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The second volume of the series Manuals in Biomedical Research, this book is aimed to be both a concise introduction to the diverse field of microscopy and a practical guide those who require the use of microscopic for methods in their research. It provides young as well as experienced scientists a state-of-the-art multidisciplinary overview of microscopic techniques, covering all the major microscopy fields in biomedical sciences and showing their application in evaluating samples ranging from molecules to cells and tissues.Microscopy has revolutionized our understanding of biological events. Within the last two decades, microscopic techniques have provided insights into the dynamics of biological processes that regulate such events. Biological discovery, to a large extent, depends on advances in imaging techniques and various microscopic techniques have emerged as central and indispensable tools in the biomedical sciences.The four authors bring with them extensive experiences spanning across disciplines such as Microbiology, Molecular and Cell Biology, Tissue Engineering, Biomedical and Regenerative Medicine and so forth, reinforcing the fact that microscopy has proven useful in countless investigations into the mysteries of life.
The second volume of the series Manuals in Biomedical Research, this book is aimed to be both a concise introduction to the diverse field of microscopy and a practical guide those who require the use of microscopic for methods in their research. It provides young as well as experienced scientists a state-of-the-art multidisciplinary overview of microscopic techniques, covering all the major microscopy fields in biomedical sciences and showing their application in evaluating samples ranging from molecules to cells and tissues.Microscopy has revolutionized our understanding of biological events. Within the last two decades, microscopic techniques have provided insights into the dynamics of biological processes that regulate such events. Biological discovery, to a large extent, depends on advances in imaging techniques and various microscopic techniques have emerged as central and indispensable tools in the biomedical sciences.The four authors bring with them extensive experiences spanning across disciplines such as Microbiology, Molecular and Cell Biology, Tissue Engineering, Biomedical and Regenerative Medicine and so forth, reinforcing the fact that microscopy has proven useful in countless investigations into the mysteries of life.
Optical-sectioning microscopes are powerful tools that can be used by biomedical scientists and clinicians to visualize and monitor subcellular features in thick tissues at relatively large depths (up to hundreds of microns). In clinical settings, such technologies could play a transformative role in detecting early-stage cancer and guiding the surgical resection of tumors. This dissertation focuses on optimizing optical-sectioning microscopes that utilize off-axis illumination and collection architectures to spatially filter out-of-focus and multiply-scattered photons. In particular, we will focus on overcoming certain challenges with using dual-axis confocal (DAC) microscopy and light-sheet microscopy to image biological specimens, such as mitigating resolution degradation caused by refractive heterogeneities within tissues and achieving rapid microscopic pathology of large clinical specimens. In the case of DAC microscopy, tissue-imaging performance is highly sensitive to the effects of refractive-index heterogeneities within biological specimens, which cause beam steering and distortion artifacts that lead to misalignment of the off-axis illumination and collection beam paths of a DAC microscope. Since the propagation-invariant and self-reconstructing features of Bessel beams have been shown to benefit microscopy of specimens with refractive heterogeneities, Bessel-beam illumination has been explored as a means to alleviate resolution degradation that occurs when performing tissue-imaging with DAC microscopy. Results indicate that DAC microscopy with Bessel illumination exhibits reduced resolution degradation from microscopic tissue heterogeneities compared to DAC microscopy with conventional Gaussian illumination. In the context of light-sheet microscopy, an open-top light-sheet (OTLS) microscope along with a novel fluorescent analog of hematoxylin and eosin (H&E) staining, has been developed and optimized for robust intraoperative pathology of lumpectomy margins. OTLS images of the surfaces of fresh breast samples were compared to gold-standard H&E histological images to showcase that the image quality of OTLS microscopy can approximate that of archival H&E histology of formalin-fixed paraffin-embedded tissues. In addition, preliminary results suggest that OTLS microscopy is compatible with downstream archival H&E histology and immunohistochemistry (IHC) analyses, which are currently relied upon for definitive clinical diagnoses. These results facilitate the clinical translation of OTLS microscopy for intraoperative guidance of breast conserving surgery as well as other surgical oncology procedures.
The natural, biological, medical, and related sciences would not be what they are today without the microscope. After the introduction of the optical microscope, a second breakthrough in morphostructural surface analysis occurred in the 1940s with the development of the scanning electron microscope (SEM), which, instead of light (i. e. , photons) and glass lenses, uses electrons and electromagnetic lenses (magnetic coils). Optical and scanning (or transmission) electron microscopes are called “far-field microscopes” because of the long distance between the sample and the point at which the image is obtained in comparison with the wavelengths of the photons or electrons involved. In this case, the image is a diffraction pattern and its resolution is wavelength limited. In 1986, a completely new type of microscopy was proposed, which, without the use of lenses, photons, or electrons, directly explores the sample surface by means of mechanical scanning, thus opening up unexpected possibilities for the morphostructural and mechanical analysis of biological specimens. These new scanning probe microscopes are based on the concept of near-field microscopy, which overcomes the problem of the limited diffraction-related resolution inherent in conventional microscopes. Located in the immediate vicinity of the sample itself (usually within a few nanometers), the probe records the intensity, rather than the interference signal, thus significantly improving resolution. Since the most we- known microscopes of this type operate using atomic forces, they are frequently referred to as atomic force microscopes (AFMs).
