No description available.

<p>Nonlinear optical microscopy serves as a great tool for biomedical imaging due to its high resolution, deep penetration, inherent three dimensional optical sectioning capabilities and superior performance in scattering media. Conventional nonlinear optical microscopy techniques, e.g. two photon fluorescence and second harmonic generation, are based on detecting a small light signal emitted at a new wavelength that is well separated from the excitation light. However, there are also many other nonlinear processes, such as two-photon absorption and self-phase modulation, that do not generate light at new wavelengths and that have not been extensively explored for imaging. This dissertation extends the accessible mechanisms for contrast to the later nonlinear optical processes by combining femtosecond laser pulse shaping and homodyne detection. We developed a rapid pulse shaper with a relatively simple and compact instrument design that modifies the spectrum of individual laser pulses from an 80 MHz mode-locked laser. The pulse shaper enables simultaneous two-photon absorption and self-phase modulation imaging of various nanoparticles in-vitro with high sensitivity. We also applied this imaging technique to study the nonlinear optical response in graphene. Because our technology detects the nonlinear signature encoded within the laser pulse itself, we achieve intrinsic contrast of biological and non-biological samples in highly scattering media. These capabilities have significant implications in biomedical imaging and nanophotonics.</p> / Dissertation

Lung carcinoma is the most prevalent type of cancer in the world, considered to be a relentlessly progressive disease, with dismal mortality rates to patients. Recent advances in targeted therapy hold the premise for the delivery of better, more effective treatments to lung cancer patients, that could significantly enhance their survival rates. Optimizing care delivery through targeted therapies requires the ability to effectively identify and diagnose lung cancer along with identifying the lung cancer cell type specific to each patient, \textit{small cell carcinoma}, \textit{adenocarcinoma}, or \textit{squamous cell carcinoma}. Label free optical imaging techniques such as the \textit{Coherent anti-stokes Raman Scattering microscopy} have the potential to provide physicians with minimally invasive access to lung tumor sites, and thus allow for better cancer diagnosis and sub-typing. To maximize the benefits of such novel imaging techniques in enhancing cancer treatment, the development of new data analysis methods that can rapidly and accurately analyze the new types of data provided through them is essential. Recent studies have gone a long way to achieving those goals but still face some significant bottlenecks hindering the ability to fully exploit the diagnostic potential of CARS images, namely, the streamlining of the diagnosis process was hindered by the lack of ability to automatically detect cancer cells, and the inability to reliably classify them into their respective cell types. More specifically, data analysis methods have thus far been incapable of correctly identifying and differentiating the different non-small cel lung carcinoma cell types, a stringent requirement for optimal therapy delivery. In this study we have addressed the two bottlenecks named above, through designing an image processing framework that is capable of, automatically and accuratly, detecting cancer cells in two and three dimensional CARS images. Moreover, we built upon this capability with a new approach at analyzing the segmented data, that provided significant information about the cancerous tissue and ultimately allowed for the automatic differential classification of non-small cell lung carcinoma cell types, with superb accuracies.

Aseptic loosening of artificial joints is caused by an osteolytic reaction to wear debris mediated by macrophages and other cells. Imaging these wear particles within cells can be a key process in understanding particle-cell interactions. However, the compounds used in surgical implants are not easily visualised as no tagging molecule can be added without altering the properties of the material. We were interested in using a label free optical technique known as coherent anti-Stokes Raman scattering spectroscopy (CARS) to image these particles in cells. In this thesis we studied how to use CARS to image physiologically relevant wear particles within cells. We characterised the responses from our CARS system and found them to be in good agreement to the Raman spectra we obtained for the same materials. We showed that the forward scattered CARS signal was consistently larger than the backwards scattered signal for the same size particles, and also generated a larger contrast, especially between sub-micron sized particles and the non-resonant background. Wear particles of polyethylene isolated from a pin-on-plate wear simulator were shown to be in a similar size range to those retrieved from revision tissue. When incubated in our model macrophage cells we were able to image areas of CARS signal that indicated the location of these particles in the cell. Furthermore, using multiple CARS images taken at different Raman resonances we were able to distinguish between three different polymeric compounds added to cells, showing the specificity of the technique. The inherent 3D sectioning capabilities of multiphoton microscopy were used to generate projected images of the cells and contents, as well as estimating the particle loads within cells. These results show that CARS could be an important tool in imaging intra-cellular polyethylene and characterising the interactions of wear particles with cells and the surrounding tissue.

