Serial optical coherence microscopy for label-free volumetric histopathology

The observation of histopathology using optical microscope is an essential procedure for examination of tissue biopsies or surgically excised specimens in biological and clinical laboratories. However, slide-based microscopic pathology is not suitable for visualizing the large-scale tissue and native 3D organ structure due to its sampling limitation and shallow imaging depth. Here, we demonstrate serial optical coherence microscopy (SOCM) technique that offers label-free, high-throughput, and large-volume imaging of ex vivo mouse organs. A 3D histopathology of whole mouse brain and kidney including blood vessel structure is reconstructed by deep tissue optical imaging in serial sectioning techniques. Our results demonstrate that SOCM has unique advantages as it can visualize both native 3D structures and quantitative regional volume without introduction of any contrast agents

Factors Affecting Human Achilles Sub-tendon Mechanical Behaviour and the Clinical Application Potential

The human Achilles tendon is composed of three twisted sub-tendons, arising from soleus and gastrocnemii muscles, but the interaction between these sub-tendons during body movements is largely unknown. The most widely reported phenomenon is the non-uniform intra-tendinous displacements during movements; however, how this non-uniformity contributes to the overall tendon mechanical behaviour has not been thoroughly studied. This thesis aims to investigate the potential factors affecting this intra-tendinous displacement non-uniformity, by conducting mechanical and material characterisation and in silico modelling on Achilles sub-tendons, and to discover possibilities to translate these findings into clinical applications. The study conducted on the equine bifurcated tendon-ligament model demonstrated that differences in the material and mechanical properties at the fascicle and sub-tendon levels may be associated with displacement non-uniformity. Human Achilles sub-tendon material properties were not statistically different but a greater cross-sectional area, failure force and displacement were found in the soleus than gastrocnemii sub-tendons. These results suggest that the material and mechanical properties of sub-tendons may affect the overall tendon level mechanical behaviour. Finite element analysis on three reconstructed Achilles sub-tendon models identified that reduced inter-sub-tendon sliding, as reported previously in aged tendons, reduced displacement non-uniformity. Comparing the results from different models further suggested that the inherent tendon morphology, with different length, twist, and sub-tendon arrangements, could result in different tendon level displacements and stress distributions. The results from Raman spectroscopy on both equine and human tendon tissues demonstrated the ability to differentiate tendon samples with known compositional differences based on spectral features alone, suggesting that Raman spectroscopy has the potential for rapid analysis of tendon compositions in the future. In conclusion, this thesis has identified three factors that could affect the overall Achilles tendon mechanical behaviour and proposed potential approaches for future clinical applications to improve tendon healthcare

Applications of raman spectroscopy in dentistry part II: Soft tissue analysis

Raman spectroscopy is rapidly moving from an experimental technique for the analysis of biological molecules to a tool for the real-time clinical diagnosis and in situ evaluation of the oral tissue in medical and dental research. The purpose of this study is to identify various applications of Raman spectroscopy, to evaluate the contemporary status and to explore future directions in the field of dentistry. Several in-depth applications are presented to illustrate Raman spectroscopy in early diagnosis of soft tissue abnormalities. Raman spectroscopy allows to analyze histological and biochemical composition of biological tissues. The technique not only demonstrates its role in the disclosure of dysplasia and malignancy but also in performing guided biopsies, diagnosing sialoliths, and assessment of surgical margins. Raman spectroscopy is used to identify the molecular structures and its components to give substantial information about the chemical structure properties of these molecules. In this paper, we acquaint the utilization of Raman spectroscopy in analyzing the soft tissues in relation to dentistry

