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 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.

Biomedical optical imaging is widely used in the research community and medical diagnosis. However, many optical imaging techniques in the biomedical field are not optimized. Developing and improving these techniques is often a complex task involving sample preparation, instrument design, and data handling. As a summary of my Ph.D. research, this dissertation discusses the design optimization strategies for biomedical optical imaging applications. It is structured with an introduction discussing the general design consideration and the design process for biomedical optical imaging, followed by detailed discussions of four biomedical optical imaging applications developed during my Ph.D. program. The dissertation has six major chapters. The first chapter summarizes the general technology development processes for biomedical optical imaging applications, It introduces the three main elements of biomedical optical imaging, which include sample preparation, imaging systems, and data handling. Following the introduction, the second chapter discusses the Pocket MUSE project, a smartphone-based fluorescence microscope that costs less than $20 to build. Other highlights of the design include a small footprint, high imaging performance, versatile sample compatibility, and simple sample preparation methods. The third chapter describes the SLIME project, an OCT-based 3D microvascular mapping technique using an injectable scattering contrast agent, developed to study the coronary vascular abnormalities in a quail embryo animal model. This technique features a novel sample preparation strategy, where the PVA-borate cross-linking (slime) chemistry is used to stabilize a low-viscosity scattering contrast agent in situ. The fourth chapter introduces the LIMPID project, a simple optical clearing technique that makes tissues transparent in a single step. This sample preparation technique is designed to enhance the imaging depth for a broad range of optical imaging applications, enabling 3D optical imaging of millimeters-thick tissue samples. The fifth chapter describes the CompassLSM project, a simple yet high-performance light sheet microscope with sub-4-μm high optical sectioning capability in a multi-millimeter large FOV. The high imaging performance is achieved by synchronizing the focus of an axially swept light sheet to the rolling shutter of a CMOS camera. The final chapter provides a summary and conclusions about this dissertation.

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.

This book presents the advances in super-resolution microscopy in physics and biomedical optics for nanoscale imaging. In the last decade, super-resolved fluorescence imaging has opened new horizons in improving the resolution of optical microscopes far beyond the classical diffraction limit, leading to the Nobel Prize in Chemistry in 2014. This book represents the first comprehensive review of a different type of super-resolved microscopy, which does not rely on using fluorescent markers. Such label-free super-resolution microscopy enables potentially even broader applications in life sciences and nanoscale imaging, but is much more challenging and it is based on different physical concepts and approaches. A unique feature of this book is that it combines insights into mechanisms of label-free super-resolution with a vast range of applications from fast imaging of living cells to inorganic nanostructures. This book can be used by researchers in biological and medical physics. Due to its logically organizational structure, it can be also used as a teaching tool in graduate and upper-division undergraduate-level courses devoted to super-resolved microscopy, nanoscale imaging, microscopy instrumentation, and biomedical imaging.

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

This book provides an introduction to design of biomedical optical imaging technologies and their applications. The main topics include: fluorescence imaging, confocal imaging, micro-endoscope, polarization imaging, hyperspectral imaging, OCT imaging, multimodal imaging and spectroscopic systems. Each chapter is written by the world leaders of the respective fields, and will cover: principles and limitations of optical imaging technology, system design and practical implementation for one or two specific applications, including design guidelines, system configuration, optical design, component requirements and selection, system optimization and design examples, recent advances and applications in biomedical researches and clinical imaging. This book serves as a reference for students and researchers in optics and biomedical engineering.