Automated multimodal spectral histopathology for quantitative diagnosis of residual tumour during basal cell carcinoma surgery

Multimodal spectral histopathology (MSH), an optical technique combining tissue auto-fluorescence (AF) imaging and Raman micro-spectroscopy (RMS), was previously proposed for detection of residual basal cell carcinoma (BCC) at the surface of surgically-resected skin tissue. Here we report the development of a fully-automated prototype instrument based on MSH designed to be used in the clinic and operated by a non-specialist spectroscopy user. The algorithms for the AF image processing and Raman spectroscopy classification had been first optimised on a manually-operated laboratory instrument and then validated on the automated prototype using skin samples from independent patients. We present results on a range of skin samples excised during Mohs micrographic surgery, and demonstrate consistent diagnosis obtained in repeat test measurement, in agreement with the reference histopathology diagnosis. We also show that the prototype instrument can be operated by clinical users (a skin surgeon and a core medical trainee, after only 1-8 hours of training) to obtain consistent results in agreement with histopathology. The development of the new automated prototype and demonstration of inter-instrument transferability of the diagnosis models are important steps on the clinical translation path: it allows the testing of the MSH technology in a relevant clinical environment in order to evaluate its performance on a sufficiently large number of patients

Improving Clinical Diagnosis of Melanocytic Skin Lesions by Raman Spectroscopy

High-quality Raman signals from melanocytic lesions compatible with a possible clinical application have not been demonstrated yet. The objectives of the work described in this thesis were: I: The development of a Raman spectroscopic prototype for objective and fast assessment of melanocytic skin lesions clinically suspicious for melanoma; II: Identification of the main spectroscopic features of melanoma and benign melanocytic lesions suspicious for melanoma; III: Assessment of the feasibility of Raman spectroscopy as an adjunct technique to improve clinical diagnosis of melanocytic skin lesions

An Investigation of the Diagnostic Potential of Autofluorescence Lifetime Spectroscopy and Imaging for Label-Free Contrast of Disease

The work presented in this thesis aimed to study the application of fluorescence lifetime spectroscopy (FLS) and fluorescence lifetime imaging microscopy (FLIM) to investigate their potential for diagnostic contrast of diseased tissue with a particular emphasis on autofluorescence (AF) measurements of gastrointestinal (GI) disease. Initially, an ex vivo study utilising confocal FLIM was undertaken with 420 nm excitation to characterise the fluorescence lifetime (FL) images obtained from 71 GI samples from 35 patients. A significant decrease in FL was observed between normal colon and polyps (p = 0.024), and normal colon and inflammatory bowel disease (IBD) (p = 0.015). Confocal FLIM was also performed on 23 bladder samples. A longer, although not significant, FL for cancer was observed, in paired specimens (n = 5) instilled with a photosensitizer. The first in vivo study was a clinical investigation of skin cancer using a fibre-optic FL spectrofluorometer and involved the interrogation of 27 lesions from 25 patients. A significant decrease in the FL of basal cell carcinomas compared to healthy tissue was observed (p = 0.002) with 445 nm excitation. A novel clinically viable FLS fibre-optic probe was then applied ex vivo to measure 60 samples collected from 23 patients. In a paired analysis of neoplastic polyps and normal colon obtained from the same region of the colon in the same patient (n = 12), a significant decrease in FL was observed (p = 0.021) with 435 nm excitation. In contrast, with 375 nm excitation, the mean FL of IBD specimens (n = 4) was found to be longer than that of normal tissue, although not statistically significant. Finally, the FLS system was applied in vivo in 17 patients, with initial data indicating that 435 nm excitation results in AF lifetimes that are broadly consistent with ex vivo studies, although no diagnostically significant differences were observed in the signals obtained in vivo.Open Acces

