Ebook: Biophotonics in Pathology

The evolution of anatomic pathology from a discipline based upon direct observation of visual light images to whole spectrum multi-modality computer-intensive analysis is entering a phase of accelerated growth. However, the technologic base is expanding more rapidly than our ability to exploit it for diagnostic and experimental pathology. While the theoretical and practical underpinnings of the new technology are complex, the underlying principles are quite simple. Energy interactions with a biological specimen basically primarily involve four kinds of effects: absorption, reflection, scatter, and secondary emission. This holds true, for example, for both a radio wave perturbing aligned molecular dipoles or UV light shining at a slide. Radiologists have been quicker than pathologists at exploiting this generalization. The reasons for this may be found in the pixel level complexity of a microscopic slide as compared to a typical radiological image. The aim in pathology is reconstructing images from the data with resolution good enough for morphologic analysis at the cellular and tissue level, whereas the aim in radiology is to get enough visualization to identify the lesion or location of a biochemical event. Paradoxically, modern radiology deals with both ends of the spectrum (molecules and organs), while pathologists remain focused (pun intended) on cells and tissue architecture, and study molecular events in that context.

The purpose of this book is to present some of the promising new image-based and related technologies for the study of visualization, characterization, and analysis of abnormal cells and tissues that have evolved over the past few years and to discuss their current and potential applications in experimental pathology and, where feasible, clinical pathologic diagnosis. Just as the flourishing of molecular biology has led to a paradigm shift that has rejuvenated our field, the convergence of biomedicine, bioengineering, and sophisticated new tools of mathematical analysis will move us towards a more quantitative and analytic discipline.

Although Photonics is a term often used in relation to light-based circuits, it is actually more inclusive, including the generation, emission, transmission, modulation, and signal processing of light. Biophotonics can therefore be used to describe the development and application of optical techniques to the study of biological molecules, cells, and tissues. This can be applied to imaging-based modalities for the analysis of non-photonic data, such as various other biophysical parameters, an example of which (impedance) is included in this volume. This is the rationale for the title of this volume: “Biophotonics in Pathology”. As will be discussed in the final chapter, we are beginning to see a convergence of radiology and pathology, and pathologists must be quick to embrace the rapidly evolving new technology if they are to remain relevant, which is the rationale for the subtitle: “Pathology at the Crossroads”. In this regard, radiologists, as well as basic scientists and engineers working in these overlapping areas, should also find the material presented here of interest, as it shows the confluence of methodologies similar to those applied in radiology with morphologic analysis at the cellular and tissue level.

Unfortunately, the technologic basis for these approaches is expanding more rapidly than our ability to exploit it for both diagnostic and experimental pathology. While the theoretical and practical underpinnings of the new technology are complex, the underlying physical principles are straightforward. Collaboration of engineers and physical scientists with biomedical researchers is slowly increasing, but it is difficult for many of the biologically oriented to feel comfortable with the concepts and approaches of the physical sciences and their mathematical underpinnings. The converse is often true as well, and many of the excellent publications in the engineering literature, for example, make use of very unsophisticated biological models.

The authors, all experts in the field, have described this technology in a manner accessible to an audience consisting of pathologists, cell biologists, and biochemists as well as biomedical engineers, using their own research as well as literature review to provide descriptions and examples of the use of this new armamentarium. Although it has been written from the prospective of pathology, it should be of interest to this wider audience as well. Moreover, as I have suggested above, it appears likely that these approaches, as well as new techniques for in situ molecular imaging that are outside the scope of this book, as well as evolving non-invasive radiological procedures for screening and diagnosis will lead to a convergence of pathology and radiology, which would make for an even more profound paradigm shift.

Some of the material in this volume has appeared previously in review format in several issues of volumes 34 and 35 of Analytical Cellular Pathology. Analytical Cellular Pathology is a journal dedicated to the publication of original articles relating to the application of physical techniques, new imaging modalities, and computational analysis in addition to molecular approaches to the study of disease. It also publishes reviews, mini-reviews, and commentaries, which are also subject to review by its Editorial Board.

