Fluorescence microscopy, analytical method

This chapter discusses the range of analytical methods which use the properties of X-rays to identify composition. The methods fall into two distinct groups those which study X-rays produced by the atoms to chemically identify the elements present, and X-ray diffraction (XRD), which uses X-rays of known wavelengths to determine the spacing in crystalline structures and therefore identify chemical compounds. The first group includes a variety of methods to identify the elements present, all of which examine the X-rays produced when vacancies in the inner electron shells are filled. These methods vary in how the primary vacancies in the inner electron shell are created. X-ray fluorescence (XRF) uses an X-ray beam to create inner shell vacancies analytical electron microscopy uses electrons, and particle (or proton) induced X-ray emission (PIXE) uses a proton beam. More detailed information on the techniques described here can be found in Ewing (1985, 1997) and Fifield and Kealey (2000). 

The most frequently applied analytical methods used for characterizing bulk and layered systems (wafers and layers for microelectronics see the example in the schematic on the right-hand side) are summarized in Figure 9.4. Besides mass spectrometric techniques there are a multitude of alternative powerful analytical techniques for characterizing such multi-layered systems. The analytical methods used for determining trace and ultratrace elements in, for example, high purity materials for microelectronic applications include AAS (atomic absorption spectrometry), XRF (X-ray fluorescence analysis), ICP-OES (optical emission spectroscopy with inductively coupled plasma), NAA (neutron activation analysis) and others. For the characterization of layered systems or for the determination of surface contamination, XPS (X-ray photon electron spectroscopy), SEM-EDX (secondary electron microscopy combined with energy disperse X-ray analysis) and... 

Other analytical methods can also be applied for the detection of F in archaeological artefacts, especially when it is possible to take a sample or to perform microdestructive analysis. These are namely the electron microprobe with a wavelength-dispersive detector (WDX), secondary ion mass spectrometry (SIMS), X-ray fluorescence analysis under vacuum (XRF), transmission electron or scanning electron microscopy coupled with an energy-dispersive detector equipped with an ultrathin window (TEM/SEM-EDX). Fluorine can also be measured by means of classical potentiometry using an ion-selective electrode or ion chromatography. 

Technical examination of objects coated with a protective covering derived from the sap of a shrubby tree produces information that can be used to determine the materials and methods of manufacture. This information sometimes indicates when and where the piece was made. This chapter is intended to present a brief review of the raw material urushi, and the history and study of its use. Analytical techniques have included atomic absorption spectroscopy, thin layer chromatography, differential thermal analysis, emission spectroscopy, x-ray radiography, and optical and scanning electron microscopy these methods and results are reviewed. In addition, new methods are reported, including the use of energy dispensive x-ray fluorescence, scanning photoacoustical microscopy, laser microprobe and nondestructive IR spectrophotometry. 

Finally we wish to introduce four new analytical methods for lacquer problems three are now used in the Detroit Institute of Arts lacquer project and the remaining method is undergoing development for lacquer use. These methods are energy dispersive x-ray fluorescence, laser microprobe, scanning photoacoustical microscopy, and nondestructive IR spectrophotometry. 

Abstract Surface analyses have been one of the key technologies for corrosion control and surface finishing. It is very important that the most appropriate apparatus for the purpose of the analyses should be selected from various analytical techniques. In this chapter, surface analytical methods for corrosion control and surface finishing, such as X-ray fluorescence analysis (XRF), X-ray diffraction analysis (XRD), X-ray photo-electron spectroscopy (XPS), scanning electron microscopy (SEM), transmission electron microscopy (TEM), Auger electron spectroscopy (AES), Secondary ion mass spectrometry (SIMS), Rutherford back-scattering spectrometry (RBS), Surface-enhanced Raman spectroscopy (SERS), Fourier-transform infrared spectroscopy (FTIR), and so on, are briefly introduced. 

X-Ray fluorescence is widely used in the analysis of modern refractories, including boron nitride, together with standard analytical methods [48]. Radiation-induced products in a-BN can be studied with scanning transmission electron microscopy (STEM) [49]. Transmission electron microscopy using bright field and dark field imaging and selected area electron diffraction was used to study a-BN ceramics formed by pyrolysis of organyl-substituted amino-borazine precursors. These ceramics are mainly amorphous with pockets of microfibrils or microcrystallites [50]. 

Most analytical methods employed to study elementary reactions and reaction intermediates provide information about ensembles of reactive molecules. However, for more than a decade now, it has been possible to monitor transformations of single molecules by means of fluorescence microscopy. This involves the labeling of at least one of the reactants with a fluorophore, which in the case of a catalytic reaction would be either the catalyst or the substrate. The visualization of a chemical reaction then relies on changes of the fluorescence induced by a transformation at the reactive center of a catalyst. Herten and coworkers present several case histories. The use of single-molecule spectroscopy and the way in which the methods developed in this field may give rise to potential single-molecule catalysis are described in Chapter 3. 

The computer age has brought about considerable innovation in the operation of laboratory instrumentation. One consequence of this is the wider acceptance and utilization of the optical microscope as a quantitative analytical instrument. A brief literature survey illustrates the diversity of disciplines and optical methods associated with the development of computer interfaced optical microscopy. This is followed by a description of how our methods of fluorescence, interferometry and stereology, nsed for characterizing polymeric foams, have incorporated computers. 

The lifetime of the excited state of fluorophores may be altered by physical and biochemical properties of its environment. Fluorescence lifetime imaging microscopy (FLIM) is thus a powerful analytical tool for the quantitative mapping of fluorescent molecules that reports, for instance, on local ion concentration, pH, and viscosity, the fluorescence lifetime of a donor fluorophore, Forster resonance energy transfer can be also imaged by FLIM. This provides a robust method for mapping protein-protein interactions and for probing the complexity of molecular interaction networks. 

Electron probe and X-ray fluorescence methods of analysis are used for rather different but complementary purposes. The ability to provide an elemental spot analysis is the important characteristic of electron probe methods, which thus find use in analytical problems where the composition of the specimen changes over short distances. The examination of the distribution of heavy metals within the cellular structure of biological specimens, the distribution of metal crystallites on the surface of heterogeneous catalysts, or the differences in composition in the region of surface irregularities and faults in alloys, are all important examples of this application. Figure 8.45 illustrates the analysis of parts of a biological cell just 1 pm apart. Combination of electron probe analysis with electron microscopy enables visual examination to be used to identify the areas of interest prior to the analytical measurement. 

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