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In this paper, we describe two methods for computerized analysis of cryo electron tomography reconstructions of biomolecules. Both methods aim at quantifying the degree of structural flexibility of macromolecules and eventually resolving the inner dynamics through automatized protocols. The first method performs a Brownian dynamics evolution of a simplified molecular model into a fictitious force field generated by the tomograms. This procedure enables us to dock the simplified model into the experimental profiles. The second uses a fuzzy framework to delineate the subparts of the proteins and subsequently determine their interdomain relations. The two methods are discussed and their complementarities highlighted with reference to the case of the immonoglobulin antibody. Both artificial maps, constructed from immunoglobulin G entries in the Protein Data Bank and real tomograms are analyzed. Robustness issues and agreement with previously reported measurements are discussed. [ABSTRACT FROM AUTHOR]

Since the late 1980s, atomic force microscopy (AFM) has been increasingly used in biological sciences and it is now established as a versatile tool to address the structure, properties and functions of biological specimens. AFM is unique in that it provides three-dimensional images of biological structures, including biomolecules, lipid films, 2D protein crystals and cells, under physiological conditions and with unprecedented resolution. A crucial prerequisite for successful, reliable biological AFM is that the samples need to be well attached to a solid substrate using appropriate, nondestructive methods. In this review, we discuss common techniques for immobilizing biological specimens for AFM studies. [ABSTRACT FROM AUTHOR]

Surface imaging techniques were combined to determine appropriate manipulation of technologically important surfaces for commercial applications. The complementarity of the microscopy methods, scanning electron microscopy, electron probe microanalysis and atomic force microscopy assessed and correlated form and function of the surface modifications. Stainless steel disks (1 cm in diameter) were laser-cut from the same sheets of stainless steel and treated by electropolishing or left untreated for controls. Each treatment was analysed separately using each technique. First, the disks were examined by visual inspection and electron probe microanalysis for surface characteristics and elemental composition, respectively. Aliquots of bacterial suspensions (saline rinses of poultry carcasses from a commercial broiler processing plant) were then diluted in broth and monitored for growth by spectrophotometry. Stainless steel disks (1 cm in diameter) were added and the cultures were grown to sufficient density to allow attachment of bacterial cells to test surfaces. Relative differences in the surface morphology shown by atomic force microscopy, including Z ranges, roughness and other measurements, corresponded by treatment with the differences in reduction of bacterial counts shown by scanning electron microscopy. A model of wet-processing conditions tested the effects of corrosive treatment of surfaces. Less bacterial attachment occurred after corrosive treatment on controls and electropolished samples. Electropolishing significantly reduced bacterial numbers and the effects of corrosive action compared to the controls. Thus, the multiple imaging techniques showed that engineered changes on stainless steel surfaces improved the resistance of the surface finish to bacterial attachment, biofilm formation, and corrosive action. [ABSTRACT FROM AUTHOR]

Summary The atomic force microscope (AFM) is an exquisitely delicate probe measuring the height of a specimen at discrete sampling points in a fixed two-dimensional (2D) raster. The resulting topograph is a 2D digital image, with each pixel representing a distinct height measurement. The height of an object is determined as the average of the maximum heights measured above the supporting surface. We show that such object heights derived from a variety of organic samples depend critically on the sampling or pixel size of the 2D raster. It is concluded that to obtain accurate specimen heights, the pixel size must be small enough to resolve submolecular structures and thus ensure representative sampling of the height variation on the surface. [ABSTRACT FROM AUTHOR]

Summary Simple ancillary metadata, such as those encompassed by the 15 elements of the Dublin Core, may be sufficient and entirely appropriate for basic coarse-granularity cross-domain resource discovery. However, they are insufficient and inappropriate for content description of complex data types such as videos, which require more detailed relational models. We propose a metadata classification schema for the characterization of items and events in videos that permits subsequent query by content. Following MPEG-7 nomenclature, metadata intrinsic to the information content of the video are defined as either structural or semantic, where structural metadata are numerical feature primitives produced by analysing the colour, shape, texture, structure and motion within the video frames, whereas semantic metadata describe the locations and timings of individual items and particular actions or events in the video, and are thus of higher information value. In this paper, the semantic metadata required to describe the visual information content of videos are defined and classified into four distinct classes: Media Entities; Content Items; Events; and Supplementary Items, and three types of property tables are defined: Identity Tables; Spatio-Temporal Position Tables; and Event Tables, in which these metadata may be stored in a relational database. [ABSTRACT FROM AUTHOR]

Summary Using the video metadata descriptors and data model defined in the accompanying paper (Shotton, D. M. et al. (2002) A metadata classification schema for semantic content analysis of videos. J. Microsc. 205 , 33–42), we discuss how analysis of the content of scientific videos, and subsequent query by content of the resulting semantic metadata, can be enhanced by the use of an object-relational database. We illustrate this by describing VANQUIS , a Web-based prototype vi deo an alysis and qu ery i nterface s ystem for the interactive spatio-temporal analysis and subsequent query by content of videos. Using VANQUIS to generate standard SQL (structured query language) statements that address complex data types stored in an object-relational database, relationships between characters and events contained within and between videos can be identified, and the appropriate video segments containing these characters and events can be retrieved for viewing. We give examples of analysis and query implementation by using VANQUIS to analyse a biological microscopy video, and discuss the wider potential of this methodology for the analysis and query by content of videos containing more general subject matter. [ABSTRACT FROM AUTHOR]