The incidence of gastrointestinal (GI) cancers has been steadily increasing in the UK since the mid 1970’s. These include cancers of the colon and oesophagus. Colon cancers have a high incidence rate, being the fourth most common cancer in the UK for both men and women. Oesophageal cancers in comparison are much rarer, however they have a poor survival rate primarily due to a late diagnosis. The key to improving survival for these cancers and many others is to detect and remove the disease at the early stages, to prevent the cancer from advancing. At present the ‘gold standard’ for diagnosis is a biopsy followed by histopathology. This technique is invasive, time consuming and highly subjective. It is therefore important to look towards non-invasive methods for early and rapid diagnosis. Optical techniques have begun to show such promise. By probing the interactions of tissues with light, diagnostic information is able to be obtained non-invasively. Techniques such as Raman spectroscopy utilise inherent molecular vibrations to extract biochemical information from tissues. Raman spectroscopy, however, is currently fundamentally limited by long acquisition times, due to the inherently weak signals produced. Using coherent Raman techniques such as coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS), the molecular vibrations are coherently driven to provide an enhancement in signal. This thesis explored spectral signatures from snap frozen oesophageal sections in the fingerprint (450 cm-1 to 1850 cm-1) and high wavenumber (2800 cm-1 to 3050 cm-1) regions using spontaneous Raman and compared with spectra from hyperspectral SRS. The diagnostic potential for each technique was assessed for four major pathology groups, normal, Barrett’s oesophagus, dysplasia and adenocarcinoma. Samples were classified using a principal component fed linear discriminant analysis (PCA-LDA) approach with a leave-one-out cross validation. Comparisons were made to haematoxylin and eosin (H&E) stained sections. Raman in the fingerprint region was found to be the most promising for diagnosis. There were minimal changes in the high wavenumber region between pathology groups which was also reflected in the SRS spectra and proved to be insufficient for classification. Further comparisons were made between spontaneous and coherent Raman techniques using frozen colon sections. The morphological and structural information available was explored using a k-means cluster analysis. Both spontaneous and coherent Raman were able to distinguish important structural features in the colon, such as the epithelial cells that form the colonic glands and surrounding connective tissue. Both are important visual markers for cancer diagnosis in the current approach. SRS demonstrated higher spatial resolution and faster acquisition times in comparison to spontaneous Raman. This work has discussed the many advantages of using coherent Raman techniques for tissue applications, but has also highlighted some of the limitations for spectral measurements, arising from the complexity of the system.

No description available.

Articular cartilage has been imaged using the following multi-photon modalities: Second Harmonic Generation (SHG), Two-photon Fluorescence (TPF) and Coherent Anti-Stokes Raman Scattering (CARS). A simple epi detection microscope was constructed for SHG and TPF imaging in the early stages of this research. Later the imaging was transferred to a new microscope system which allowed simultaneous forwards and epi detection and combined CARS imaging with TPF and SHG. Multiphoton spectroscopic studies were conducted on both intact tissue samples and the major components of the extracellular matrix, in order to identify sources of TPF. Fluorescence was detected from type II collagen, elastin and samples of purified collagen and elastin crosslinks. Age related glycation crosslinks of collagen may be a significant source of TPF. No fluorescence was detected from proteoglycans. In intact, unfixed healthy articular cartilage the cells were observed via CARS, surrounded in their pericellular matrix which is characterised by an increase in TPF. The collagen of the extra cellular matrix showed up clearly in the SHG images. Diseased cartilage was also imaged revealing microscopic lesion at the articular surface in early osteoarthritis and highly fibrous collagen structures and cell clusters in more advanced degeneration. In young healthy cartilage a network of elastin fibres were found lying parallel to the articular surface in the most superficial 50μm of the tissue. Regional variations in these fibres were also investigated. The fibres appeared mainly long and straight suggesting that they may be under tension, further work is needed to identify whether they have a mechanical function. The polarization sensitivity of the SHG from collagen has been investigated for both cartilage and tendon. In the most superficial tissue these measurements can be used directly to determine the collagen fibre orientation. However at increasing depths the effects of biattenuation and birefringence must be considered. Healthy cartilage has a characteristic pattern of polarization sensitivity with depth and this changes at lesions indicating a disruption of the normal collagen architecture. The methods developed in this thesis demonstrate the use of non-linear microscopy to visualise the structure of the extracellular matrix and cells in intact unstained tissue. They should also be appropriate in many areas of cell and matrix biology.

