Attenuated total reflection 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 

Uke germanium, siHcon also has a fairly high refractive index (n= 3.4). Silicon is rarely used for the fabrication of large IREs (for which the optical path may be 

Contracted Onginal beam size beam size Refractive index of hemisphere 

Similar to germanium, silicon also has a fairly high refractive index ( = 3.4). Silicon is rarely used for the fabrication of large IREs, for which the optical path may be several centimeters, because the presence of trace impurities leads to excessive absorption of the radiation below about 1200 cm . With the very short path through the IRE installed in a microscope objective, however, this is no longer a problem. Thus, either silicon or germanium can be used productively as the IRE in an ATR objective. 

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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]. 

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). 

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. 

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. 

Sommer, A. J., Tsinger, L. G., Marcott, C. and Story, G. M. (2001) Attenuated total internal reflection infrared mapping microspectroscopy using an imaging microscope. Appl. 

Lewis, L.L. and Sommer, A.J. (2000) Attenuated total internal reflection infrared mapping microspectroscopy of soft materials. Appl. Spectrosc., 54 (2), 324-330. 

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