Ebook: Vibrational Spectroscopy in Diagnosis and Screening

All over the world, scientists working in academia, government and industry are actively engaged in the characterisation, screening and diagnosis of different pathological conditions. In addition, they use an array of analytical techniques to investigate molecular changes associated with the interaction of drugs, chemicals and environmental factors on different organisms. Ideally, the aim is to monitor the system of interest, without disturbing it, in a sensitive, rapid and automated manner at minimum cost. Vibrational spectroscopy is one of the few analytical techniques that meets all of these requirements. It offers both advantages and tremendous potential, but also has limitations and challenges.

The goal of this book is to show how a range of vibrational spectroscopic techniques can be used in the screening and diagnosis of different systems and conditions and to provide information about the latest experimental and computational approaches in current use. This book not only covers the well known near infrared (NIR), mid-infrared and Raman spectroscopic methods, but also includes a new vibrational technique, Terahertz (THz) spectroscopy. With the developments in technology for the recording of spectra and the analysis of data using chemometric methods, vibrational spectroscopy has proved attractive to scientists engaged in the study of complex systems such as biological cells, tissues and foodstuffs.

In contrast to other books in the field, which mainly discuss the applications of vibrational spectroscopy in biomedical and food sciences, this book includes several chapters related to the diagnosis and screening of different biological systems such as calcified tissues, dental tissues, stem cells, and forensic and aquatic science.

The organization of this book is as follows:

After a short introduction, some historical background and information on application trends in the first chapter, the second chapter focuses on the background to methodological approaches from experimental to computational analysis in vibrational spectroscopy and microspectroscopy. The next chapter examines the analysis of protein structure, with particular attention for the screening of proteins in cells and tissues by vibrational spectroscopy. The use of near-infrared spectroscopy for the characterisation of single molecules in complex biological fluids is presented in chapter 4.

One of the important advantages of vibrational spectroscopy, and one which makes it ideal for the study of a diverse range of complex systems, is the capacity to record spectra of solids/aggregates/suspensions. These cannot be readily analysed using other techniques, which require homogeneous solutions or well defined crystals etc. Vibrational spectroscopy has therefore been invaluable in the characterisation of the protein aggregates often associated with neurodegenerative protein misfolding diseases such as prion disease, Alzheimer's disease, Parkinson's disease, Huntington's disease etc. The use of infrared spectroscopy in neurodegenerative protein-misfolding diseases is discussed in chapter 5.

In recent years the immense medical potential of stem cells for treating diseases has been a major development. The characterisation of stem cells is very important for quality control and structure-function studies. Vibrational spectroscopy offers the possibility of characterising stem cells, and examples of such studies are provided in chapter 6. The potential of infrared spectroscopy in the diagnosis and screening of cancer has been an active field of research for at least the last two decades. Much progress has been made in this field and this is discussed in chapters seven, eight and nine.

The use of vibrational spectroscopy for imaging cells and tissues is another emerging field of research activity. The power of this approach is increasingly appreciated, and it may not be too long before vibrational spectral imaging becomes a specialist tool in hospitals for screening and diagnosis. Chapter 8 gives an example of how FTIR and FPA methods are being used to discriminate breast tissular structure.

The application of infrared spectroscopy in the screening and diagnosis of diabetes is covered in chapter 10. Characterisation of bone, cartilage and dental tissues by vibrational spectroscopy is covered in chapters 11 and 12, respectively. The usefulness of vibrational spectroscopy in the diagnosis and screening of aquatic environments is covered in chapter 13. The next chapter of the book discusses the applications of vibrational spectroscopy for the screening and characterisation of tissues in relation to forensics research. The emerging field of synchrotron-based vibrational spectroscopy and the use of synchrotron radiation based infrared spectroscopy in the diagnosis and screening of feed and food quality is presented in chapter 15.

Our choice of topics will hopefully give readers a general overview with a broad perspective of the investigation of tissues and cells for screening and diagnosis purposes using different vibrational spectroscopic methods. We also believe that the book will provide knowledge about how these techniques can be used in the early diagnosis of disease, which is a very important issue.

