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"The goal of this engineering thesis was to restore and upgrade a legacy electro-optical microscopy system for use in experimental research. This was accomplished through the application of a broad range of engineering disciplines and problem solving skills. The operation of the legacy system was assessed as a whole, and the performance of each component was characterized and compared to the design requirements. Malfunctioning technology was repaired, when possible, and new devices and software were implemented to enhance the capabilities of the original design. The resulting imaging system is capable of producing data on par with the legacy system and serves as a base for implementing additional microscopy techniques and future upgrades"--Author's abstract.
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
This book covers important aspects of modern optical microscopy and image restoration technologies. Instead of pure optical treatment, the book is delivered with the consideration of the scientists who utilize optical microscopy in their daily research. However, enough details are provided in basic imaging principles, optics and instrumentation in microscopy, spherical aberrations, deconvolution and image restoration. A number of microscopic technologies such as polarization, confocal and multi-photon microscopy are highlighted with their applications in biological and materials sciences/engineering.
"Biological and biomedical research is often contingent upon microscopy techniques for observation and studying of biological features and processes, and subsequent analysis. For many applications, it is necessary that the selected imaging system provide high spatial resolution and large field-of-view, in order to be able to visualize individual biological structures or agents within the sample, while capturing an area large enough, where meaningful analysis, such as particle tracking, could be performed within a single frame. Various lens-based and lens-free imaging platforms, each with their own sets of advantages and disadvantages, offer different imaging modalities suitable for different specimens and applications, but they all suffer from a main limitation: the trade-off between spatial resolution and field-of-view. This competition cannot be eliminated but could be optimized, based on the chosen imaging system specifications. This work addresses the restrictive trade-off, and introduces a mobile phone-based illumination-imaging platform that maximizes the attainable field-of-view at high resolution, and expands the use of phone screen illumination to a lens-free platform.The thesis transitions from a broad introduction to microscopy in the biological and biomedical fields into a general protocol for identification of imaging system requirements for a targeted application, modelled after a specific example for imaging of a biocomputational microfluidic device that utilizes microorganisms as exploratory problem-solving agents. The following chapters introduce the aforementioned dual-phone system, which uses a phone camera with an external lens for imaging, to achieve a spatial resolution of at least 2 [mu]m, and a large field-of-view of 3.6 × 2.7mm. For illumination, it uses the screen of another phone to project multi-modal illumination patterns, including but not limited to bright-field, dark-field, Rheinberg illumination, point illumination, fluorescence, and differential phase contrast. Put together, this illumination-imaging system forms a novel, inexpensive, compact, portable, and versatile microscope for use in low-resource environments. It could be used in research, medical, educational, and environmental settings for both qualitative and quantitative imaging of cells, microorganisms, and other micron-sized objects. The adaptability of phone screen illumination allows it to be further integrated into lens-free imaging platforms, as well as conventional microscopes"--
This book covers various aspects of modern microscopy, with emphasis on multidimensional (three-dimensional and higher) and multimodality microscopy. The topics discussed include multiphoton fluorescent microscopy, confocal microscopy, x-ray microscopy and microtomography, electron microscopy, probe microscopy and multidimensional image processing for microscopy. In addition, there are chapters demonstrating typical microscopical applications, both biological and material.
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.
Once the second edition was safely off to the printer, the 110 larger world of micro-CT and micro-MRI and the smaller world authors breathed a sigh of relief and relaxed, secure in the belief revealed by the scanning and transmission electron microscopes. that they would “never have to do that again. ” That lasted for 10 To round out the story we even have a chapter on what PowerPoint years. When we ?nally awoke, it seemed that a lot had happened. does to the results, and the annotated bibliography has been In particular, people were trying to use the Handbook as a text- updated and extended. book even though it lacked the practical chapters needed. There As with the previous editions, the editor enjoyed a tremendous had been tremendous progress in lasers and ?ber-optics and in our amount of good will and cooperation from the 124 authors understanding of the mechanisms underlying photobleaching and involved. Both I, and the light microscopy community in general, phototoxicity. It was time for a new book. I contacted “the usual owe them all a great debt of gratitude. On a more personal note, I suspects” and almost all agreed as long as the deadline was still a would like to thank Kathy Lyons and her associates at Springer for year away.
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 (