Label-free polarisation-resolved optical imaging of biological samples

Myelin is a biological structure present in all the gnathostomata. It is a highly- ordered structure, in which many lipid-enriched and densely compacted phospho- lipid bilayers are rolled up in a cylindrical symmetry around a subgroup of axons. The myelin sheath increases the electrical transverse resistance and reduces the ca- pacitance making the saltatory conduction of action potentials possible and therefore leading to a critically improved performance in terms of nervous impulse conduc- tion speeds and travel lengths. Myelin pathologies are a large group of neurological diseases that often result in death or disability. In order to investigate the main causes of myelin damage and its temporal progression many microscopy techniques are currently employed, such as electron microscopy and histochemistry or fluorescence imaging. However, electron microscopy and histochemistry imaging require complex sample prepara- tion and are therefore unsuitable for live imaging. Fluorescence imaging, as well as its derivatives, confocal and two-photon imaging, relies on the use of fluorescent probes to generate the image contrast but fluorophores and the associated sample processing, when applicable to living specimens, might nonetheless modify the bi- ological properties of the target molecule and perturb the whole biological process under investigation; moreover, fluorescent immunostaining still requires the fixation of the cells. Coherent anti-Stokes Raman Scattering (CARS) microscopy, on the other hand, is a powerful and innovative imaging modality that permits the study of liv- ing specimens with excellent chemical contrast and spatial resolution and without the confounding and often tedious use of chemical or biological probes. This is par- ticularly important in clinical settings, where the patient biopsy must be explanted in order to stain the tissue. In these cases it may be useful to resort to a set of label-free microscopy techniques. Among these, CARS microscopy is an ideal tool to investigate myelin morphology and structure, thanks to its abundance of CH2 bonds. The chemical selectivity of CARS microscopy is based on the properties of the contrast-generating CARS process. This is a nonlinear process in which the energy difference of a pair of incoming photons (\u201cpump\u201d and \u201cStokes\u201d) matches the energy of one of the vibrational modes of a molecular bond of interest. This vibrational excited state is coherently probed by a third photon (\u201cprobe\u201d) and anti-Stokes radi- ation is emitted. In this thesis I shall discuss the development of a multimodal nonlinear opti- cal setup implementing CARS microscopy together with general Four-Wave Mix- ing, Second Harmonic Generation and Sum Frequency Generation microscopies. Moreover, I shall present a novel polarisation-resolved imaging scheme based on the CARS process, which I named Rotating-Polarisation (RP) CARS microscopy and implemented in the same setup. This technique, using a freely-rotating pump-and- probe-beam-polarisation plane, exploits the CARS polarisation-dependent rules in order to probe the degree of anisotropy of the chemical-bond spatial orientations inside the excitation point-spread function and their average orientation, allowing at the same time the acquisition of large-field-of-view images with minimal polarisa- tion distortions. I shall show that RP-CARS is an ideal tool to investigate the highly- ordered structure of myelinated nervous fibres thanks to the strong anisotropy and symmetry properties of the myelin molecular architecture. I shall also demonstrate that this technique allows the fully label-free assessment of the myelin health status both in a chemical model of myelin damage (lysophos- phatidylcholine-exposed mouse nerve) and in a genetic model (twitcher mouse) of a human leukodystrophy (Krabbe disease) while giving useful insights into the pathogenic mechanisms underlying the demyelination process. I shall also discuss the promises of this technique for applications in optical tractography of the nerve fibres in the central nervous system and for the investigation of the effects of ageing on the peripheral nervous system. Moreover, I shall demonstrate by means of numer- ical simulations that RP-CARS microscopy is extremely robust against the presence of scatterers (such as lipid vesicles, commonly found in the peripheral nervous sys- tem). Finally, I shall discuss the results of the exploitation of my multimodal setup in a different area at the boundary of biophysics and nanomedicine: the observation of the internalization of different kinds of nanoparticles (boron-nitride nanotubes, barium-titanate nanoparticles and barium-titanate-core/gold-shell nanoparticles) by cultured cells and the demonstration of the nanopatterned nature of a structure built with two-photon lithography

