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Results for “Biological Resolution”

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PubMed1976

Identification of biological molecules in situ at high resolution via the fluorescence excited by a scanning electron beam.

Proteins, nucleic acids, and fluorescein-conjugated antibody are shown to be identifidable in situ via the fluorescence excited by the focused electron beam of a canning electron microscope. A molecular species is identified by its characteristic fluorescence spectrum and by a characteristic alteration of the spectrum with time under the electron beam. Primary protein fluorescence is relatively rapidly destroyed by the beam, but protein photoproduct fluorescence is more rugged and will in some cases permit detection of small numbers of protein molecules. Nucleic acid fluorescence is extremely long-lived and will permit detection of small numbers of nucleic acid residues. The theoretical resolution limit for localization of a particular molecular species -- about 20 A--is determined by the known maximum distance for molecular excitation by fast electrons. Drect extapolation from an observed resolution of 900 A in the localization of nucleic acid using a low-efficiency detector leads to an experimental resolution limit of less than 60 A. Fluorescence is strongly quenched by residual water in the specimen. Similar quenching is produced by some macromolecular associations and so may serve to localize such associations.

Animals↗
PubMed2002

Photoacoustic tomography of biological tissues with high cross-section resolution: reconstruction and experiment.

A modified back-projection approach deduced from an exact reconstruction solution was applied to our photoacoustic tomography of the optical absorption in biological tissues. Pulses from a Ti:sapphire laser (4.7 ns FWHM at 789.2 nm) were employed to generate a distribution of photoacoustic sources in a sample. The sources were detected by a wide-band nonfocused ultrasonic transducer at different positions around the imaging cross section perpendicular to the axis of the laser irradiation. Reconstructed images of phantoms made from chicken breast tissue agreed well with the structures of the samples. The resolution in the imaging cross section was experimentally demonstrated to be better than 60 microm when a 10 MHz transducer (140% bandwidth at -60 dB) was employed, which was nearly diffraction limited by the detectable photoacoustic waves of the highest frequency.

Acoustics↗
PubMed2004

Concepts for nanoscale resolution in fluorescence microscopy.

Spatio-temporal visualization of cellular structures by fluorescence microscopy has become indispensable in biology. However, the resolution of conventional fluorescence microscopy is limited by diffraction to about 180 nm in the focal plane and to about 500 nm along the optic axis. Recently, concepts have emerged that overcome the diffraction resolution barrier fundamentally. Formed on the basis of reversible saturable optical transitions, these concepts might eventually allow us to investigate hitherto inaccessible details within live cells.

Animals↗
PubMed1989

Crystal dissolution of biological and ceramic apatites.

High resolution transmission electron microscopy (Hr TEM) studies on biological and synthetic calcium phosphate have provided information on the dissolution process at the crystal level. The purpose of this study was to investigate the dissolution of ceramic hydroxyapatite (HA) after implantation using Hr TEM. Recovered HA ceramic implanted in bony and nonbony sites in animals and in periodontal pockets in humans were used for the study. For comparison, sections of human fluorotic enamel with caries and sections of shark enameloid previously exposed to 0.1 HCl were similarly investigated. Hr TEM studies demonstrated that in both the biological and ceramic apatites, the lattice and atomic defects were the starting points in the dissolution process. However, significant differences in the process of dissolution were observed: (1) biological apatite crystals showed preferential core dissolution whereas ceramic apatite crystals showed nonspecific dissolution at the cores and at the surfaces; (2) the dissolution of biological apatites appeared to consistently extend along the crystal's c-axis whereas dissolution of the ceramic HA did not appear to be correlated with the crystal's c-axis. The observed differences in crystal dissolution between biological and ceramic apatites may be attributed to the following: (1) the unique crystal/protein interaction present with biological apatites but absent in ceramic HA; (2) differences in defect distribution between biological and ceramic apatites which are due to the differences in the original of these defects; and (3) the longer morphological c-axis of biological apatites compared with that of ceramic apatites.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Prospects for using an IVEM with a FEG for imaging macromolecules towards atomic resolution.

Specimen preparation and imaging techniques for biological macromolecules have been improved to the point where attention to the electron-optical imaging conditions becomes a significant factor for achieving high resolution. A field emission gun (FEG) can provide an illumination source with a better spatial and temporal coherence suitable for imaging near atomic resolution. Our computational analysis of carbon film images taken between Scherzer focus and 1.1 microns underfocus (20x Scherzer focus) with the Hitachi 200 kV microscope with a cold field emission gun shows detectable contrast beyond 3.5 A resolution. In biological imaging, a large defocus is often used to optimize the low-resolution contrast in order to facilitate the subsequent steps in computer reconstruction. An intermediate-voltage electron microscope (IVEM) would optimize the contrast at high resolution by reducing the temporal coherent effects. In theory, the IVEM would give a greater depth of field so that large macromolecular assemblies such as viruses and cellular structures can be interpreted and reconstructed reliably using the projection approximation. These experimental and theoretical considerations provide a rationale for designing a future IVEM with a FEG suitable for biological macromolecule imaging close to atomic resolution.

Fourier Analysis↗
PubMed2000

Structure determination of biological macromolecules in solution using nuclear magnetic resonance spectroscopy.

