An improved method for 3D reconstruction based on uniform point drift registration estimation

A common requirement for both Atom Probe Field-Ion Microscopy (APFIM) and Scanning Tunnelling Microscopy (STM) is a sharp pointed tip for use as either the specimen (APFIM) or the probe (STM). Traditionally, tips have been prepared by either chemical or electropolishing techniques. Recently, ion-milling has been successfully employed in the production of APFIM tips [1]. Conventional electropolishing techniques are applicable to a wide variety of metals, but generally require careful manual adjustments during the polishing process and may also be time-consuming. In order to reduce the time and effort involved in the preparation process, a compact, self-contained polishing unit has been developed. This system is based upon the conventional two-stage electropolishing technique in which the specimen/tip blank is first locally thinned or “necked”, and subsequently electropolished until separation occurs.[2,3] The result of this process is the production of two APFIM or STM tips. A mechanized polishing unit that provides these functions while automatically maintaining alignment has been designed and developed.

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Immunoelectron microscopic localization of elastic tissue in archival material using an improved method

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100161539 ◽

1990 ◽

Vol 48 (3) ◽

pp. 794-795

Author(s):

J. C. Fanning ◽

J. F. White ◽

R. Polewski ◽

E. G. Cleary

Keyword(s):

Normal Animal ◽

Animal Tissue ◽

Elastic Tissue ◽

Human Tissues ◽

Improved Method ◽

Tissue Samples ◽

Archival Material ◽

Immuno Electron Microscopy ◽

Arteries And Veins ◽

Biochemical Examination

Elastic tissue is an important component of the walls of arteries and veins, of skin, of the lungs and in lesser amounts, of many other tissues. It is responsible for the rubber-like properties of the arteries and for the normal texture of young skin. It undergoes changes in a number of important diseases such as atherosclerosis and emphysema and on exposure of skin to sunlight.We have recently described methods for the localizationof elastic tissue components in normal animal and human tissues. In the study of developing and diseased tissues it is often not possible to obtain samples which have been optimally prepared for immuno-electron microscopy. Sometimes there is also a need to examine retrospectively samples collected some years previously. We have therefore developed modifications to our published methods to allow examination of human and animal tissue samples obtained at surgery or during post mortem which have subsequently been: 1. stored frozen at -35° or -70°C for biochemical examination; 2.

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Detection, Averaging, and 3D Reconstruction of Biological Specimens on Hypercubes (Transputer-based) Computers

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100181026 ◽

1990 ◽

Vol 48 (1) ◽

pp. 454-455

Author(s):

Jose-Maria Carazo ◽

I. Benavides ◽

S. Marco ◽

J.L. Carrascosa ◽

E.L. Zapata

Keyword(s):

3D Reconstruction ◽

3D Structure ◽

Three Dimensional ◽

Software Tools ◽

Mapping Method ◽

Computer Architectures ◽

Parallel Computer ◽

Reconstruction Process ◽

Computing Power ◽

Order Of Magnitude

Obtaining the three-dimensional (3D) structure of negatively stained biological specimens at a resolution of, typically, 2 - 4 nm is becoming a relatively common practice in an increasing number of laboratories. A combination of new conceptual approaches, new software tools, and faster computers have made this situation possible. However, all these 3D reconstruction processes are quite computer intensive, and the middle term future is full of suggestions entailing an even greater need of computing power. Up to now all published 3D reconstructions in this field have been performed on conventional (sequential) computers, but it is a fact that new parallel computer architectures represent the potential of order-of-magnitude increases in computing power and should, therefore, be considered for their possible application in the most computing intensive tasks.We have studied both shared-memory-based computer architectures, like the BBN Butterfly, and local-memory-based architectures, mainly hypercubes implemented on transputers, where we have used the algorithmic mapping method proposed by Zapata el at. In this work we have developed the basic software tools needed to obtain a 3D reconstruction from non-crystalline specimens (“single particles”) using the so-called Random Conical Tilt Series Method. We start from a pair of images presenting the same field, first tilted (by ≃55°) and then untilted. It is then assumed that we can supply the system with the image of the particle we are looking for (ideally, a 2D average from a previous study) and with a matrix describing the geometrical relationships between the tilted and untilted fields (this step is now accomplished by interactively marking a few pairs of corresponding features in the two fields). From here on the 3D reconstruction process may be run automatically.

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Structural Studies on the Eukaryotic Ribosome

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100180033 ◽

1990 ◽

Vol 48 (1) ◽

pp. 256-257

Author(s):

Adriana Verschoor ◽

Ronald Milligan ◽

Suman Srivastava ◽

Joachim Frank

Keyword(s):

Chick Embryo ◽

3D Reconstruction ◽

Single Particle ◽

Mass Density ◽

Particle Surface ◽

Uranyl Acetate ◽

Electron Microscopic ◽

Eukaryotic Ribosome ◽

Negative Stain ◽

Reconstruction Methods

We have studied the eukaryotic ribosome from two vertebrate species (rabbit reticulocyte and chick embryo ribosomes) in several different electron microscopic preparations (Fig. 1a-d), and we have applied image processing methods to two of the types of images. Reticulocyte ribosomes were examined in both negative stain (0.5% uranyl acetate, in a double-carbon preparation) and frozen hydrated preparation as single-particle specimens. In addition, chick embryo ribosomes in tetrameric and crystalline assemblies in frozen hydrated preparation have been examined. 2D averaging, multivariate statistical analysis, and classification methods have been applied to the negatively stained single-particle micrographs and the frozen hydrated tetramer micrographs to obtain statistically well defined projection images of the ribosome (Fig. 2a,c). 3D reconstruction methods, the random conical reconstruction scheme and weighted back projection, were applied to the negative-stain data, and several closely related reconstructions were obtained. The principal 3D reconstruction (Fig. 2b), which has a resolution of 3.7 nm according to the differential phase residual criterion, can be compared to the images of individual ribosomes in a 2D tetramer average (Fig. 2c) at a similar resolution, and a good agreement of the general morphology and of many of the characteristic features is seen.Both data sets show the ribosome in roughly the same ’view’ or orientation, with respect to the adsorptive surface in the electron microscopic preparation, as judged by the agreement in both the projected form and the distribution of characteristic density features. The negative-stain reconstruction reveals details of the ribosome morphology; the 2D frozen-hydrated average provides projection information on the native mass-density distribution within the structure. The 40S subunit appears to have an elongate core of higher density, while the 60S subunit shows a more complex pattern of dense features, comprising a rather globular core, locally extending close to the particle surface.

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