Throughout the book we have taken care to use the nomenclature recommended in the journal of Applied Spectroscopy, which can be found via the link below:

http://www.s-a-s.org/media/pdf/2010/03/17/apls-64-01-136.pdf.

We hope that the materials presented in this book offer a glimpse of the power of NIR mid-FTIR Raman THz spectroscopy for the screening diagnosis of pathological and environmental conditions, as well as for understanding the molecular basis of disease processes.

We would like to thank all the authors who contributed to this book. Finally, we would like to thank Nihal Simsek Ozek (PhD student) and Dr. Ceren Aksoy and Dr. Sara Banu Akkas (former PhD students) of Feride Severcan's laboratory, for their valuable support; they were always eager to help us by finding references, drawing diagrams, preparing the cover of the book etc.

Feride Severcan (Ankara, Turkey)

Parvez I. Haris (Leicester, UK)

This Chapter introduces the topic of vibrational spectroscopy in diagnosis and screening. A brief background to the key vibrational spectroscopic methods that are in current use is discussed. The historical dimension to the use of vobrational spectroscopy in diagnosis and screening is addressed. Finally, the recent trend in the use of mid-infrared, near-infrared, Raman and Terahertz spectroscopy in various areas of diagnosis and screening is presented.

The success of vibrational spectroscopy in diagnosis and screening is closely dependent on the selection of right methodological approaches, which include correct sample preparation and spectral acquisition, as well as accurate data analysis, including pre-processing, statistical analysis, and complementing with different chemometric methods. Throughout this book, various aspects within these steps of infrared (IR) and Raman spectroscopy and microspectroscopy, as well as Terahertz spectroscopy, are discussed extensively. Taking this into consideration, the main concern of this chapter is to summarize methodological approaches and address a variety of practical points that are frequently encountered and need clarification during vibrational spectroscopic studies. Emphasis on computational analysis is also provided due to a lack of such a guide in the biospectroscopic literature especially targeting new starters. The focus is on describing univariate statistical analyses frequently used in biospectroscopy. Less emphasis is given on multivariate statistical analyses, because these subjects are thoroughly discussed with examples in the following chapters.

Discovering effective solutions to cope with the problems associated with human health issues necessitates the use of state of the art techniques in the fields of medicinal, industrial, and service-providing applications. Proteins are undoubtedly the work-horse of biological systems and play vital roles in a wide variety of important processes. Thus monitoring of proteins in cells and tissues using vibrational spectroscopy can be valuable for diagnosis and screening. Vibrational spectroscopy is particularly attractive as it can be used to probe proteins in complex systems including cells, tissues, biofluids and even whole organisms without the need for potentially perturbing probe molecules. Thanks to developments in instrumentation and data processing tools, it provides the researchers and laboratory technicians with relative ease to overcome the hurdles associated with the biological specimen preparations for protein research and handling of the data that is collected from a large number of samples and a huge variety of sources. In this chapter, besides experimental techniques and methods used in protein screening, applications of vibrational spectroscopy to different biological systems will be discussed.

As water is a strong absorber in the near infrared (NIR) region, the most efficient way to analyze single molecules in complex fluids is to bind the analyte of interest with high efficiency and selectivity to a well-designed carrier material. This strategy enables on one side the direct measurement and on the other side an increase in sensitivity due to the appearing pre-concentrating effects. In the present contribution, a strategy for the analysis of low and high density lipoprotein (LDL and HDL) in human serum applying NIR spectroscopy and multivariate calibration techniques is described. During method development it is useful to evaluate the feasibility of NIRS for classifying and identifying different analytes by establishing a qualitative principal component analysis (PCA) based cluster model. In case of LDL and HDL analysis, titanium oxide (TiO₂) beads offer an efficient material for selective immobilization, including incubation and a washing step. For quantification, a principal component regression (PCR) model of standards in a range from 500–3000 ppm (clinical value is 1500 ppm) and a partial least squares regression (PLSR) model of HDL standards in a range from 100–1000 ppm (clinical value is 400 ppm) are highly efficient. Wavenumber region selection allowed gaining main spectral information between 4000 and 7240 cm⁻¹. For the analysis of real samples it is necessary to analyze HDL and LDL in chronological order by employing precipitation. It is demonstrated that this NIRS method is a highly useful potential alternative or even supplementary clinical method for the fast determination of single molecules in complex biological fluids.

