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fixed usingfreeze substitution methods. Here fixatives are infused into the preparation

at low temperature, after which the specimen is slowly warmed to room temperature.

Usingcryo-electron tomography (Cryo-ET)the 3D structure of cells and macromol-

ecules can be visualised at 5–8 nm resolution. Cells are typically rapidly frozen, fixed by

freeze substitution and embedded in epoxy resin. Thick 200 nm sections are cut and

imaged in the TEM equipped with a tilting stage. A typical tilt series of 100 or so images

is collected in a digital form and exported to a computer reconstruction program for

analysis. By using electron tomography, a 2D digital EM image is converted into a high-

resolution 3D representation of the specimen (Fig. 4.24, see also colour section). The

method is especially useful for imaging the fine connections within cells especially the

cytoskeleton and nuclear pores and elucidating the surface structures of viruses.

4.6.4 Integrated microscopy

The same specimen can be viewed in the light microscope and subsequently in the EM.

This approach is calledintegrated microscopy. The correlation of images of the same

cell collected using the high temporal resolution of the light microscope and the high

spatial resolution of the EM gives additional information to imaging using the two

techniques separately (Fig. 4.25). The integrated approach also addresses the problem

of artifacts. Probes are now available that are fluorescent in the light microscope and

are electron dense in the EM.

4.7 Image archiving

Most images produced by any kind of modern microscope are collected in a digital

form. In addition to greatly speeding up the collection of the images (and experiment

times), the use of digital imaging has allowed the use of digital image databases and

Fig. 4.23Immunoelectron microscopy. Scanning electron microscope (SEM) image of microbesEnterococcus

faecalislabelled with 10 nm collidal gold for the surface adhesion protein ‘aggregation substance’. This protein

facilitates exchange of DNA during conjugation. The gold labels appear as white dots on the surface of the

bacteria. (Image kindly provided by the late Stan Erlandsen, University of Minnesota, USA.)

133 4.7 Image archiving