ATR microspectroscopy

It can be seen from Equations 1.2 and 1.3 that the spatial resolution of infrared microspectroscopy can be improved by immersing the sample in a medium of high refractive index. This exactly what is done in attenuated total reflection (ATR) spectroscopy using a single-reflection hemispherical internal reflection element (IRE). Eor example, if a germanium n = 4.0) hemispherical IRE is used, not only... 

The technique of FT-IR internal ATR has been developed to the point that, today, ATR mirror lenses are available for an IR microscope. Furthermore, a newly developed, dedicated diamond internal reflection instrument, the lUuminatIR (Smith s Detection, Shelton, CT, USA) has now joined the ranks of microspectroscopy. This instrument incorporates a small, horizontally mounted diamond, on the surface of which is placed the material to be examined. In this way, the material is in optical contact with the diamond, and is held in place by a shaft pressing down from above. In this case, the radiation enters from beneath the instrument at an appropriate angle, and internally reflected rays are subsequently collected. The specimen is illuminated from beneath with a near-IR source that is detected and displayed on a video screen. With this optical arrangement, it is possible to locate a particular part of the material in the field of view and to interrogate it Such an arrangement is particularly user friendly, and indeed it is mostly used by... 

Sample preparation is perhaps the most critical part of a successful IR microspectroscopy experiment In the same way that the IR microscope can be used in a number of ways to collect spectra, so too can sample preparation can approached in a variety of ways. As biological materials are most frequently probed in either transmission mode or reflection mode, these methods will be described here. Other methods are also available, however, such as grazing incidence and attenuated total reflection (ATR) [1]. 

A wide range of compounds can be investigated by IR microscopy. The broad scale of sampling accessories for IR microspectroscopy even includes objectives for ATR or grazing angle measurements. 

Infrared microspectroscopy has been applied to the study of macerals (organic fractions) from a wide range of coals [106,116] and oxidized coal [117]. It may be performed either in transmission or reflection [106]. In the former case, sample preparation is tedious, and transmission IR microspectroscopy is seldom used. The preparation of samples for reflection measurements is simpler for example, the technique of attenuated total reflectance (ATR) has been applied to the study of coal [106]. Indeed, if the standard ATR accessories (which have been available for many years) were not suitable for block coal samples, recently, an ATR lens, equipped with a silicon (or germanium) internal reflection element, has become available for use with IR microscopes, and the technique was successfully applied... 

Another application of microspectroscopy is characterization of laminated polymer films. Multilayer polymer films of different fimctionality are common in the packaging industry and identification of the different layers is of commercial importance. The layers vary from about 1 /xm in thickness (adhesive) to 10-50 /zm and there are often several layers. If there are only two layers, attenuated total reflectance (ATR) methods can be used for identification. For films with more than two layers, the normal procedure is to microtome a 10-20 fj,m cross section for edgewise analysis using microspectroscopy. A comparison of infrared and Raman microspectroscopies for some multilayer films has been pubhshed (Fig. 25) (193). 

Figure 14.33 a) ATR spectra of single nylon carpet fiber untreated (top), treated (middle), and the result of spectral subtraction (bottom), b) Bicomponent fiber transmission spectrum (top), ATR spectrum of Nylon 6 sheath (middle), and difference spectrum of PET core (bottom).Reproduced with permission from Cho, L, et a ., "Single Fiber Analysis by Internal Reflection Infrared Microspectroscopy," iourna of Forensic Sciences 46 (2001), 1309-1314. Copyright 2001, ASTM International. 

FTIR has been used extensively for identification of coatings. Important methods for the study of paint material are KBr pellets prepared from scratched off paint material, ATR measurement of coated surfaces and measurements on cross sections of a coating with FTIR microspectroscopy. FTIR is an excellent way of obtaining information quickly about the basic chemical class of a binding material. Samples are placed in a diamond anvil cell and compressed to a thin film a beam condenser focuses the IR beam to an area of 1.0 mm [99]. Qther methods for determining the binder structure of cross-linked systems comprise PyGC. 