Neurodegenerative diseases are characterized by the malfunction and/or death of nerve cells, i.e. neurons, in the central and peripheral nervous system. Many neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, Huntington's disease, and the prion diseases, involve the misfolding and aggregation of naturally occurring proteins. These aggregates are thought to be toxic to cells, playing a critical role in neurodegeneration. This chapter focuses on how FTIR spectroscopy and microspectroscopy have been used to evaluate the structural changes in these disease-related proteins. It also examines the effects of lipid peroxidation and breakdown, and the disease-related metabolic changes in carbohydrate and nucleic acid composition. Methods for examining these biochemical changes in vitro, in cell culture, and in tissue are described. Finally, recent applications to a wide range of neurodegenerative diseases will be profiled with an outlook toward future advances and uses of FTIR spectroscopic methods for understanding, diagnosing, and treating neurodegenerative diseases.

Stem cell studies hold enormous potential for development of new therapies with unique differentiation ability of stem cells into various cell types. In this manner, investigation of stem cells in normal developmental and physiological states as well as in pathological conditions may lead to understanding of disease pathogenesis and development of new cellular therapies. The chemical changes responsible for a stem cell's unique physiological properties have been explored by various invasive techniques. The biological techniques being used for stem cell analyses involve fixing, staining, and chemical drying. All these steps have damaging effect on life processes of stem cells and they do not provide real-time information about cellular conditions. Therefore; newly non-destructive research methods that enable real-time chemical monitoring, high-quality data collection with less experimental complexity and expense must be used for stem cell characterization. In this context, vibrational spectroscopic appoaches can provide promissing alternatives to get new information into the field of stem cell biology, and they can be successfully used for chemical analysis, quantification and imaging of stem cells. The application of new complimentary vibrational spectroscopic aproaches will shed light on stem cell biology and will provide new insigth into stem cell reseaches for future therapeutic and regenerative medicines.

IR spectrum of a cell provide an accurate fingerprint of cell metabolism at the moment of the measure. This contribution explores the feasibility and the limits of using IR spectroscopy for the selection of new drugs based purely on their mode of action.

The recent development of Fourier Transform Infrared spectroscopic imaging devices provides a new imaging approach for cell and tissue pathology as distribution and structure of cellular components can be observed without sample staining. However, in order to translate this emerging technology to the clinic, optimization of the acquisition, classification and validation techniques is a prerequisite step. The information extracted from the spectral data largely depends on the quality of the raw spectra but also on the corrections and processing methods. In this chapter, some of the guidelines for the recording and preprocessing methods will be presented. Unsupervised statistical approaches for tissue type discrimination illustrate the potential of this technology on histopathology.

Infrared and Raman spectroscopy, the most frequently applied vibrational spectroscopic techniques, have advanced in recent years with increasing use both in academia and industry. This is due largely to steady improvements in instrumentation and the availability of chemometrics to assist in the analysis of data. Applications of infrared and Raman spectroscopy in cancer research have developed similarly and this contribution will focus on those applications.

Diabetes mellitus is one of the common metabolic diseases which severely affect many organs in the body. It involves perturbation of carbohydrate, fat and protein metabolisms and affects a great amount of the world's population. In this chapter, the application of vibrational spectroscopy, namely Infrared and Raman spectroscopy together with microspectroscopy, and Terahertz spectroscopy to the screening and diagnosis of diabetes will be discussed. In the first section, molecular mechanism and the pathophysiology of diabetes are summarized. In the following parts, brief information about the non-invasive blood glucose measurement techniques are mentioned, and the applications of vibrational spectroscopy to the diagnosis of diabetes are given. Vibrational spectroscopic applications are generally used together with different statistical analysis methods and chemometric tools, such as cluster analysis and artificial neural networks which provide determination of diabetes induced changes in various tissue macromolecules. Based on these variations, the discrimination of diseased state from the healthy subjects will become possible. Spectroscopic techniques have growing applicability due to their advantages over conventional techniques. They allow the visualization of the presence and state of the diseases depending on specific spectral database. By this way, the early diagnosis of metabolic diseases, such as diabetes will be possible by analyzing the variations in spectral parameters. Thus, understanding the basis of these spectroscopic techniques become an important issue in terms of the disease recognition.

