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Ex-vivo and In-vivo Optical Molecular Pathology

Edited by

Jürgen Popp

 

 

 

 

 

Title Page

List of Contributors

  1. Laurence Maximillian Almond
  2. Biophotonics Research Unit
  3. Leadon House
  4. Gloucestershire Royal Hospital
  5. Great Western Road
  6. Gloucester GL1 3NN
  7. UK
  1. Hugh Barr
  2. Biophotonics Research Unit
  3. Leadon House
  4. Gloucestershire Royal Hospital
  5. Great Western Road
  6. Gloucester GL1 3NN
  7. UK
  1. Benjamin Bird
  2. Northeastern University
  3. Laboratory for Spectral Diagnosis (LSpD)
  4. Department of Chemistry and Chemical Biology
  5. Huntington Ave
  6. Boston
  7. Massachusetts 02115
  8. USA
  1. Thomas Bocklitz
  2. Friedrich-Schiller University Jena
  3. Institute of Physical Chemistry
  4. Helmholtzweg 4
  5. D-07743 Jena
  6. Germany
  1. Riccardo Cicchi
  2. National Institute of Optics
  3. National Research Council
  4. Largo E. Fermi 6
  5. Florence
  6. Italy
  1. and
  1. European Laboratory for Non-linear Spectroscopy (LENS)
  2. Via N. Carrara 1
  3. Sesto-Fiorentino
  4. Italy
  1. Max Diem
  2. Northeastern University
  3. Laboratory for Spectral Diagnosis (LSpD)
  4. Department of Chemistry and Chemical Biology
  5. Huntington Ave
  6. Boston
  7. Massachusetts 02115
  8. USA
  1. Joerg Felber
  2. Friedrich-Schiller-Universität Jena
  3. Abteilung für Gastroenterologie
  4. Hepatologie und Infektiologie
  5. Klinik für Innere Medizin II
  6. Erlanger Allee 101
  7. D-07740 Jena
  8. Germany
  1. Jennifer Fore
  2. Northeastern University
  3. Laboratory for Spectral Diagnosis (LSpD)
  4. Department of Chemistry and Chemical Biology
  5. Huntington Ave
  6. Boston
  7. Massachusetts 02115
  8. USA
  1. Martin Goetz
  2. Universitätsklinikum Tübingen
  3. Klinik für Innere Medizin 1
  4. Otfried-Müller-Str 10
  5. D-72076 Tübingen
  6. Germany
  1. Joanne Hutchings
  2. Biophotonics Research Unit
  3. Leadon House
  4. Gloucestershire Royal Hospital
  5. Great Western Road
  6. Gloucester GL1 3NN
  7. UK
  1. Charlotte Kallaway
  2. Biophotonics Research Unit
  3. Leadon House
  4. Gloucestershire Royal Hospital
  5. Great Western Road
  6. Gloucester GL1 3NN
  7. UK
  1. Catherine Kendall
  2. Biophotonics Research Unit
  3. Leadon House
  4. Gloucestershire Royal Hospital
  5. Great Western Road
  6. Gloucester GL1 3NN
  7. UK
  1. Christoph Krafft
  2. Leibniz Institute of Photonic Technology
  3. Jena
  4. Albert-Einstein-Str. 9
  5. D-07745 Jena
  6. Germany
  1. Kathleen Lenau
  2. Northeastern University
  3. Laboratory for Spectral Diagnosis (LSpD)
  4. Department of Chemistry and Chemical Biology
  5. Huntington Ave
  6. Boston
  7. Massachusetts 02115
  8. USA
  1. Christian Matthäus
  2. Friedrich-Schiller University Jena
