ABSTRACT
The fundamental vibrations of the molecules that are most relevant to biology or medicine may be investigated using electromagnetic radiation in the mid-infrared spectral range. However, because water constitutes an essential ingredient of biomedical samples and exhibits a strong absorption in the mid-infrared spectral region, means had to be found to mitigate or avoid the pronounced spectral contribution of water. The use of small sample thicknesses, the application of intense light sources, the well-defined drying of the sample, and alternative techniques of vibrational spectroscopy (such as Raman spectroscopy) have all been tried. Today, biomedical vibrational spectroscopy is capable of supporting medical diagnostics by, for example, simultaneously determining the concentration of multiple analytes or by direct attribution of a sample’s spectrum to a particular disease under investigation. Since the spectroscopic tools as well as the algorithms for analysis are readily available, this chapter concludes with a note of caution
on evaluation processes and stresses the role of independent validation. 2.1 IntroductionWhen H. H. Mantsch inaugurated a series of conferences on “biomedical vibrational spectroscopy” in 1998,1 strong technical advances had already enabled the sensitive and reproducible study of biomedical samples by means of the spectroscopy of molecular vibrations. These advances have nowadays found their continuation in, for example, the availability of mid-infrared detector arrays or the commercialization of quantum cascade lasers. Many review articles, special issues, conference proceedings, and books testify to the growing importance of this vivid field of research.2-11 In order to elucidate the key factors of biomedical vibrational spectroscopy, this chapter focuses on the analysis of biofluids. Most of the concepts and approaches are also being used for tissue diagnostics and breath analysis. 2.2 On the Role of WaterSince the spring constants of typical bonds in organic molecules such as C-C, C-H, or O-H are on the order of 500 to 2000 N/m, the fundamental frequencies of simple diatomic stretch vibrations correspond to wavelengths of electromagnetic radiation around, say, 3 to 12 µm, which lies in the mid-infrared spectral range. Biological processes strongly depend on the fact that the relevant biochemistry takes place in water. For example, water contributes approximately 60% to the total mass of the human body. Thus, the spectroscopy of molecular vibrations in biological or medical samples is strongly related to the spectroscopic properties of water. The fundamental vibrations of water correspond to wavenumbers (i.e., inverse wavelengths) around 3700 cm-1 and 1600 cm-1 such that electromagnetic radiation with wavelengths in the mid-infrared region is strongly absorbed by the molecular vibrations of water (see Fig. 2.1). Given the abundance of water, the spectroscopy of fundamental vibrations of biomolecules in
cells, tissue, microorganisms, body fluids, and so on will be dominated by the absorption of water, while the biomolecules of interest will hide their smaller absorption signals in the “water background.” Since the mid-infrared spectroscopy of molecules is based on changing the dipole moment of the molecule, the fairly high dipole moment of water worsens the issue. An example is shown in Fig. 2.2, which shows the relative transmission of mid-infrared laser light through a thin microcavity filled with air (a) and which illustrates the 80% decrease of the transmission if the cavity is filled with water (b, solid line). If glucose is added to the water at a concentration of 500 mg/dL (which would be a pathologically high glucose level in blood, five times above the normal glucose concentration), only a very small further signal change is observed.12
