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P J Koeck

Publications and source records attributed to P J Koeck.

5 recordsLinked to original sources

Ins and outs of digital electron microscopy.

In order to do any sort of computer processing of the images produced by an electron microscope they have to be digitized. Therefore, any form of microscopy that involves computer processing has to be considered as digital microscopy independently of whether the original images were recorded on film and scanned or recorded directly on a digital medium such as the charge coupled device (CCD). As soon as one digitizes images, one encounters a range of effects and artefacts specific to the digital world such as aliasing, quantization errors, and ambiguities in the modulation transfer function. Even a quick discussion of these effects makes it clear that what comes out of a digitizer is not related in a simple way to what goes in. After introducing the commonly used recording media, I will discuss the most important effects of digitization and then compare the limitations and advantages of the different recording media for various applications.

Automation↗

Evaluation of scanners and CCD cameras for high-resolution TEM of protein crystals and single particles.

The modulation transfer function (MTF) and the geometric errors of two flatbed scanners, a slow-scan CCD (SSC) camera and film, have been measured and compared. The geometric errors of the SSC camera and film have been measured using diffraction spots from a lipid crystal. The SSC camera was shown to have the smallest geometric errors while film had the best MTF. Even though film had the best MTF, this is significantly reduced when scanning the film, so that the MTF of the film and scanner combined are comparable to the MTF of the SSC camera.

Biophysical Phenomena↗

Two-dimensional crystals of reconstituted beta-subunits of the chaperonin TF55 from Sulfolobus shibatae.

We have obtained 2-dimensional crystals of the beta-subunits of the chaperonin TF55 from Sulfolobus shibatae reconstituted into oligomers in the absence of alpha-subunits. The subunits form rings with 9-fold rotational symmetry which arrange themselves in a trigonal lattice. From electron micrographs of negatively stained specimens we have calculated a projection map in plane group p312 showing the rings in top-view.

Archaeal Proteins↗

Two-dimensional crystallization of the chaperonin TF55 from the hyperthermophilic archaeon Sulfolobus solfataricus.

Oligomers of the chaperonin TF55 from Sulfolobus solfataricus have been successfully crystallized in two dimensions via their interaction with a phospholipid monolayer at the air/liquid interface. Oligomer orientation was dependent upon the lipid headgroup used. A neutral lipid monolayer gave rise to small paracrystalline areas of TF55 side views, whereas a negatively charged lipid monolayer resulted in large coherent crystalline areas of the chaperonin in an end-on orientation. These 2D crystals had p312 symmetry (a = b = 162 A, gamma = 60 degrees). Two-dimensional projection structures of the end-on arrays were produced by electron microscopy and image processing techniques. Under the conditions used to grow the crystals, the protein formed complexes of two stacked nine-subunit rings with threefold symmetry.

Archaeal Proteins↗

Improved immuno double labelling for cell and structural biology.

Colloidal gold is the most widely used electron dense marker in biological electron microscopy. The development of procedures for making gold particles of very defined sizes has made double or even multiple labelling possible using gold of two or more different sizes. Lately a new type of electron dense marker has been developed consisting of ligand-stabilized metal atom clusters rather than colloidal particles. The differences between these two types of markers are highlighted and the advantages of using metal atom clusters for immuno labelling of certain biological specimens are discussed. Possible methods of distinguishing two such cluster labels in double labelling experiments are reviewed.

Artifacts↗