  3. Institute of Physical Chemistry
  4. Helmholtzweg 4
  5. D-07743 Jena
  6. Germany
  1. Antonella Mazur
  2. Northeastern University
  3. Laboratory for Spectral Diagnosis (LSpD)
  4. Department of Chemistry and Chemical Biology
  5. Huntington Ave
  6. Boston
  7. Massachusetts 02115
  8. USA
  1. Miloš Miljkoviimage
  2. Northeastern University
  3. Laboratory for Spectral Diagnosis (LSpD)
  4. Department of Chemistry and Chemical Biology
  5. Huntington Ave
  6. Boston
  7. Massachusetts 02115
  8. USA
  1. Masoud Mireskandari
  2. Universitätsklinikum Jena
  3. Institut für Pathologie
  4. Ziegelmühlenweg 1
  5. D-07740 Jena
  6. Germany
  1. Francesco S. Pavone
  2. European Laboratory for Non-linear Spectroscopy (LENS)
  3. Via N. Carrara 1
  4. Sesto-Fiorentino
  5. Italy
  1. and
  1. University of Florence
  2. Department of Physics and Astronomy
  3. Via G. Sansone 1
  4. Sesto Fiorentino
  5. Italy
  1. Iver Petersen
  2. Universitätsklinikum Jena
  3. Institut für Pathologie
  4. Ziegelmühlenweg 1
  5. D-07740 Jena
  6. Germany
  1. Jürgen Popp
  2. Leibniz Institute of Photonic Technology
  3. Jena
  4. Albert-Einstein-Str. 9
  5. D-07745 Jena
  6. Germany
  1. and
  1. Institute of Physical Chemistry & Abbe Center of Photonics
  2. Friedrich-Schiller University Jena
  3. Helmholtzweg 4
  4. D-07743 Jena
  5. Germany
  1. Eric Potma
  2. University of California
  3. School of Physical Sciences
  4. Beckman Laser Institute and Medical Clinic
  5. Health Sciences Road
  6. Irvine
  7. CA 92617
  8. USA
  1. Michael Schmitt
  2. Friedrich-Schiller University Jena
  3. Institute of Physical Chemistry
  4. Helmholtzweg 4
  5. D-07743 Jena
  6. Germany
  1. Jen Schubert
  2. Northeastern University
  3. Laboratory for Spectral Diagnosis (LSpD)
  4. Department of Chemistry and Chemical Biology
  5. Huntington Ave
  6. Boston
  7. Massachusetts 02115
  8. USA
  1. Andreas Stallmach
  2. Friedrich-Schiller-Universität Jena
  3. Abteilung für Gastroenterologie
  4. Hepatologie und Infektiologie
  5. Klinik für Innere Medizin II
  6. Erlanger Allee 101
  7. D-07740 Jena
  8. Germany
  1. Nick Stone
  2. University of Exeter
  3. College of Engineering
  4. Mathematics and Physical Sciences
  5. Streatham Drive
  6. Exeter EX4 4QL
  7. UK
  1. and
  1. Biophotonics Research Unit
  2. Leadon House
  3. Gloucestershire Royal Hospital
  4. Great Western Road
  5. Gloucester GL1 3NN
  6. UK
  1. Jeffrey L. Suhalim
  2. University of California
  3. School of Physical Sciences
  4. Beckman Laser Institute and Medical Clinic
  5. Health Sciences Road
  6. Irvine
  7. CA 92617
  8. USA
  1. James Wood
  2. Biophotonics Research Unit
  3. Leadon House
  4. Gloucestershire Royal Hospital
  5. Great Western Road
  6. Gloucester GL1 3NN
  7. UK