Stanley Cohen, M.D.

Director, The Center for Biophysical Pathology

UMDNJ–New Jersey Medical Center

Newark, N.Y.

E-mail: cohenst@ umdnj.edu

The conventional optical microscopes have been used widely in scientific research and in clinical practice. The modern digital microscopic devices combine the power of optical imaging and computerized analysis, archiving and communication techniques. It has a great potential in pathological examinations for improving the efficiency and accuracy of clinical diagnosis. This chapter reviews the basic optical principles of conventional microscopes, fluorescence microscopes and electron microscopes. The recent developments and future clinical applications of advanced digital microscopic imaging methods and computer assisted diagnosis schemes are also discussed.

It requires a good deal of will power to resist hyperbole in considering the advances that have been achieved in fluorescence microscopy in the last 25 years. Our effort has been to survey the modalities of microscopic fluorescence imaging available to cell biologists and perhaps useful for diagnostic pathologists. The gamut extends from established confocal laser scanning through multiphoton and TIRF to the emerging technologies of super-resolution microscopy that breech the Abbe limit of resolution. Also considered are the recent innovations in structured and light sheet illumination, the use of FRET and molecular beacons that exploit specific characteristics of designer fluorescent proteins, fluorescence speckles, and second harmonic generation for native anisometric structures like collagen, microtubules and sarcomeres.

Spectral imaging methods are attracting increased interest from researchers and practitioners in basic science, preclinical and clinical arenas. A combination of better labeling reagents and better optics creates opportunities to detect and measure multiple parameters at the molecular and cellular level. These tools can provide valuable insights into the basic mechanisms of life, and yield diagnostic and prognostic information for clinical applications. There are many multispectral technologies available, each with its own advantages and limitations. This chapter will present an overview of the rationale for spectral imaging, and discuss the hardware, software and sample labeling strategies that can optimize its usefulness in clinical settings.

Much of the difficulty in reaching consistent evaluations of radiology and pathology imaging studies arises from subjective impressions of individual observers. Developing strategies that can reliably transform complex visual observations into well-defined algorithmic procedures is an active area of exploration which can advance clinical practice, investigative research and outcome studies. The literature shows that when characterizations are based upon computer-aided analysis, objectivity, reproducibility and sensitivity improve considerably. Advanced imaging and computational tools could potentially enable investigators to detect and track subtle changes in measurable parameters leading to the discovery of novel diagnostic and prognostic clues which are not apparent by human visual inspection alone. The overarching objective of this book chapter is to provide readers with a summary of the origin, evolution and future directions for the fields of automated image interpretation and computer-assisted diagnostics. The chapter begins with a high-level overview of the fields of image processing, pattern recognition, and computer vision followed by a description of how these disciplines relate to the more comprehensive fields of computer-assisted diagnostics and image guided decision support. Throughout the remainder of the chapter we have supplied multiple illustrative examples demonstrating how recent advances and innovations in each of these areas have impacted clinical and research activities throughout pathology and radiology including high-throughput tissue microarray analysis, multi-spectral imaging, and image co-registration.

In pathology, histological examination of the tissue is the “gold standard” to diagnose various diseases. It has contributed significantly toward identifying the abnormalities in tissues and cells, but has inherent drawbacks when used for fast and accurate diagnosis. These limitations include the lack of in vivo observation in real time and sampling errors due to limited number and area coverage of tissue sections. Its diagnostic yield also varies depending on the ability of the physician and the effectiveness of any image guidance technique that may be used for tissue screening during excisional biopsy. In order to overcome these current limitations of histology-based diagnostics, there are significant needs for either complementary or alternative imaging techniques which perform non-destructive, high resolution, and rapid tissue screening. Optical coherence tomography (OCT) is an emerging imaging modality which allows real-time cross-sectional imaging with high resolutions that approach those of histology. OCT could be a very promising technique which has the potential to be used as an adjunct to histological tissue observation when it is not practical to take specimens for histological processing, when large areas of tissue need investigating, or when rapid microscopic imaging is needed. This review will describe the use of OCT as an image guidance tool for fast tissue screening and directed histological tissue sectioning in pathology.