One of the strengths of FTIR spectroscopy is its diverse range of sampling techniques. Examples include attenuated total reflectance (ATR), diffuse reflectance (DRIFT), photoacoustic (PA), grazing angle, microspectroscopy and more specialised techniques such as synchrotron-radiation-based FTIR (SR-FTIR) microspectroscopy 11-12). The following section outlines ATR and SR-FTIR microspectroscopy in more detail and then provides specific applications of their use for the analysis of C. neoformans, S. favosa and L crassa. 

As noted above, the best spatial resolution of a microscope is ultimately determined by diffraction of the radiation. Thus, the spatial resolution is limited by the radius r of the Airy disk for the longest wavelength in the spectrum and hence depends on n, the refractive index of the medium in which the optics are immersed, for example, 1.0 for air and up to 1.56 for oils. Oil immersion is almost never used for infrared microspectroscopy because of absorption by the oil but has occasionally been used to improve the spatial resolution in Raman microspectroscopy. Immersion oils have been shown to be essential in order to obtain good depth resolution with confocal Raman microscopy [21]. Of greater importance from a practical standpoint for infrared microspectroscopy is the improvement in spatial resolution that is achieved in an attenuated total reflection (ATR) measurement with a hemispherical IRE, especially when the IRE is fabricated from germanium ( = 4.0) or silicon (n = 3.4.)... 

In order to enhance spatial resolution, it is necessary to make the NA of the objective larger, as is clear from Equation (17.3) that is, either n or 6, or both of them should be increased. Due to the optical geometry of a microscope, there is an upper limit for 0. On the other hand, it is possible practically to increase n by introducing an attenuated total reflection (ATR) accessory into a microscope (see Chapter 13 for the ATR method this is a frequently used accessory for many recent infrared microspectroscopy absorption measurements). The refractive index n of Ge, which is a commonly used material for an internal reflection element (IRE) in the ATR method, is about 4, and the NA when using a Ge IRE exceeds 2. This means that, if an ATR accessory with a Ge IRE is combined with a microscope, the theoretical spatial resolution is enhanced about four times that of the conventional reflection measurement. In fact, in the FT-IR microspectroscopic imaging measurement with a Ge ATR accessory, it has been confirmed that a spatial resolution comparable to the infrared wavelength used for the measurement is realized, and thus a higher spatial resolution may be attainable. 

See also ATR and Reflectance IR Spectroscopy, Applications High Resolution Electron Energy Loss Spectroscopy, Applications Inelastic Neutron Scattering, Applications Inelastic Neutron Scattering, Instrumentation IR Spectroscopy, Theory Raman and IR Microspectroscopy Surface-Enhanced Raman Scattering (SERS), Applications. 

The application of IR spectroscopy with respect to the characterisation of cellulosic (plant) fibres is demonstrated. The ability to characterise fibres is of importance to textile conservators, as this information aids in the determination of the age and origin of the artefact from which they are taken, and may influence the choice of treatment. The fibres under examination are taken largely from the bast group (flax, hemp, jute and ramie) in addition, sisal and cotton are compared. FT-IR microspectroscopy and ATR techniques are employed. To complement the conventional use of these methods, the inherent polarisation effects of the equipment are exploited to record polarised IR spectra. Jute, sisal and cotton are readily differentiated, but flax, hemp and ramie prove more difficult to distinguish. Peak ratio techniques are apphed in the latter case. 2 refs. 

The composition at various positions on the lens was measured by attenuated total reflectance Fourier transform infrared (ATR-FTIR) microspectroscopy. The composition was determined from the normalized intensity of characteristic peaks for SAN17 at 698 cm and for PMMA at 1727 cm Figure 2a. Data were collected with a Nexus 870 FT-IR bench coupled to a continuum microscope (Thermo Nicolet, Madison, WI). Spectra were collected at a resolution of 2 cm" for 32 scans. Individual spectra were collected at 320 oon intervals over the lens surface. From the compositions, the refractive index at each point was calculated. 

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