlated cryo-fluorescence light microscopy, and even super-resolution light microscopy (e.g. cryo-PALM), and CryoET. In these techniques, a sample containing a fluorescently-tagged protein of interest is plunge-frozen and first imaged in a light microscope equipped with a special stage to allow the sample to be kept at sub-crystallization temperatures (< −150 °C). The location of the fluorescent signal is identified and the sample is transferred to the CryoTEM, where the same location is then imaged at high resolution by CryoET. See also Electron microscopy Electron tomography Transmission electron cryomicroscopy Transmission electron microscopy References External links Getting started in cryo-EM course (Caltech) Cell biology Electron microscopy techniques