Three-Dimensional Microscopy

There are two ways to generate 3D images (mechanical or optical) serial sectioning and tomography. Mechanical sectioning has been used to image metal alloys [137-139] and is in principle applicable to polymeric materials. 

By using a higher X-ray flux (i.e., a synchrotron source), distinct polymer phases can be detected using phase contrast. The interaction of electromagnetic radiation (X-rays) 

The first report of TEMT on block copolymer nanostructures, by Spontak [164], appeared in 1988. This was followed by three morphological smdies carried out in the 1990s [165-167]. Only recently has TEMT become more popular in characterizing polymer nanostructures, including block copolymers [134, 164, 167-173], nanocomposites [174, 175], and polymer nanocomposites [176]. Kawase et al. [177] recently presented a protocol to perform complete rotation (i.e., 90°) on a Zr02/polymer nanocomposite, by which they achieved truly quantitative TEMT for the first time. 

Interface anisotropy is another important parameter in multiphase fluids. This is mostly relevant to relate rheological properties with microstructure [192]. The interface anisotropy can be quantified using the interface tensor q j, defined as qy = fs i j where W  

The three components of the normal vector cannot be computed from 2D micrographs without previous knowledge (or assumption) of the interface shape. For simple shapes with axial symmetry that can be described with simple functions, such as ellipsoidal droplets and cylindrical threads, analytical expressions for (and qy) have been obtained from 2D images [193-195]. However, for complex interfaces (e.g., cocontinuous structures), which are not axisymmetric and cannot be described by any analytical equation, this approach is not applicable. 

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Straub, M. and Hell, S. W. (1998). Fluorescence lifetime three-dimensional microscopy with picosecond precision using a multifocal multiphoton microscope. Appl. Phys. Lett. 73, 1769-71. 

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M.G. Gustafsson, D.A. Agard, J.W. Sedat. 3D widefield microscopy with two objective lenses experimental verification of improved axial resolution, in Three-Dimensional Microscopy Image Acquisition and Processing 111, Proceedings of SPIE, 1996... 

Chen. H., Hughes. D. D., Chan. T., Sedat. J. W., and Agard. D. A. (1996). IVE (Image Visualization Environment) A software platform for all three-dimensional microscopy applications. J. Struct. Biot 116,. 56-60. 

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One more significant aspect of modem microscopy is the quantitative interpretation of the images in terms of the microstmcture of the object. Although most microscopes include or can be combined with powerful image processing systems, the interpretation of the contrast is still the main problem. On the other hand, reliable micromorpbological information could be easily obtained from a set of thin flat cross sections which reveal only density information, from which case accurate two- and three-dimensional numerical parameters of the internal microstmcture could be calculated. 

Considering existing microscopical techniques, one can find that non-destmctive information from the internal stmcture of an object in natural conditions can be obtained by transmission X-ray microscopy. Combination of X-ray transmission technique with tomographical reconstmction allows getting three-dimensional information about the internal microstmcture [1-3]. In this case any internal area can be reconstmcted as a set of flat cross sections which can be used to analyze the two- and three-dimensional morphological parameters [4]. For X-ray methods the contrast in the images is a mixed combination of density and compositional information. In some cases the compositional information can be separated from the density information [5]. Recently there has been a... 

A first example of application of microtomography is taken from life sciences. Here X-ray microscopy and microtomography allows to reconstruct the internal three-dimensional microstructure without any preparation and sometimes even of living objects. Fig. la shows an X-ray transmission microscopical image of bone (femoral head). Several reconstructed cross-sections are shown in Fig.lb. Fig.lc shows the three-dimensional reconstruction of this bone. 

Frank J 1996 Three-Dimensional Electron Microscopy of Macromolecular Assemblies (New York Aoademio)... 

Ruiz T 1998 Conferenoe talk Gordon Conf. on Three-Dimensional Electron Microscopy... 

Cork T and Kino G S 1996 Confocal Scanning Optical Microscopy and Related Imaging Systems (New York Academic) Gu Min 1996 Principles of Three Dimensional Imaging In Confocal Microscopes (Singapore World Scientific)... 

Bhawalkar J D, Swiatkiewicz J, Pan S J, Samarabandu J K, Liou W S, He G S, Berezney R, Cheng P C and Prasad P N 1996 Three-dimensional laser scanning two-photon fluorescence confocal microscopy of polymer materials using a new, efficient upconverting fluorophore Scanning 18 562-6... 

Figure Bl.22.11. Near-field scanning optical microscopy fluorescence image of oxazine molecules dispersed on a PMMA film surface. Each protuberance in this three-dimensional plot corresponds to the detection of a single molecule, the different intensities of those features being due to different orientations of the molecules. Sub-diffraction resolution, in this case on the order of a fraction of a micron, can be achieved by the near-field scaiming arrangement. Spectroscopic characterization of each molecule is also possible. (Reprinted with pennission from [82]. Copyright 1996 American Chemical Society.)...

Although experimental studies of DNA and RNA structure have revealed the significant structural diversity of oligonucleotides, there are limitations to these approaches. X-ray crystallographic structures are limited to relatively small DNA duplexes, and the crystal lattice can impact the three-dimensional conformation [4]. NMR-based structural studies allow for the determination of structures in solution however, the limited amount of nuclear overhauser effect (NOE) data between nonadjacent stacked basepairs makes the determination of the overall structure of DNA difficult [5]. In addition, nanotechnology-based experiments, such as the use of optical tweezers and atomic force microscopy [6], have revealed that the forces required to distort DNA are relatively small, consistent with the structural heterogeneity observed in both DNA and RNA. 

This electron microscopy reconstruction has since been extended to high resolution (3 A) where the connections between the helices and the bound retinal molecule are visible together with the seven helices (Figure 12.3c). The helices are tilted by about 20° with respect to the plane of the membrane. This is the first example of a high-resolution three-dimensional protein structure determination using electron microscopy. The structure has subsequently been confirmed by x-ray crystallographic studies to 2 A resolution. 

The three-dimensional structure of the bacterial membrane protein, bac-teriorhodopsin, was the first to be obtained from electron microscopy of two-dimensional crystals. This method is now being successfully applied to several other membrane-bound proteins. 

Henderson, R., Unwin, RN.T. Three-dimensional model of purple membrane obtained by electron microscopy. Nature 257 28-32, 1975. 

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