Microscopy limitations

II. Transmission Electron Microscopy (Limitations of Conventional TEM in the Characterization of Catalysts)... 

Electron diffraction studies are usually limited to transferred films (see Chapter XV), One study on Langmuir films of fatty acids has used cryoelectron microscopy to fix the structures on vitrified water [179], Electron diffraction from these layers showed highly twinned structures in the form of faceted crystals. 

Transmission electron microscopy (TEM) can resolve features down to about 1 nm and allows the use of electron diffraction to characterize the structure. Since electrons must pass through the sample however, the technique is limited to thin films. One cryoelectron microscopic study of fatty-acid Langmuir films on vitrified water [13] showed faceted crystals. The application of TEM to Langmuir-Blodgett films is discussed in Chapter XV. 

One of the more recent advances in XPS is the development of photoelectron microscopy [ ]. By either focusing the incident x-ray beam, or by using electrostatic lenses to image a small spot on the sample, spatially-resolved XPS has become feasible. The limits to the spatial resolution are currently of the order of 1 pm, but are expected to improve. This teclmique has many teclmological applications. For example, the chemical makeup of micromechanical and microelectronic devices can be monitored on the scale of the device dimensions. 

Raman microscopy is more developed than its IR counterpart. There are several reasons for this. First, the diffraction limit for focusing a visible beam is about 10 times smaller than an IR beam. Second, Raman spectroscopy can be done in a backscattering geometry, whereas IR is best done in transmission. A microscope is most easily adapted to a backscattermg geometry, but it is possible to do it in transmission. 

B1.18.5.5 CONTRAST ENHANCEMENT AND PRACTICAL LIMITS TO CONFOCAL ONE-PHOTON-EXCITATION FLUORESCENCE MICROSCOPY... 

Hell S W and Kroug M 1995 Ground-state-depletion fluorescence microscopy a concept for breaking the diffraction resolution limit Appl. Phys. B 60 495-7... 

While the spatial resolution in classical microscopy is limited to approximately X/2, where X is the optical wavelength (tlie so-called Abbe Limit [194], -0.2 pm with visible light), SNOM breaks through this barrier by monitoring the evanescent waves (of high spatial frequency) which arise following interaction with an... 

Henderson R 1995 The potential and limitations of neutrons, electrons and x-rays for atomic resolution microscopy of unstained biological molecules Q. Rev. Biophys. 28 171-93... 

Abstract. Molecular dynamics (MD) simulations of proteins provide descriptions of atomic motions, which allow to relate observable properties of proteins to microscopic processes. Unfortunately, such MD simulations require an enormous amount of computer time and, therefore, are limited to time scales of nanoseconds. We describe first a fast multiple time step structure adapted multipole method (FA-MUSAMM) to speed up the evaluation of the computationally most demanding Coulomb interactions in solvated protein models, secondly an application of this method aiming at a microscopic understanding of single molecule atomic force microscopy experiments, and, thirdly, a new method to predict slow conformational motions at microsecond time scales. 

A completely new method of determining siufaces arises from the enormous developments in electron microscopy. In contrast to the above-mentioned methods where the surfaces were calculated, molecular surfaces can be determined experimentally through new technologies such as electron cryomicroscopy [188]. Here, the molecular surface is limited by the resolution of the experimental instruments. Current methods can reach resolutions down to about 10 A, which allows the visualization of protein structures and secondary structure elements [189]. The advantage of this method is that it can be apphed to derive molecular structures of maaomolecules in the native state. 

The electron micrographs of Fig. 4.11 are more than mere examples of electron microscopy technique. They are the first occasion we have had to actually look at single crystals of polymers. Although there is a great deal to be learned from studies of single crystals by electron microscopy, we shall limit ourselves to just a few observations ... 

X-Ray Microscopy. Because of the short wavelength of x-rays, they have, for nearly 100 years, held out the hope of being utilized in order to significantly lower the diffraction limit of resolution when visible light is used. The difficulties of focusing x-rays and the relative weakness of x-ray sources have, until recently, fmstrated efforts to teach that goal (25). 

Soft x-rays with wavelengths of 1—10 nm ate used for scanning x-ray microscopy. A zone plate is used to focus the x-ray beam to a diameter of a few tens of nanometers. This parameter fixes and limits the resolution. Holographic x-ray microscopy also utilizes soft x-rays with photoresist as detector. With a strong source of x-rays, eg, synchrotron, resolution is in the 5—20-nm range. Shadow projection x-ray microscopy is a commercially estabflshed method. The sample, a thin film or thin section, is placed very close to a point source of x-rays. The "shadow" is projected onto a detector, usually photographic film. The spot size is usually about 1 ]lni in diameter, hence the resolution cannot be better than that. 

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. 

The classical polarizing light microscope as developed 150 years ago is still the most versatile, least expensive analytical instrument in the hands of an experienced microscopist. Its limitations in terms of resolving power, depth of field, and contrast have been reduced in the last decade, in which we have witnessed a revolution in its evolution. Video microscopy has increased contrast electronically, and thereby revealed structures never before seen. With computer enhancement, unheard of resolutions are possible. There are daily developments in the X-ray, holographic, acoustic, confocal laser scanning, and scanning tunneling micro-... 

Other artifacts that have been mentioned arise from the sensitivity of STM to local electronic structure, and the sensitivity of SFM to the rigidity of the sample s surface. Regions of variable conductivity will be convolved with topographic features in STM, and soft surfaces can deform under the pressure of the SFM tip. The latter can be addressed by operating SFM in the attractive mode, at some sacrifice in the lateral resolution. A limitation of both techniques is their inability to distinguish among atomic species, except in a limited number of circumstances with STM microscopy. 

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