Deep and High-Resolution Three-Dimensional Tracking of Single Particles using Nonlinear and Multiplexed Illumination

This tutorial will discuss the methodology of low dose electron diffraction and imaging of crystalline biological objects, the problems of data interpretation for two-dimensional projected density maps of glucose embedded protein crystals, the factors to be considered in combining tilt data from three-dimensional crystals, and finally, the prospects of achieving a high resolution three-dimensional density map of a biological crystal. This methodology will be illustrated using two proteins under investigation in our laboratory, the T4 DNA helix destabilizing protein gp32*I and the crotoxin complex crystal.

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3-D reconstruction of molecular shape from microcrystal thin sections

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100077311 ◽

1983 ◽

Vol 41 ◽

pp. 748-749

Author(s):

S. Cusack ◽

J.-C. Jésior

Keyword(s):

Three Dimensional ◽

Molecular Shape ◽

Biological Molecules ◽

Fourier Space ◽

Single Particles ◽

Thin Sections ◽

Standard Methods ◽

X Ray ◽

X Ray Crystallography ◽

Dimensional Reconstruction

Three-dimensional reconstruction techniques using electron microscopy have been principally developed for application to 2-D arrays (i.e. monolayers) of biological molecules and symmetrical single particles (e.g. helical viruses). However many biological molecules that crystallise form multilayered microcrystals which are unsuitable for study by either the standard methods of 3-D reconstruction or, because of their size, by X-ray crystallography. The grid sectioning technique enables a number of different projections of such microcrystals to be obtained in well defined directions (e.g. parallel to crystal axes) and poses the problem of how best these projections can be used to reconstruct the packing and shape of the molecules forming the microcrystal.Given sufficient projections there may be enough information to do a crystallographic reconstruction in Fourier space. We however have considered the situation where only a limited number of projections are available, as for example in the case of catalase platelets where three orthogonal and two diagonal projections have been obtained (Fig. 1).

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Electron crystallographic determination of the 3-D structure of staurolite at atomic resolution

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010008701x ◽

1991 ◽

Vol 49 ◽

pp. 540-541

Author(s):

Kenneth H. Downing ◽

Hu Meisheng ◽

Hans-Rudolf Went ◽

Michael A. O'Keefe

Keyword(s):

High Resolution ◽

Three Dimensional ◽

High Resolution Electron Microscopy ◽

Atomic Resolution ◽

Dimensional Structure ◽

Structure Information ◽

Resolution Electron ◽

Scattering Effects ◽

High Resolution Images ◽

Effects Of Radiation

With current advances in electron microscope design, high resolution electron microscopy has become routine, and point resolutions of better than 2Å have been obtained in images of many inorganic crystals. Although this resolution is sufficient to resolve interatomic spacings, interpretation generally requires comparison of experimental images with calculations. Since the images are two-dimensional representations of projections of the full three-dimensional structure, information is invariably lost in the overlapping images of atoms at various heights. The technique of electron crystallography, in which information from several views of a crystal is combined, has been developed to obtain three-dimensional information on proteins. The resolution in images of proteins is severely limited by effects of radiation damage. In principle, atomic-resolution, 3D reconstructions should be obtainable from specimens that are resistant to damage. The most serious problem would appear to be in obtaining high-resolution images from areas that are thin enough that dynamical scattering effects can be ignored.

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Computer Assisted Image Reconstruction of Oligomer Structure

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100092323 ◽

1976 ◽

Vol 34 ◽

pp. 472-473

Author(s):

A.M. Jones ◽

A. Max Fiskin

Keyword(s):

Three Dimensional ◽

Depth Of Field ◽

Computer Assisted ◽

Projection Data ◽

Two Dimensional ◽

Single Particles ◽

Dimensional Reconstruction ◽

Computer Assisted Image ◽

The Fourier Transform ◽

Space Approach

If the tilt of a specimen can be varied either by the strategy of observing identical particles orientated randomly or by use of a eucentric goniometer stage, three dimensional reconstruction procedures are available (l). If the specimens, such as small protein aggregates, lack periodicity, direct space methods compete favorably in ease of implementation with reconstruction by the Fourier (transform) space approach (2). Regardless of method, reconstruction is possible because useful specimen thicknesses are always much less than the depth of field in an electron microscope. Thus electron images record the amount of stain in columns of the object normal to the recording plates. For single particles, practical considerations dictate that the specimen be tilted precisely about a single axis. In so doing a reconstructed image is achieved serially from two-dimensional sections which in turn are generated by a series of back-to-front lines of projection data.

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Three-Dimensional Immunoelectron Microscopy of Yeast Cells by a New High-Resolution Replica Method

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100119594 ◽

1985 ◽

Vol 43 ◽

pp. 562-563

Author(s):

Hirano T. ◽

M. Yamaguchi ◽

M. Hayashi ◽

Y. Sekiguchi ◽

A. Tanaka

Keyword(s):

High Resolution ◽

Plasma Polymerization ◽

Three Dimensional ◽

Freeze Fracture ◽

Replica Method ◽

Yeast Cells ◽

Dynamic Aspect ◽

Replica Technique ◽

Gaseous Hydrocarbon ◽

Transmission Electron

A plasma polymerization film replica method is a new high resolution replica technique devised by Tanaka et al. in 1978. It has been developed for investigation of the three dimensional ultrastructure in biological or nonbiological specimens with the transmission electron microscope. This method is based on direct observation of the single-stage replica film, which was obtained by directly coating on the specimen surface. A plasma polymerization film was deposited by gaseous hydrocarbon monomer in a glow discharge.The present study further developed the freeze fracture method by means of a plasma polymerization film produces a three dimensional replica of chemically untreated cells and provides a clear evidence of fine structure of the yeast plasma membrane, especially the dynamic aspect of the structure of invagination (Figure 1).

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