The aim of this chapter is to review the application of vibrational spectroscopy and microspectroscopy, namely FTIR and Raman spectroscopy and microspectroscopy in the diagnosis of drug- or disease-induced bone and cartilage disorders. The first section gives an introduction on the structure and composition as well as diseases of bone and cartilage and summarizes diagnostic techniques used to quantify and qualify these changes. This section also mentions the advantages and disadvantages of currently used diagnostic techniques and vibrational spectroscopy and microspectroscopy. The second and third sections describe protocols for tissue preparation, data processing and analysis for FTIR and Raman spectroscopy and microspectroscopy. The last section presents the application of these vibrational techniques for detailed molecular investigation of the disorders in bone and cartilage. Specific examples are provided to illustrate the application of cluster analysis, analysis of secondary structure, etc.

Fourier Transform infrared/Raman spectroscopy and microscopy are highly valuable for conducting research on dental applications. In this chapter, the structural characterization of dentin and enamel, the biophysical and biochemical changes induced by diseases, diagnosis of dental caries, dentin-enamel junction (DEJ), effects of dental etchants on teeth and adhesive/dentin interface are reviewed.

Vibrational spectroscopy offers ecologists the opportunity to perform sensitive and non-destructive analyses, rapidly. The main purpose of this chapter is to illustrate some of many emerging applications of vibrational spectroscopy in the field of diagnosis and screening of aquatic environments. The sections of this chapter will summarize many innovative studies utilizing vibrational spectroscopy in order to answer ecologically based questions on aquatic species. The advantages of this technique will be presented thoroughly by revealing the diversity of aquatic species investigated with this tool. This summary of the work done in the field will benefit aquatic ecologists trying to go beyond their boundaries.

This literature review is aimed at discussing advanced vibrational spectroscopic techniques for the examination of evidence in a forensic investigation. Such evidence includes fingerprints, hair, body fluids and, bones. Recent developments in the fields of infrared and Raman spectroscopy have allowed for forensic evidence to be analyzed efficiently with minimal destruction to the sample. With the development of sophisticated techniques such as synchrotron radiation-Fourier transform infrared microscopy and spatially offset Raman spectroscopy in conjunction with advanced statistical analyses, chemical properties of bulk and micro-sized particles can be identified. Vibrational spectroscopy allows for characteristic structural information to be obtained for the molecule of interest and advancements in infrared and Raman spectroscopy in particular have been increasingly important to forensic science due to their ability to detect even trace amounts of evidence.

Synchrotron light is million-times brighter than sunlight. Synchrotron-based infrared light is thousands times than globar-sourced infrared light. With synchrotron-radiation based infrared microspectroscopy (SR-IMS), a non-invasive and most powerful research tool has been developed, which can be used to study inherent structure and biopolymer conformations of biomaterials at cellular and molecular levels. The objective of this research program was to use synchrotron-based SR-IMS with molecular chemistry imaging technique plus multivariate spectral analyses to study various biopolymers distribution and intensity at four different locations in a modeled feed tissue (sorghum bicolor L.) within intact tissue. The biopolymers investigated are highly associated with feed and food nutrient availability and included both easily-digested and digestion-resistant biopolymers in complex plant seed systems. The experiment of imaging in complex tissue system was conducted at the U2B beamline station at National Synchrotron Light Sources, Brookhaven National Laboratory (U.S. Dept of Energy). The unique chemical functional groups which are associated with biopolymers were analyzed using molecular spectroscopic technique. The results showed that the architecture of the complex feed system and distribution of biopolymers intensity and their ratios within four different locations in the modeled seed tissue could be revealed at an ultra-spatial resolution within cellular dimensions. The multivariate molecular spectral analyses are conclusive in showing that they can discriminate and classify the different inherent structures within the seed tissues designed for feed and food purpose. Other applications and future direction in diagnosis and screening of feed and food quality are also discussed.