A detailed understanding of the function of a biological macromolecule requires knowledge of its three-dimensional structure. Most atomic-resolution structures of biological macromolecules have been solved either by X-ray diffraction in single crystals or by nuclear magnetic resonance (NMR) in solution. This review surveys the method of NMR structure determination. First, a brief introduction to NMR and its basic concepts is presented. The main part of the article deals with the individual steps necessary for an NMR structure determination. At the end, the discussion turns to considerations on the influence of the molecular size of the macromolecules on the structure determination by NMR. New techniques are discussed that greatly enhance the possibilities of applying NMR to large molecular systems.

Humans↗
PubMed1999

Electrostatically balanced subnanometer imaging of biological specimens by atomic force microscope.

To achieve high-resolution topographs of native biological macromolecules in aqueous solution with the atomic force microscope (AFM) interactions between AFM tip and sample need to be considered. Short-range forces produce the submolecular information of high-resolution topographs. In contrast, no significant high-resolution information is provided by the long-range electrostatic double-layer force. However, this force can be adjusted by pH and electrolytes to distribute the force applied to the AFM tip over a large sample area. As demonstrated on fragile biological samples, adjustment of the electrolyte solution results in a local reduction of both vertical and lateral forces between the AFM tip and proteinous substructures. Under such electrostatically balanced conditions, the deformation of the native protein is minimized and the sample surface can be reproducibly contoured at a lateral resolution of 0.6 nm.

Bacterial Proteins↗
PubMed1983

A high-resolution electron microscope study of synthetic and biological carbonated apatites.

A series of synthetic carbonated apatites and human dental enamels characterized by chemical analysis, infrared spectroscopy, and X-ray diffraction was studied using high-resolution transmission electron microscopy. Steps on the surfaces of apatite crystals, often only a few unit cells high and occasionally one unit cell high, were observed by their Fresnel diffraction contrast. Highly substituted synthetic carbonated apatites appeared to have more irregular and finer-textured surface features than materials with less carbonate substitution. The surface features of enamel apatite crystal were also irregular, but surface steps were less frequently aligned in crystallographic directions. Complex strain fields due to radiation damage centers were observed in some crystals and the fine structure of dislocations and grain boundaries in synthetic apatites was resolved at high magnification. Experimental lattice-image contrasts, in favorable circumstances, could be matched to computer-simulated images and were found to contain detail at near atomic resolution, around 2.0-2.5 A.

Adult↗
PubMed1999

Review: electron crystallography: present excitement, a nod to the past, anticipating the future.

From a modest beginning with negatively stained samples of the helical T4 bacteriophage tail, electron crystallography has emerged as a powerful tool in structural biology. High-resolution density maps, interpretable in terms of an atomic structure, can be obtained from specimens prepared as well-ordered, two-dimensional crystals, and the resolution achieved with helical specimens and icosahedral viruses is approaching the same goal. A hybrid approach to determining the molecular structure of complex biological assemblies is generating great interest, in which high-resolution structures that have been determined for individual protein components are fitted into a lower resolution envelope of the large complex. With this as background, how much more can be anticipated for the future? Considerable scope still remains to improve the quality of electron microscope images. Automation of data acquisition and data processing, together with the emergence of computational speeds of 10(12) floating point operations per second or higher, will make it possible to extend high-resolution structure determination into the realm of single-particle microscopy. As a result, computational alignment of single particles, i.e., the formation of "virtual crystals," can begin to replace biochemical crystallization. Since single-particle microscopy may remain limited to "large" structures of 200 to 300 kDa or more, however, smaller proteins will continue to be studied as helical assemblies or as two-dimensional crystals. The further development of electron crystallography is thus likely to turn increasingly to the use of single particles and small regions of ordered assemblies, emphasizing more and more the potential for faster, higher throughput.

Crystallography↗
PubMed2001

Determining the structure of biological macromolecules by transmission electron microscopy, single particle analysis and 3D reconstruction.

Single particle analysis and 3D reconstruction of molecules imaged by transmission electron microscopy have provided a wealth of medium to low resolution structures of biological molecules and macromolecular complexes, such as the ribosome, viruses, molecular chaperones and photosystem II. In this review, the principles of these techniques are introduced in a non-mathematical way, and single particle analysis is compared to other methods used for structural studies. In particular, the recent X-ray structures of the ribosome and of ribosomal subunits allow a critical comparison of single particle analysis and X-ray crystallography. This has emphasised the rapidity with which single particle analysis can produce medium resolution structures of complexes that are difficult to crystallise. Once crystals are available, X-ray crystallography can produce structures at a much higher resolution. The great similarities now seen between the structures obtained by the two techniques reinforce confidence in the use of single particle analysis and 3D reconstruction, and show that for electron cryo-microscopy structure distortion during sample preparation and imaging has not been a significant problem. The ability to analyse conformational flexibility and the ease with which time-resolved studies can be performed are significant advantages for single particle analysis. Future improvements in single particle analysis and electron microscopy should increase the attainable resolution. Combining single particle analysis of macromolecular complexes and electron tomography of subcellular structures with high-resolution X-ray structures may enable us to realise the ultimate dream of structural biology-a complete description of the macromolecular complexes of the cell in their different functional states.

Bacteria↗