Written by leading optical phase microscopy experts, this book is a comprehensive reference to phase microscopy and nanoscopy techniques for biomedical applications, including differential interference contrast (DIC) microscopy, phase contrast microscopy, digital holographic microscopy, optical coherence tomography, tomographic phase microscopy, spectral-domain phase detection, and nanoparticle usage for phase nanoscopy The Editors show biomedical and optical engineers how to use phase microscopy for visualizing unstained specimens, and support the theoretical coverage with applied content and examples on designing systems and interpreting results in bio- and nanoscience applications. Provides a comprehensive overview of the principles and techniques of optical phase microscopy and nanoscopy with biomedical applications. Tips/advice on building systems and working with advanced imaging biomedical techniques, including interpretation of phase images, and techniques for quantitative analysis based on phase microscopy. Interdisciplinary approach that combines optical engineering, nanotechnology, biology and medical aspects of this topic. Each chapter includes practical implementations and worked examples.
In the quest for better and faster images of cellular and subcellular structures, biology-oriented optical microscopes have advanced significantly in the last few decades. Novel microscopy techniques such as non-linear microscopy (NLM), including two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) microscopy, and light-sheet fluorescence microscopy (LSFM) are emerging as alternatives that overcome some the intrinsic limitations of standard microscopy systems. In this thesis I aimed to advance such techniques even more, and combine them with other photonic technologies to provide novel tools that would help to address complex biological questions. This thesis is organized in two main parts. The first part is dedicated to applications involving femtosecond lasers that are employed for precise microsurgery. For that, damage assessment methodologies based on NLM were developed and tested in relevant biomedical models. In the second part, wavefront engineering methods were employed to enhance the imaging capabilities of light-sheet microscopy systems. These novel methodologies were tested as well in relevant biological applications. This thesis is, therefore, organized as follows: In chapter 1, a brief and comprehensive review of the basic microscopy techniques employed in this thesis is presented, together with the challenges and achievements of this thesis in sequential order. In chapter 2, a multimodal imaging methodology for the assessment of laser induced collateral damage is presented. This was specifically developed for the control of the damage in femtosecond-laser dissection of single axons within a living Caenorhabditis elegans (C. elegans). Here, it is shown that collateral damages at the level of the myosin structure of the muscles adjacent to the axon, can be readily detected. In chapter 3, the optimized multimodal methodology developed in the chapter 2 was employed for minimally invasive dissection of axons of D-type motoneurons in C elegans. Here, a microfluidic chip for C elegans immobilization and a detailed protocol was employed to evaluate the axon regeneration of such neurons. The potential of such platform for testing drugs with regeneration-enhancing capabilities is also presented. In chapter 4, a novel use of TPEF microscopy is presented to characterize and fine tune the laser for photodisruption of excised human crystalline lens samples. In chapter 5, a thorough description of the implementation of a multimodal Digital Scanned Light-Sheet Microscope (DSLM) able to work in the linear and nonlinear regimes under either Gaussian or Bessel beam excitation schemes, is presented. The enhanced capabilities of the developed system is evaluated using in vivo C. elegans samples and multicellular tumor spheroids In chapter 6, the development of a completely new concept in light sheet-based imaging is presented. This is based on the extension of the depth-of-field of the lens in the emission path of the microscope by using wavefront coding (WFC) techniques. Furthermore, I demonstrate the application of the developed methodology for fast volumetric imaging of living biological specimens and 3D particle tracking.
Biomedical Applications of Microprobe Analysis is a combination reference/laboratory manual for the use of microprobe analysis in both clinical diagnostic and research settings. Also called microchemical microscopy, microprobe analysis uses high-energy bombardment of cells and tissue, in combination with high resolution EM or confocal microscopy to provide a profile of the ion, metal, and mineral concentrations present in a sample. This allows insight into the physiology and pathophysiology of a wide variety of cells and tissues.This book describes methods for obtaining detailed information about the identity and composition of particles too small to be seen with the naked eye and describes how this information can be useful in diagnostic and biomedical research. Up-to-date review of electron microprobe analysis Detailed descriptions of sample preparation techniques Recent technologies including confocal microscopy, infrared microspectroscopy, and laser raman spectroscopy Over 100 illustrations with numerous specific applications Contributions by world-renowned experts in the field Brief summary of highlights precedes each chapter
Ideal for cell biologists, life scientists, biomedical engineers, and clinicians, this handbook provides comprehensive treatment of the theories, techniques, and biomedical applications of nonlinear optics and microscopy.
Nonlinear optical microscopy techniques have emerged as a set of successful tools within the biomedical research field. These techniques have been successfully used to study autofluorescence signals in living tissues, structural protein arrays, and to reveal the presence of lipid bodies inside the tissular volume. In the first section, the nonlinear contrast technique foundations is described, and also, a practical approach about how to build and combine this setup on a single confocal system platform shall be provided. In the next section, examples of the usefulness of these approaches to detect early changes associated with the progression of different epithelial and connective tissular diseases are presented.
Written by leading optical phase microscopy experts, this book is a comprehensive reference to phase microscopy and nanoscopy techniques for biomedical applications, including differential interference contrast (DIC) microscopy, phase contrast microscopy, digital holographic microscopy, optical coherence tomography, tomographic phase microscopy, spectral-domain phase detection, and nanoparticle usage for phase nanoscopy The Editors show biomedical and optical engineers how to use phase microscopy for visualizing unstained specimens, and support the theoretical coverage with applied content and examples on designing systems and interpreting results in bio- and nanoscience applications. Provides a comprehensive overview of the principles and techniques of optical phase microscopy and nanoscopy with biomedical applications Tips/advice on building systems and working with advanced imaging biomedical techniques, including interpretation of phase images, and techniques for quantitative analysis based on phase microscopy Interdisciplinary approach that combines optical engineering, nanotechnology, biology and medical aspects of this topic. Each chapter includes practical implementations and worked examples