Transflectance spectrum

FIGURE 17.12 Influence of a 3°C change in room temperature. Hay absorbance spectra displayed as fourth-order derivatives with a fourth-order derivative transflectance spectrum of pure water included for reference. 

Individual cells may also be deposited onto a Cap2 window support, but perhaps the more practical and most appropriate (in terms of its match to cytological practices) method nowadays is to use a low-e glass slide and record a transflection spectrum, see next section. 

We should note that interference fringes are sometimes seen in transflection spectra. When the film is smooth but the substrate is rough, the full intensity of the beam that has been reflected from the front surface of the film will be measured at the detector. However, only a fraction of the beam that has been diffusely reflected from the substrate will be focused on the detector element. In this case, the fringes are not completely canceled, and the baseline of the transflectance spectrum exhibits a sinusoidal modulation. This effect can often be seen in the transflectance spectrum... 

Vibrational microspectrometry will undoubtedly be applied to medical diagnosis in the near future. One particularly important application of microspectrometry is for the characterization of tissue samples. Tissue samples can be mounted on a water-insoluble infrared-transparent window such as ZnSe, but these windows are expensive and not conducive to visual examination (e.g., after staining of the tissue). A convenient alternative to transmission spectrometry is the measurement of the transflectance spectrum (see Section 13.5) of tissue samples mounted on low-emissivity glass slides [4]. These slides are transparent to visible light but highly reflective to mid-infrared radiation. 

The final type of measurement that can be made with the microscope in its reflection mode is diffuse reflection (DR) spectroscopy. Today, very few appHca-tions of mid-lR microspectroscopy of neat samples are available, because for mid-IR DR spectrometry the samples should be diluted to a concentration of between 0.5 and 5% with a nonabsorbing diluent (e.g., KBr powder) to preclude band saturation and severe distortion by reflection from the front surface of the particles. However, this mode has substantial application for NIR measurements, where sample dilution is not needed. Because the absorption of NIR radiation by most samples is rather weak, they must either be at least 1 mm thick or be mounted on a reflective or diffusing substrate, such as a ceramic or Teflon disk. In the latter case, the spectrum is caused by a combination of diffuse reflection, transflection and front-surface reflection (hopefully with diffuse reflection being the dominant process). 

One hundred and thirty-eight (138) oil samples were analyzed with visible (vis) and near-infrared (NIR) transflectance spectroscopy. Forty-six of them were Greek pure extra virgin olive oils and the same oils adulterated with 1% (w/w) and 5% (w/w) sunflower oil. However, no significant difference was found between the spectrum of pure sunflower oil and that of olive oil, which can be detected by the naked eye. Olive and sunflower oils differ in their composition principally in their content of linoleic and oleic acids. Accordingly, typical figures for olive oil were quoted at 12.3% and 66.3%, respectively, while for sunflower oil the corresponding mean values of 66.2% and 25.1%, respectively. 

Figure 4.2 Schematic diagrams of some reflection spectroscopy techniques (a)-(c), internal reflection spectroscopy (a) and (b), single-reflection ATR (c), multiple internal reflection (MIR) (d) transflectance . Notes (i) collimated beam with ATR hemicylinder (ii) the component of specular reflectance (dotted line) will be superimposed on the double-pass transmission spectrum of the film in the transflectance measurement.

Another type of transflectance spectroscopy that is commonly used for measurements of near-infrared spectra falls somewhere between specular reflection and diffuse reflection spectroscopy (see Chapter 16). Because overtone and combination bands are weak, anomalous dispersion is usually negligibly small in the near infrared. It is quite common for the NIR spectra of liquids to be measured by transflection using the same optical geometry used for diffuse reflection. In this case, the sample is loaded in a cell whose rear face is a ceramic disk. Radiation that has passed through the sample is scattered back through the sample, emerges from the window, and is focused onto the detector. This is a particularly useful way of measuring the spectrum of turbid samples, such as milk. 

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