Ultrastructural examination by transmission and scanning electron microscopy involves a series of specialized preparation steps which may introduce artefacts in the micrographs. X-ray microscopy can take instant images of speci-mens but is mostly restricted to a few synchrotron X-ray sources. We have utilized a bench-top nanosecond laser-plasma to produce a single-shot source of nanosecond X-rays tuned for maximum contrast with carbon-rich material. To examine the ultrastructure by absorption profiles, we utilized a laser-produced plasma generated by a single-shot laser (1.06 μm wavelength, 5 × 1012 W cm−2 intensity) focused on to a silicon target as an X-ray source for high-resolution X-ray microscopy. This approach eliminates the specimen preparation steps. Whole hydrated cells of Escherichia coli and purified preparations of lipopolysaccharide (LPS) and chromosomal DNA (cDNA) were streaked onto poly(methyl methacrylate) (PMMA)-coated grids (resist). This resist was exposed to X-rays under vacuum at a distance of 2.5 cm from the target disc. The silicon plasma produced by a 10-ns burst of laser energy (at 20 J) radiates strong emission lines in the region of 300 eV. The X-rays penetrate the sample and their absorption profile is transferred on to the resist where PMMA acts as a negative to generate an image. By atomic force microscopy imaging of this photoresist we have visualized layers around cells of E. coli, darker areas inside the cell probably corresponding to cDNA, and preliminary images of LPS and DNA molecules. This technique has resolution at the 100 Å level, produces images similar to the space-filling models of macromolecules and may be of great value in the study of the ultrastructure of hydrated live biological specimens. [ABSTRACT FROM AUTHOR]

The structures of the large and small ribosomal subunits of Escherichia coli were reconstructed using spectroscopic electron microscopy and quaternion-assisted angular reconstitution to resolutions of better than 4 nm. In addition, the distributions of phosphorus within these complexes were reconstructed. The three-dimensional reconstruction of the distribution of this atomic element is an extension of microanalysis (in two dimensions) for phosphorus identification and mapping, as a signature of the arrangement of the phosphate backbones of the constituent ribosomal RNAs. The results on both the phosphorus reconstructions and the total reconstructions (protein and ribosomal RNA) reveal several passageways through both subunits. The structures correspond favourably with other independent reconstructions of the whole E. coli ribosome from cryoelectron micrographs and their accompanying models of translation (Frank et al., Nature, 376, 441-444, 1995; Stark et al., Structure, 3, 815-821, 1995). The overall reconstructions in conjunction with the phosphorus (rRNA) distributions are the first to be achieved synchronously for this nucleoprotein complex. [ABSTRACT FROM AUTHOR]

Scanning (atomic) force microscopy (SFM) permits high-resolution imaging of a biological specimen in physiological solutions. Untreated extracellular haemoglobin molecules of the common North American earthworm, Lumbricus terrestris, were imaged in NH4Ac solution using calibrated SFM. Individual molecules and their top and side views were clearly identified and were comparable with the images of the same molecule obtained by scanning transmission electron microscopy (STEM). A central depression, the presumed mouth of the hole, was detected. We analysed 75 individual molecules for their lateral dimensions. Compression varied for different molecules, presumably because of the variation of the interaction between the SFM tip and the protein molecule. Two effective heights which correspond to the heights of the points of the haemoglobin molecules first and last touched by the tip, h1 and h2, respectively, were measured for each protein and ranged between 1.58 and 16.2 nm for h1 and 1.23 and 13.6 nm for h2. The apparent diameter was measured and ranged from 44.9 to 86.6 nm (63.2±10.5 nm, n =75), which is about twice the diameter of the molecule reported by STEM for the top view orientation. The higher the measured effective heights, the worse was the tip convolution effect. In order to determine the tip parameters (semivertical angle, curvature of radius and the cut-off height) and to calibrate images of earthworm haemoglobin molecules, spherical gold particles were scanned as standards. The tip sectional radii at distances of h1 and h2 above the tip apex were subtracted from the apparent diameter of the protein. The calibrated lateral dimension was 29.1 ±3.85 nm, which is close to the reported scanning transmission electron microscopy data 30.0 ±0.8 nm. The results presented here demonstrate that the calibration approach of imaging gold particles is practical and relatively accurate. Calibrated SFM imaging can be applied to the study of other biomacromolecules. [ABSTRACT FROM AUTHOR]

A computer model based on the elastic properties of rubber is introduced for the evaluation of the lateral resolution in atomic force microscopy of deformable specimens. The computational results show that, if the full width at half-height can be defined as the lateral resolution, it is continuously improved at greater probe forces, at the expense of a reduced molecular height. In fact, even for a probe that is bigger than the molecule, the real size of the molecule can be 'recovered' at about 25% compression. This result demonstrates that for a better lateral resolution, a greater probe force can be beneficial, provided that the molecule is not moved or damaged and the response remains elastic. Measurements on isolated low-density lipoproteins (LDL) show that with 26% vertical compression, the lateral size measured in atomic force microscopy is only about 72% of the value predicted by a simple convolution, and is only slightly larger (≈ 13%) than the known size of LDL. Therefore, the results on LDL provide a direct support for the conclusions of the computational model. [ABSTRACT FROM AUTHOR]