Single particle analysis

[1] These methods were developed to improve and extend the information obtainable from TEM images of particulate samples, typically proteins or other large biological entities such as viruses.

Single particle analysis methods are, in general, reliant on the sample being homogeneous, although techniques for dealing with conformational heterogeneity[5] are being developed.

Images (micrographs) are taken with an electron microscope using charged-coupled device (CCD) detectors coupled to a phosphorescent layer (in the past, they were instead collected on film and digitized using high-quality scanners).

This low dose, as well as variations in the metal stain used (if used)[6] means images have high noise relative to the signal given by the particle being observed.

The algorithms make use of fast Fourier transforms (FFT), often employing Gaussian shaped soft-edged masks in reciprocal space to suppress certain frequency ranges.

This, along with features inherent in the microscope's lens system, creates blurring of the collected images visible as a point spread function.

Proteins in vitreous ice ideally adopt a random distribution of orientations (or viewing angles), allowing a fairly isotropic reconstruction if a large number of particle images are used.

Filtered back projection is a commonly used method of generating 3D reconstructions in single particle analysis, although many alternative algorithms exist.

Tilt methods are best suited to negatively stained samples, and can be used for particles that adsorb to the carbon support film in preferred orientations.

These often enable the user to manually dock in protein coordinates (structures from X-ray crystallography or NMR) of subunits into the electron density.

He first tested this new methodology at the Forel Institute of the University of Geneva and presented this new analytical approach at the 'Colloid 2oo2' symposium during the spring 2002 meeting of the EMRS, and in the proceedings in 2003.

[18] This study presents the theory of SP ICP-MS and the results of tests carried out on clay particles (montmorillonite) as well as other suspensions of colloids.