The study of intact, living cells using non-invasive optical spectroscopic methods offers the opportunity to assess cellular structure and organization in a way that is not possible with commonly used cell biology imaging techniques. We have developed a novel spectroscopic technique for diagnosing disease at the cellular level based on using low-coherence interferometry (LCI) to detect the angular distribution of scattered light. Angle-resolved LCI (a/LCI) combines the ability of LCI to isolate scattering from sub-surface tissue layers with the ability of light scattering spectroscopy to obtain structural information on sub-wavelength scales. In application to examining cellular structure, a/LCI enables quantitative measurements of changes in the size and texture of cell nuclei. These quantitative measurements are characteristic of different pathological states. The capabilities of a/LCI were demonstrated using a clinical system that can be applied in endoscopic surveillance of esophageal tissue, producing high sensitivity and specificity for detecting dysplastic tissues in vivo. Experiments with in vitro cell samples also show the utility of a/LCI in observing structural changes due to environmental stimuli as well as detecting apoptosis due to chemotherapeutic agents.

MRI, one of the major clinical imaging modalities, has gained an important role in studying small animal models, e.g., rats and mice. But imaging rodents comes with challenges, since the image resolution needs to be ∼ 3000-times higher to resolve anatomical details at a level comparable to clinical imaging. A resolution on the order of 100 microns or less redefines MR imaging as MR microscopy. We discuss in this chapter the basic components of the MR imaging chain, with a particular emphasis on small animal imaging demands: from hardware design to basic physical principles of MR image formation, and contrast mechanisms. We discuss special considerations of animal preparation for imaging, and staining methods to enhance contrast. Attention is given to factors that increase sensitivity, including exogenous contrast agents, high performance radiofrequency detectors, and advanced MR encoding sequences. Among these, diffusion tensor imaging and tractography add novel information on white matter tracts, helping to better understand important aspects of development and neurodegeneration. These developments open avenues for efficient phenotyping of small animal models, in vivo – to include anatomical as well as functional estimates, or ex-vivo – with exquisite anatomical detail. The need for higher resolution results in larger image arrays that need to be processed efficiently. We discuss image-processing approaches for quantitative characterization of animal cohorts, and building population atlases. High throughput is essential for these methods to become practical. We discuss current trends for increasing detector performance, the use of cryoprobes, as well as strategies for imaging multiple animals at the same time. Ultimately, the development of highly specific probes, with the possibility to be used in multimodal imaging, will offer new insights into histology. MRM, alone or in combination with other imaging modalities, will increase the knowledge of fundamental biological processes, help understanding the genetic basis of human diseases, and test pharmacological interventions.

Biological organisms and their component organs, tissues and cells have unique electrical impedance properties. Impedance properties often change with changes in structure, composition, and metabolism, and can be indicative of the onset and progression of disease states. Over the past 100 years, instruments and analytical methods have been developed to measure the impedance properties of biological specimens and to utilize these measurements in both clinical and basic science settings. This chapter will review the applications of impedance measurements in the biomedical sciences, from whole body analysis to impedance measurements of single cells and cell monolayers, and how cellular impedance measuring instruments can now be used in high throughput screening applications.

Raman scattering is the inelastic scattering of light by chemical bonds, and can therefore show molecular specificity. It can be used both in pure spectroscopy mode, and in imaging mode. While many applications of Raman spectroscopy and imaging in the biomedical field have been so far demonstrated, the use of this technology for pathology applications is still in its early stages. In this paper we review some of the most important recent developments in this field, including a description of relevant technologies, applications to molecular sensing, characterization of cells and tissues of interest, and disease detection via Raman scattering.