Preface

Spectroscopy, in general, plays an invaluable role in clinical diagnostics. The principle that all of these analytical techniques have in common is the interaction of electromagnetic radiation with the human body or a sample biopsy. All these techniques can be further divided into invasive and noninvasive methods. Long established are colorimetric methods that are based on absorption in the visible region of the spectrum, fluorescence, and optoacoustic techniques. A typical application of fluorescence is, for instance, flow cytometry. Optoacoustic effects are mainly employed for imaging, such as, for instance, the oxygenation level of blood. Examples that utilize light in the near-infrared (NIR) are optical coherence tomography (OCT) and NIR spectroscopy. OCT has become very popular in ophthalmology, dermatology, and cardiology. NIR spectroscopy is used, for instance, to measure blood sugar or to determine the saturation level of hemoglobin.

Visualization techniques commonly used in everyday clinical routines are often based on white light and are essential for the detection and diagnosis of diseases, as well as for the estimation of the risks associated with the disease. Also, for monitoring the development during treatment, these visualization techniques are essential. Endoscopy, for example, can visualize the inside of various organs such as the esophagus, the stomach, or the colon, and is therefore one of the most important techniques in internal medicine. Also very important in pathology are microscopic techniques. Bright-field microscopy is still the most widely used type of microscopy to investigate pathological biopsy samples. It is essential for the evaluation of cancer types, cancer grades, various inflammatory diseases, or changes associated with genetic disorders. There is no doubt that conventional pathology will always be based on these basic visualization techniques. However, by simply looking at structural changes in tissues and cells, there are often diagnostic questions that a pathologist cannot answer with absolute certainty. For instance, it is often very difficult to distinguish between certain cancer types. A better evaluation would often have consequences not only for the diagnosis but also for the treatment of the patient. Another important aspect is the time lag between the inspection of the patient and the preparation of the pathological report. By the application of new technologies, it is possible to have the routinely stained slides of small biopsy samples for microscopic evaluation on the same day of specimen acquisition. In uncomplicated cases, the final diagnosis can be made and the pathology report finalized on the same day. Problems occur if the specimen needs to be evaluated by ancillary methods (immunohistochemistry, molecular analysis) to reach a final and definite diagnosis. These ancillary tests can take a few days to be accomplished. The large resection specimens have to be fixed properly before processing and need to be sectioned cautiously before microscopic evaluations. A time frame of a few days to a week is needed for a complete evaluation of large samples. This unacceptable but legitimate delay contributes sometimes to the discomfort of the patient. Another aspect in pathology is the objectivity of the diagnosis itself. Although nothing can ever replace the trained eye of the pathologist, the evaluation of a pathological sample remains a matter of subjectivity. The same sample given to different pathologists may result in conflicting diagnoses. Interobserver variability is a well-known phenomenon in diagnostic pathology. Although false diagnoses by an experienced pathologist are generally rare, they are not impossible. In general, it would be advantageous if the pathologist could obtain more information about the sample or diseased area he is looking at. There are several imunohistochemical-based protocols, but they are usually limited to the analysis of a single protein. The inspection of multiple biomarkers simultaneously is rare. In addition, it may become expensive and time consuming to investigate a panel of markers. Spectroscopy offers great advantage to actually obtain chemical information about the sample. In other words, qualitative and quantitative biochemical information can be correlated with the pathology. The potential of such a combination is as wide as the field of pathology itself. Knowledge of the chemical composition of the sample would improve the evaluation of the state of the disease: for instance, what grade of malignancy the cancer has reached. It would also be possible to learn more about the origin of a cancer in the case of metastases. A great advantage over histopathology would be a faster pre-evaluation. Spectroscopic screening techniques can be, to a great extent, automated and can be operated by clinical personnel. Therefore, by screening the samples it would be possible to sort the potentially more dangerous cases before evaluation by the pathologist. This could be done on the same day, immediately after the inspection. The time-saving aspect would improve the diagnosis enormously. Additionally, the objective chemical information can be combined with the pathological diagnosis. Another great advantage is that potentially many spectroscopic techniques can be applied in vivo. Therefore, a coupling of spectroscopic fibers with endoscopes is possible. In addition to the visual image, the pathologist obtains valuable information about the chemical composition of the critical area at the same time. The potential to introduce new spectroscopic techniques into the operating theater could have great consequences for improving the decision making of the surgeon.

More recently, molecular spectroscopic techniques that are based on molecular vibrations have been applied to biomedical problems. The concept again is to combine well-established methods from analytical chemistry with optical techniques well established in medicine. The two main vibrational spectroscopy techniques are infrared (IR) spectroscopy, which is based on light absorption within the wavelength range of 2.5–25 μ m, and Raman spectroscopy, which is based on inelastic light scattering in the visible range. The coupling of these techniques to optical instrumentation has led to a tremendous growth within the field of vibrational spectroscopy. For clinical applications, the analytical instrumentation can be combined with either microscopes or endoscopes. The obtained spectral information is in comparison with, for instance, fluorescence very rich and specific. Both IR and Raman microscopy have been successfully employed to study compositional changes associated with various diseases. The two major advantages of both techniques are that they are noninvasive or minimally invasive and can be applied completely label free. Conventional histo- or imunohistochemical pathology and cytology rely on often poorly standardized staining protocols and the trained eye of the pathologist. Conceptually, IR and Raman microscopy can be automated, which would allow faster diagnosis. Because both techniques are noninvasive, the samples can be counterstained after the measurement and compared with standard histopathology. Over the past 10 years, IR and Raman microscopy have been applied to biopsy samples from virtually every organ.

When compared with each other, both techniques have advantages and disadvantages. Absorption measurements in the IR can be obtained relatively fast, which makes it possible to scan whole tissue sections within a reasonable time frame. On the other hand, the penetration depth is relatively small, so that the samples have to be cut into 5–10 μ m thin sections. Also problematic is the high absorption coefficient of water. These facts make IR microscopy very feasible to complement common histopathology and histocytology, but they hinder applications in vivo. Raman measurements can be performed under in vivo conditions, but require longer illumination times.