Lipid-membrane interactions of the bovine eye lens

The lens is a transparent, biconvex structure within the eye that has a very high refractive index. It focuses light onto the retina and enables vision. It is able to elastically deform to adjust the focal length of the eye for observing objects at varying distances, a process known as accommodation. With age or damage, the lens becomes stiffer and more likely to develop opaque regions called cataracts. Levels of oxidised lipids such as 7-ketocholesterol increase with age and raised levels of these oxysterols are observed in cataractous lenses. The goal of this project is to investigate the effect of raised levels of 7-ketocholesterol on the biophysical properties of lens membranes, which could in turn affect the structure of lens fibre cells and thereby impact on their individual function and the lens structure as a whole. Nanomechanical measurements of the lens elasticity with Atomic Force Microscopy (AFM) show that the Young’s Modulus of model and extracted bovine lens lipid membranes are lower when incubated with biologically-relevant levels of 7-ketocholesterol, signalling decreased membrane stiffness. In addition to this, the force required to extract individual thiol-tagged cholesterols from the model membranes was observed to decrease with the presence of 7-ketocholesterol, indicating this is consistent with Fluorescence Recovery After Photobleaching observations of model membranes which support a more fluid membrane with 7-ketocholesterol present as the diffusion coefficient (recovery rate of the membrane) is higher. Taken together, these findings show how the biophysical properties of the lipid membranes are altered by the presence of 7-ketocholesterol and provide insight for further investigations to examine how the eye lens is affected with age or oxidative damage. A novel model membrane for the lipids of the bovine eye lens was also created and tested, which could be used in further experiments

Advancing combined radiological and optical scanning for breast-conserving surgery margin guidance

Breast cancer is one of the most common types of cancer worldwide, and standard-of-care for early-stage disease typically involves a lumpectomy or breast-conserving surgery (BCS). BCS involves the local resection of cancerous tissue, while sparring as much healthy tissue as possible. State-of-the-art methods for intraoperatively evaluating BCS margins are limited. Approximately 20% of BCS cases result in a tissue resection with cancer at or near the resection surface (i.e., a positive margin). A two-fold increase in ipsilateral breast cancer recurrence is associated with the presence of one or more positive margins. Consequently, positive margins often necessitate costly re-excision procedures to achieve a curative outcome. X-ray micro-computed tomography (CT) is emerging as a powerful ex vivo specimen imaging technology, as it provides robust three-dimensional sensing of tumor morphology rapidly. However, X-ray attenuation lacks contrast between soft tissues that are important for surgical decision making during BCS. Optical structured light imaging, including spatial frequency domain imaging and active line scan imaging, can act as adjuvant tools to complement micro-CT, providing wide field-of-view, non-contact sensing of relevant breast tissue subtypes on resection margins that cannot be differentiated by micro-CT alone. This thesis is dedicated to multimodal imaging of BCS tissues to ultimately improve intraoperative BCS margin assessment, reducing the number of positive margins after initial surgeries and thereby reducing the need for costly follow-up procedures. Volumetric sensing of micro-CT is combined with surface-weighted, sub-diffuse optical reflectance derived from high spatial frequency structured light imaging. Sub-diffuse reflectance plays the key role of providing enhanced contrast to a suite of normal, abnormal benign, and malignant breast tissue subtypes. This finding is corroborated through clinical studies imaging BCS specimen slices post-operatively and is further investigated through an observational clinical trial focused on combined, intraoperative micro-CT and optical imaging of whole, freshly resected BCS tumors. The central thesis of this work is that combining volumetric X-ray imaging and sub-diffuse optical scanning provides a synergistic multimodal imaging solution to margin assessment, one that can be readily implemented or retrofitted in X-ray specimen imaging systems and that could meaningfully improve surgical guidance during initial BCS procedures

