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Multimodal non-linear imaging techniques provide non-invasive and potentially in vivo means to investigate tissue with cellular resolution. A particularly promising approach that has garnered attention as of late is the combination of coherent antiStokes Raman scattering (CARS), second harmonic generation (SHG) and two photon excited autofluorescence (TPEF) microscopy. In the first section of this thesis, the diagnostic potential of multimodal non-linear imaging has been demonstrated in the case of head and neck squamous cell carcinoma. The second part of this thesis investigates the feasibility of CARS microscopy for imaging intense bands in the finger-print region (800-1800 cm-1) wherein the presence of multiple overlapping peaks and interference with non-resonant background present challenges. Specifically, the emphasis is on imaging the prominent peaks arising from conjugated C=C double bonds in retinol, tretinoin, [beta]-carotene, and various microalgal pigments. The first CARS fingerprint imaging application in the thesis is concerned with the vitamin A content of liver tissue. Analogously, in a uni-cellular application, CARS has been employed to image carotenoids in the diatoms D. brightwellii and S. turris. As part of the effort in transferring multimodal microscopic technologies to the enduser, the third part of the thesis examines two beam excitation and demultiplexed detection as a means of doubling the speed of laser scanning microscopes based on compact fiber laser sources. Another area of improvement explored is the resolution of the CARS microscopic setup wherein, based on results from numerical studies, a Bessel like beam was employed as one of the excitation arms in the setup to enhance lateral resolution.
This book provides an in-depth description and discussion of different multi-modal diagnostic techniques for cancer detection and treatment using exact optical methods, their comparison, and combination. Coverage includes detailed descriptions of modern state of design for novel methods of optical non-invasive cancer diagnostics; multi-modal methods for earlier cancer diagnostic enhancing the probability of effective cancer treatment; modern clinical trials with novel methods of clinical cancer diagnostics; medical and technical aspects of clinical cancer diagnostics, and long-term monitoring. Biomedical engineers, cancer researchers, and scientists will find the book to be an invaluable resource. Introduces optical imaging strategies; Focuses on multimodal optical diagnostics as a fundamental approach; Discusses novel methods of optical non-invasive cancer diagnostics.
For a host of reasons, nonlinear optical spectroscopy is a valuable tool for biochemical applications where minimally invasive diagnostics is desired. Biochemical Applications of Nonlinear Optical Spectroscopy presents the latest technological advances and offers a perspective on future directions in this important field. Written by an international panel of experts, this volume begins with a comparison of nonlinear optical spectroscopy and x-ray crystallography. The text examines the use of multiphoton fluorescence to study chemical phenomena in the skin, the use of nonlinear optics to enhance traditional optical spectroscopy, and the multimodal approach, which incorporates several spectroscopic techniques in one instrument. Later chapters explore Raman microscopy, third-harmonic generation microscopy, and non-linear Raman microspectroscopy. The text explores the promise of beam shaping and the use of broadband laser pulse generated through continuum generation and an optical pulse shaper. Lastly, the book discusses the effects of spatial beam shaping on the generated nonlinear Raman signals in a tightly focused geometry and provides insight into the extension of nonlinear optical spectroscopy to the nanoscale through the use of plasmonic tip-enhanced arrangement. With novel experimental approaches to this technology expanding day-by-day, the book’s balanced coverage from a wide range of international contributors not only elucidates important achievements, but also outlines future directions in this dynamic and promising field.
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 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.
This monograph focuses on modern femtosecond laser microscopes for two photon imaging and nanoprocessing, on laser tweezers for cell micromanipulation as well as on fluorescence lifetime imaging (FLIM) in Life Sciences. The book starts with an introduction by Dr. Wolfgang Kaiser, pioneer of nonlinear optics and ends with the chapter on clinical multiphoton tomography, the novel high resolution imaging technique. It includes a foreword by the nonlinear microscopy expert Dr. Colin Sheppard. Contents Part I: Basics Brief history of fluorescence lifetime imaging The long journey to the laser and its use for nonlinear optics Advanced TCSPC-FLIM techniques Ultrafast lasers in biophotonics Part II: Modern nonlinear microscopy of live cells STED microscopy: exploring fluorescence lifetime gradients for super-resolution at reduced illumination intensities Principles and applications of temporal-focusing wide-field two-photon microscopy FLIM-FRET microscopy TCSPC FLIM and PLIM for metabolic imaging and oxygen sensing Laser tweezers are sources of two-photon effects Metabolic shifts in cell proliferation and differentiation Femtosecond laser nanoprocessing Cryomultiphoton imaging Part III: Nonlinear tissue imaging Multiphoton Tomography (MPT) Clinical multimodal CARS imaging In vivo multiphoton microscopy of human skin Two-photon microscopy and fluorescence lifetime imaging of the cornea Multiscale correlative imaging of the brain Revealing interaction of dyes and nanomaterials by multiphoton imaging Multiphoton FLIM in cosmetic clinical research Multiphoton microscopy and fluorescence lifetime imaging for resection guidance in malignant glioma surgery Non-invasive single-photon and multi-photon imaging of stem cells and cancer cells in mouse models Bedside assessment of multiphoton tomography
Despite a number of books on biophotonics imaging for medical diagnostics and therapy, the field still lacks a comprehensive imaging book that describes state-of-the-art biophotonics imaging approaches intensively developed in recent years. Addressing this shortfall, Advanced Biophotonics: Tissue Optical Sectioning presents contemporary methods and applications of biophotonics imaging. Gathering research otherwise scattered in numerous physical, chemical, biophysical, and biomedical journals, the book helps researchers, bioengineers, and medical doctors understand major recent bioimaging technologies and the underlying biophotonics science. Well-known international experts explore a variety of "hot" biomedical optics and biophotonics problems, including the use of photoacoustic imaging to investigate the molecular and cellular processes in living systems. The book also covers Monte Carlo modeling, tissue optics and tissue optical clearing, nonlinear optical microscopy, various aspects of optical coherence tomography, multimodal tomography, adaptive optics, and signal imaging. With 58 color images, this book represents a valuable contribution to the biomedical and biophotonics literature. Designed for researchers and practitioners in biophotonics, the book is also a useful resource for scientists in laser physics and technology, fiber optics, spectroscopy, materials science, biology, and medicine as well as students studying biomedical physics and engineering, biomedical optics, and biophotonics.