Other spectroscopic imaging approaches utilize nonlinear optical effects. Under illumination with pulsed laser radiation, molecules show various properties, which are, again, molecule specific and can therefore be used for characterization and identification. Today, most established for biological and medical applications is two-photon excited fluorescence (TPEF) spectroscopy. In TEPF, two photons of relatively long wavelength are used for excitation. Because of the application of the longer wavelength, the method is less invasive and leads to deeper penetration depths. As a consequence, TPEF can be applied in vivo and has been, for instance, used to image neurons. Another two-photon effect is second harmonic generation (SHG), which is ideal for imaging of centrosymmetric molecules such as collagen. Related to vibrational spectroscopy are the nonlinear Raman phenomena of coherent anti-Stokes Raman scattering (CARS) microscopy and stimulated Raman scattering (SRS). CARS microscopy allows rapid imaging of single molecular vibrations over fairly large areas. All these nonlinear imaging techniques can be combined, which is often referred to as multimodal imaging.

This book “Ex Vivo and In Vivo Optical Pathology” aims at introducing all the vibrational spectroscopic and nonlinear techniques in combination with modern pathology and illustrates their enormous potential for clinical applications. In particular, it addresses pathologists and medical doctors, as they are in the key positions to implement any new technical devices associated with the development of new instrumentation. The book should be a motivation to clinicians working in the field of pathology to be open to new technology. As the field of optical pathology is in a state of preclinical development, further collaboration between pathologists, natural scientists, and engineers is essential. It is therefore written in a way to be comprehensible to a very broad audience. Because the communication between experts of the above-mentioned fields is very important, the book addresses current problems from the clinical point of view, as well as technical issues associated with the development. Generally, the contents of the book should be of interest to spectroscopists who work with biological applications in particular. It should furthermore serve as a review and novel update for readers who work in the field of biophotonics, biophysics, and related areas. Last but not least, it should be a motivation for students, especially of the more advanced graduate levels who show an interest in these fields. As biophotonics becomes more and more an independent subject and is meanwhile taught at various universities and institutions, the book may also serve as a reference for instructors.

All contributors to this book are experts in their fields. The book is a consequence of long collaborations between the authors. Because the particular aim of the book is to improve pathology, it is structured in the following way: Chapters 1 and 2 are written by pathologists and clinicians to introduce the “gold standard” methodologies in the clinics and to point out current limitations. Chapter 1 introduces pathology as a diagnostic tool. First, a historical review is given. Standard histopathological routines are then described in detail. The chapter concludes with a list of the most commonly used terminology. Chapter 2 addresses commonly employed endoscopy. It also starts with a historical overview. Common types of clinical endoscopy, such as white-light endoscopy, chromoendoscopy, and endomicroscopy, are explained and compared. This is followed by a brief overview about the spectroscopic methods that have been combined with endoscopy. The chapter concludes with an outlook on future developments. Chapter 3 is a very detailed review of molecular pathology using IR and Raman microscopy. At the beginning, common histopathological techniques are described and compared with regard to their limitations. The spectroscopic basics for IR and Raman spectroscopy are explained, and examples of spectroscopic information and the generation of images are described. In the following, many examples of IR and Raman imaging of pathological tissue and cells are illustrated and how it is possible todistinguish normal from diseased, as for instance, cancer, tissue, and normal from abnormal cells. The novel techniques employed, such as micro-fluidics, are explained. Chapters 4 and 5 introduce nonlinear spectroscopic imaging techniques and their applications. Chapter 4 introduces CARS and SRS imaging and compares both techniques with spontaneous Raman spectroscopy. Examples of cell and tissue imaging in vivo and ex vivo are presented. Finally, current technical aspects and challenges are addressed. Chapter 5 focuses on multimodal imaging and introduces techniques such as TPEF, SHG, fluorescence life time imaging (FLIM), and CARS. Chapter 6 discusses the advantages and challenges of spectroscopy coupled with endoscopy that is referred to as endomicrospectroscopy. Chapter 7 deals with the implementation of modern computational statistics for diagnostic applications, because, for a careful analysis of disease-induced microspectroscopic image alterations (e.g., identification of disease-specific IR or Raman signatures), powerful image processing or chemometric approaches are required. This chapter briefly summarizes the general concepts of chemometric cell/tissue classification procedures or image analysis routines required for a successful realization of optical pathology by the spectroscopic imaging modalities presented in Chapters 3–7.

With this contribution, the authors hope to have accomplished a comprehensive overview of the current status of research in the field of optical pathology and to motivate the readers for further development and clinical applications.

Christian Matthäus, Michael Schmitt, and Jürgen Popp
Friedrich-Schiller University Jena
Institute of Physical Chemistry
Jena
Germany

Iver Petersen and Masoud Mireskandari
University Hospital Jena
Institute of Pathology
Jena
Germany


Jürgen Popp
Leibniz Institute of Photonic Technology Jena
Jena
Germany