Miniature microscopes are being developed to examine tissue in situ for early anatomic and molecular indicators of disease, in real time, and at cellular resolution. These new devices will lead to a shift from the current diagnostic paradigm of biopsy followed by histopathology and recommended therapy, to one of non-invasive point-of-care diagnosis with the possibility of treatment in the same session. This potential revolution in disease management may have a major impact on the training of future physicians to include the use and interpretation of real-time in vivo microscopic data, and will also affect the emerging fields of telepathology and telemedicine. Implementation of new technologies into clinical practice is a complex process that requires multidisciplinary communication and collaboration among clinicians, engineers and scientists. As such, our aim is to provide a forward-looking view of the critical issues facing the development of new technologies and directing clinical education. Here, we focus on the use of in vivo microscopy for detection of malignant and pre-malignant lesions as well as for guiding therapy. We will highlight some of the areas in which in vivo microscopy could address unmet clinical needs, and then review the technological challenges that are being addressed, or need to be addressed, for in vivo microscopy to become an effective clinical tool.

In vivo optical imaging is being conducted in a variety of medical applications, including optical breast cancer imaging, functional brain imaging, endoscopy, exercise medicine, and monitoring the photodynamic therapy and progress of neoadjuvant chemotherapy. In the past three decades, in vivo diffuse optical breast cancer imaging has shown promising results in cancer detection, and monitoring the progress of neoadjuvant chemotherapy. The use of near infrared spectroscopy for functional brain imaging has been growing rapidly. In fluorescence imaging, the difference between autofluorescence of cancer lesions compared to normal tissues were used in endoscopy to distinguish malignant lesions from normal tissue or inflammation and in determining the boarders of cancer lesions in surgery. Recent advances in drugs targeting specific tumor receptors, such as monoclonal antibodies (mAb), has created a new demand for developing non-invasive in vivo imaging techniques for detection of cancer biomarkers, and for monitoring their down regulations during therapy. Targeted treatments, combined with new imaging techniques, are expected to potentially result in new imaging and treatment paradigms in cancer therapy. Similar approaches can potentially be applied for the characterization of other disease-related biomarkers. In this chapter, we provide a review of diffuse optical and fluorescence imaging techniques with their application in functional brain imaging and cancer diagnosis.

The recent revolution in digital technologies and information processing methods present important opportunities to transform the way optical imaging is performed, particularly toward improving the throughput of microscopes while at the same time reducing their relative cost and complexity. Lensfree computational microscopy is rapidly emerging toward this end, and by discarding lenses and other bulky optical components of conventional imaging systems, and relying on digital computation instead, it can achieve both reflection and transmission mode microscopy over a large field-of-view within compact, cost-effective and mechanically robust architectures. Such high throughput and miniaturized imaging devices can provide a complementary toolset for telemedicine applications and point-of-care diagnostics by facilitating complex and critical tasks such as cytometry and microscopic analysis of e.g., blood smears, Papanicolaou (Pap) tests and tissue samples. In this article, the basics of these lensfree microscopy modalities will be reviewed, and their clinically relevant applications will be discussed.

Science advances both by conceptual leaps and by improved observational and analytic tools. Mechanism and function in biological systems can best be understood in the context of the complex microenvironments in which they occur, and for this purpose morphologic analysis can be critical. The technological advances in cell and tissue imaging described in this book are currently finding application in a wide variety of basic, translational, and clinical biomedical studies. We have chosen some specific approaches that illustrate the various categories of imaging methodologies available. Many other ways of applying modern morphology-based interrogation of cells and tissues have already been described and are continuously evolving. This chapter provides examples of some of these. On the clinical front, radiologists have embraced new imaging technique to a greater extent than have pathologists. This chapter discusses some of the factors responsible for this, and suggests that pathology and radiology are converging towards a more holistic approach to diagnostic imaging.