Microscopes represent tools of the utmost importance for a wide range of disciplines. Without them, it would have been impossible to stand where we stand today in terms of understanding the structure and functions of organelles and cells, tissue composition and metabolism, or the causes behind various pathologies and their progression. Our knowledge on basic and advanced materials is also intimately intertwined to the realm of microscopy, and progress in key fields of micro- and nanotechnologies critically depends on high-resolution imaging systems. This volume includes a series of chapters that address highly significant scientific subjects from diverse areas of microscopy and analysis. Authoritative voices in their fields present in this volume their work or review recent trends, concepts, and applications, in a manner that is accessible to a broad readership audience from both within and outside their specialist area.
This book is a printed edition of the Special Issue "Recent Advances in Scar Biology" that was published in IJMS
Nonlinear multiphoton multimodal microscopy (NMMM) used in biological imaging is a technique that explores the combinatorial use of different multiphoton signals, or modalities, to achieve contrast in stained and unstained biological tissues. NMMM is a nonlinear laser-matter interaction (LMI), which utilizes multiple photons at once (multiphoton processes, MP). The statistical probability of multiple photons arriving at a focal point at the same time is dependent on the two-photon absorption (TPA) cross-section of the molecule being studied and is incredibly difficult to satisfy using typical incoherent light, say from a light bulb. Therefore, the stimulated emission of coherent photons by pulsed lasers are used for NMMM applications in biomedical imaging and diagnostics.In this dissertation, I hypothesized that due to the near-IR wavelength of the Ytterbium(Yb)-fiber laser (1070 nm), the four MP-two-photon excited fluorescence (2PEF), second harmonic generation (SHG), three-photon excited fluorescence (3PEF) and third harmonic generation (THG), generated by focusing this ultrafast laser, will provide contrast to unstained tissues sufficient for augmenting current histological staining methods used in disease diagnostics. Additionally, I hypothesized that these NMMM images (NMMMIs) can benefit from computational methods to accurately separate their overlapping endogenous MP signals, as well as train a neural network for image classification to detect neoplastic, inflammatory, and healthy regions in the human oral mucosa. Chapter II of this dissertation explores the use of NMMM to study the effects of storage on donated red blood cells (RBCs) using non-invasive 2PEF and THG without breaching the blood storage bag. Unlike the lack of RBC fluorescence previously reported, we show that with two-photon (2P) excitation from an 800 nm source, and three-photon (3P) excitation from a 1060 nm source, there was sufficient fluorescent signal from hemoglobin as well as other endogenous fluorophores. Chapter III employs NMMM to establish the endogenous MP signals present in healthy excised and unstained mouse and Cynomolgus monkey retinas using 2PEF, 3PEF, SHG, and THG. We show the first epi-direction detected cross-section and depth-resolved images of unstained isolated retinas obtained using NMMM with an ultrafast fiber laser centered at 1070 nm and a ~38 fs pulse. Two spectrally and temporally distinct regions were shown; one from the nerve fiber layer (NFL) to the inner receptor layer (IRL), and one from the retinal pigmented epithelium (RPE) and choroid. Chapter IV focuses on the use of minimal NMMM signals from a 1070 nm Yb-fiber laser to match and augment H&E-like contrast in human oral squamous cell carcinoma (OSCC) biopsies. In addition to performing depth-resolved (DR) imaging directly from the paraffin block and matching H&E-like contrast, we showed how the combination of characteristic inflammatory 2PEF signals undetectable in H&E stained tissues and SHG signals from stromal collagen can be used to analytical distinguish healthy, mild and severe inflammatory, and neoplastic regions and determine neoplastic margins in a three-dimensional (3D) manner. Chapter V focuses on the use of computational methods to solve an inverse problem of the overlapping endogenous fluorescent and harmonic signals within mouse retinas. The least-squares fitting algorithm was most effective at accurately assigning photons from the NMMMIs to their source. This work, unlike commercial software, permits using custom signal source reference spectra from endogenous molecules, not from fluorescent tags and stains. Finally, Chapter VI explores the use of the OSCC images to train a neural network image classifier to achieve the overall goal of classifying the NMMMIs into three categories-healthy, inflammatory, and neoplastic. This work determined that even with a small dataset (