Master of Science

thesisHemodialysis vascular access, the interface between a dialysis patient and a dialysis machine, is quite literally the lifeblood of a patient's health. Vascular access dysfunction is the leading cause of hospitalization in hemodialysis patients. The occlusive growth of neointimal hyperplasia (NH) in expanded polytetrafluoroethylene (ePTFE) ringed grafts is the primary cause of failure. To further develop a proposed thermal ultrasound treatment to reduce or prevent NH in arteriovenous vascular grafts, the acoustic properties of ePTFE were studied in water and alcohol solutions. Previous reports of ePTFE acoustic properties are critiqued. It was found that the acoustic transmission and attenuation through ePTFE, and therefore the potential for an ultrasound-based therapy for NH, are heavily dependent on the medium in which the graft is immersed, suggesting that the acoustic properties of implanted grafts will change as grafts mature in vivo. The acoustic impedance and attenuation of water-soaked ePTFE were 0.478 ± 1.43 × 10-2 MRayl and 1.78 ± 0.111 Np/cm·MHz, respectively, while the acoustic impedance and attenuation of ePTFE in alcohol were 1.49 ± 0.149 MRayl and 0.77 ± 1.1 × 10-2 Np/cm·MHz, respectively. The use of focused ultrasound to heat implanted ringed ePTFE grafts was numerically modeled from 1.35- and 1.443-MHz transducers for in vitro geometries. Power deposition and heating, in turn, differed by an order of magnitude between various graft acoustic properties. Graft rings were predicted to be substantial absorbing and iv scattering features. In vitro phantom models were constructed: one with and one without thermocouples. At 1 W of acoustic power, the maximum temperature rise was 8˚ C. The thermocouple model containing a water-soaked graft did not experience heating in the far graft wall. The MRTI model confirmed that the graft rings are an absorbing/scattering feature. Heating was not prevented in the presence of water flow through the graft. Water was not heated significantly. Overall, results suggest ultrasound exposure can be used to generate temperature rises corresponding with the potential prevention or inhibition of NH in ringed ePTFE vascular grafts. A hybrid therapeutic/diagnostic transducer design with a therapeutic semi-annular array surrounding a diagnostic linear array is presented. Compared to a solid transducer of the same dimensions, there were only marginal aberrations in the focal plane. Numerical optimization of the element drive configuration indicated that the least distorted focal plane was produced by uniform phase and magnitude at each element

Using Raman Spectroscopy for Intraoperative Margin Analysis in Breast Conserving Surgery

Breast Conserving Surgery (BCS) in the treatment of breast cancer aims to provide optimal oncological results, with minimal tissue excision to optimise cosmetic outcome. Positive margins due to an inadequate resection occurs in 17% of UK patients undergoing BCS and prompts recommendation for further tissue re-excision to reduce recurrence risk. A second operation causes patient anxiety and significant healthcare costs. This issue could be resolved with accurate intra-operative margin analysis (IMA) to enable excision of all cancerous tissue at the index procedure. High wavenumber Raman Spectroscopy (HWN RS) is a vibrational spectroscopy highly sensitive to changes in protein/lipid environment and water content –biochemical differences found between tumour and normal breast tissue. We proposed that HWN RS could be used to differentiate between tumour and non-tumour breast tissue with a view to future IMA. This thesis presents the development of a Raman system to measure the HWN region capable of accurately detecting changes in protein, lipid and water content, in the presence of highly fluorescent surgical pigments such as blue dye that are present in surgically excised specimens. We investigate the relationship between changes in the HWN spectra with changes in water content in constructed breast phantoms to mimic protein and lipid rich environments and biological tissue. Human breast tissue of paired tumour and non-tumour samples were then measured and analysed. We found that breast tumour tissue is a protein rich, high water, low fat environment and that non-tumour is a low protein, fat rich environment with a low water content, and this can be used to identify breast cancer using HWN RS with excellent accuracy of over 90%. This thesis demonstrates a HWN RS Raman system capable of differentiating between tumour and non-tumour tissue in human breast tissue, and this has the potential to provide IMA in BCS