Technological progress in commercializing ultrafast lasers and detectors has allowed realization of cooperative processes on an ultrashort time scale, which demand a re-evaluation of the conventional cooperative phenomena with a new insight. Ultrafast cooperative phenomena in coherently prepared media and various applications of superfluorescence and coherent Raman scattering are studied in this dissertation. In particular, a simple theoretical testimony on analogy between a cooperative emission and coherent Raman scattering is presented by offering an opportunity to perform parallel research on these two processes from a unified point of view. On one hand, the superfluorescent pulse with a time duration of a few tens of picoseconds (ps) from alkali metal vapor is observed for the first time, even though cooperative phenomena in atomic vapor have been extensively studied for more than five decades. A dense rubidium vapor pumped by ultrashort (100 femtosecond, fs) pulses allows a realization of the ultrafast superfluorescence while a time-resolved study of superfluorescence is accomplished by using a streak camera with 2 ps time resolution. Experimental research on quantum nature of cooperative emissions has been “frozen” over the years (three decades) possibly because of the technical difficulties. Quantum fluctuations of superfluorescence development are explored experimentally by taking advantage of the ultra fast streak camera. Presumable applications of the superfluorescent pulse in e.g., a remote sensing, and an ultraviolet upconversion of the input infrared laser pulse are presented. The quantum interference due to different excitation pathways is revealed by the temporal coherent control technique while observing interferometric signals from alkali metal vapors. On the other hand, a new spectroscopic technique based on ultrafast coherent Raman scattering is developed. The key advantage of the presented technique is to suppress the non-resonant background noise which usually obscures possible applications of the other conventional coherent Raman techniques in practice. A reduction of the background noise is achieved by shaping and delaying the third pulse which probes the coherence of the medium (i.e., an enhancement of specific vibrations of the target molecules in unison) firstly prepared by two broadband pulses. We demonstrate a robustness and superiority of signal-to-noise ratio of the developed technique by identifying as few as 10000 bacterial spores at a single laser shot level. Finally, several comparative studies between cooperative and uncooperative processes are presented. A picosecond cooperative phenomenon in a three-photon resonant medium induced by a single as well as two-color ultrashort pulses is investigated. A time-resolved study shows that a picosecond cooperative effect is crucial in the well-established fields of resonant-enhanced multiphoton ionizations and harmonic generations. We also present a quantitative analysis for spontaneous versus broadband coherent Raman scattering on pyridine molecules. The spontaneous Raman signal is enhanced by 5 orders as a result of cooperative phenomena.

Over the last 50 years, flow cytometry has evolved from a modest cell counter into an invaluable analytical tool that measures an ever-expanding variety of phenotypes. Flow cytometers interrogate passing samples with laser light and measure the elastically scattered photons to ascertain information about sample size, granularity, and basic morphology. Obtaining molecular information, however, requires the addition of exogenous fluorescent labels. These labels, although a power tool, have numerous challenges and limitations such as large emission spectra and cellular toxicity. To move beyond fluorescent labels in microscopy, a variety of techniques that probe the intrinsic Raman vibrations within a sample have been developed, such as coherent anti-Stokes Raman scattering (CARS) and Raman microspectroscopy. In this dissertation, I present the first development of a label-free flow cytometer that measures the elastically scattered photons and probes the intrinsic Raman vibrations of passing samples using multiplex coherent anti-Stokes Raman scattering (MCARS). MCARS, a coherent Raman technique that probes a large region of the Raman spectrum simultaneously, provides rich molecularly-sensitive information. Furthermore, I present its application to sorting polymer microparticles and its use in two example biological applications: monitoring lipid bodies within cultures of Saccharomyces cerevisiae, a model yeast with numerous human homologs, and monitoring the affect of nitrogen starvation on Phaeodactylum tricornutum, a diatom, which is being genetically engineered to efficiently produce biofuels.

Microscopy techniques have been developed and refined over multiple decades, but innovation around single photon modalities has slowed. The advancement of the utility of information acquired, and minimum resolution available is seemingly reaching an asymptote. The fusion of light microscopy and well-studied nonlinear processes has broken through this barrier and enabled the collection of vast amounts of additional information beyond the topographical information relayed by traditional microscopes. Through nonlinear imaging modalities, chemical information can also be extracted from tissue. Nonlinear microscopy also can beat the resolution limit caused by diffraction, and offers up three-dimensional capabilities. The power of nonlinear imaging has been demonstrated by countless research groups, solidifying it as a major player in biomedical imaging. The value of a nonlinear imaging system could be enhanced if a reduction in size would permit the insertion into bodily cavities, as has been demonstrated by linear imaging endoscopes. The miniaturization of single photon imaging devices has led to significant advancements in diagnostics and treatment in the medical field. Much more information can be extracted from a patient if the tissue can be imaged in vivo, a capability that traditional, bulky, table top microscopes cannot offer. The development of new technologies in optics has enabled the miniaturization of many critical components of standard microscopes. It is possible to combine nonlinear techniques with these miniaturized elements into a portable, hand held microscope that can be applied to various facets of the biomedical field. The research demonstrated in this thesis is based on the selection, testing and assembly of several miniaturized optical components for use as a nonlinear imaging device. This thesis is the first demonstration of a fibre delivered, microelectromechanical systems mirror with miniaturized optics housed in a portable, hand held package. Specifically, it is designed for coherent anti-Stokes Raman scattering, second harmonic generation, and two-photon excitation fluorescence imaging. Depending on the modality being exploited, different chemical information can be extracted from the sample being imaged. This miniaturized microscope can be applied to diagnostics and treatments of spinal cord diseases and injuries, atherosclerosis research, cancer tumour identification and a plethora of other biomedical applications. The device that will be revealed in the upcoming text is validated by demonstrating all designed-for nonlinear modalities, and later will be used to perform serialized imaging of myelin of a single specimen over time.