Methods and instrumentation for raman characterization of bladder cancer tumor

High incidence and recurrence rates make bladder cancer the most common malignant tumor in the urinary system. Cystoscopy is the gold standard test used for diagnosis, nevertheless small flat tumors might be missed, and the procedure still represents discomfort to patients and high recurrence can result from of urethral injuries. During cystoscopy, suspicious tumors are detected through white light endoscopy and resected tissue is further examined by histopathology. after resection, the pathologist provides information on the differentiation of the cells and the penetration depth of the tumor in the tissue, known as grading and staging of tumor, respectively. During cystoscopy, information on tumor grading and morphological depth characterization can assist onsite diagnosis and significantly reduce the amount of unnecessarily resected tissue. Recently, new developments in optical imaging and spectroscopic approaches have been demonstrated to improve the results of standard techniques by providing real-time detection of macroscopic and microscopic biomedical information. Different applications to detect anomalies in tissues and cells based on the chemical composition and structure at the microscopic level have been successfully tested. There is, nevertheless, the need to cope with the demands for clinical translation. This doctoral thesis presents the investigations, clinical studies and approaches applied to filling the main open research questions when applying Raman spectroscopy as a diagnostic tool for bladder cancer tumor grading and general Raman spectroscopy-based oncological clinical studies

A dark field illumination probe linked to Raman spectroscopy for non-invasivety determination of ocular biomarkers

For early and effective diagnosis of eye diseases, acquiring biochemical information in the eye is preferred. However, it is obtained by performing a biopsy of the eye tissue. This poses a risk to the integrity of the eye and cannot be performed on a regular basis. Raman spectrometry is a potential and powerful tool for the non-invasive investigation of biochemical information. The challenge to use it in an ophthalmic application is the essential of a high-power laser direct shining through the eye, which raises safety concerns for potential retinal damage .In this thesis, biomedical applications of Raman spectroscopy are explored for eye disease biomarkers and ocular drug measurements in ex vitro, in vitro and in vivo. To ensure a safety measurement by projecting a laser in the eye, two types of dark-field illumination probes are designed, manufactured and validated in conjunction with confocal Raman spectroscopy (CRS) to avoid light damage of the retina. Furthermore, a non-contact dark-field illumination method for the same purpose is proposed and theoretically validated

Developing endoscopic instrumentation and techniques for in vivo fluorescence lifetime imaging and spectroscopy

Confocal fluorescence endomicroscopes employ fibre optics along with miniaturised scanning and focussing mechanisms to allow microscopic investigation of remote tissue samples with sub-cellular resolution. For this reason they are widely used in biomedical research, both in clinical studies and in small animal imaging experiments. Fluorescence lifetime imaging microscopy (FLIM) has been shown to provide contrast between normal and unhealthy tissue in several diseases including gastro-intestinal (GI) cancer. As such, there is significant interest in developing instrumentation that will allow endoscopic confocal FLIM as this would permit the in vivo investigation of human GI tissue. This thesis describes the development and use of several instruments and techniques aimed at clinically viable in vivo fluorescence lifetime spectroscopy and confocal endomicroscopy. This research has consisted of two broad branches: the study of the fluorescence signature of healthy and diseased tissue both ex vivo and in vivo; and the development of a novel method for achieving beam scanning in confocal endomicroscopy. Firstly the tissue studies are discussed. This begins with the application of a compact steady-state diffuse reflectance/fluorescence spectrometer and a fibre-optic-coupled time-resolved spectrofluorometer to an in vivo investigation of the spectral signatures of skin cancer. This study – which involved the interrogation of 27 clinically diagnosed lesions – was carried out in collaboration with researchers at Lund University in Sweden and revealed significant differences between healthy and diseased tissue both in terms of fluorescence lifetime and steady state reflectance and fluorescence spectra. Further to this study, work is presented charting the development of a clinically viable spectrometer, which measures time-resolved fluorescence spectra with two excitation wavelengths (375 nm and 435 nm) as well as diffuse reflectance spectra. The entire system is contained within a compact trolley (120 x 70 x 55 cm) for easy transportation and safe use in a clinic. It utilises a fibre optic probe to deliver/collect light that can be inserted into the working channel of a medical endoscope meaning that the system can be used to measure diffuse reflectance and time-resolved fluorescence spectra in the GI tract in vivo. The development and testing of this system are discussed and data are presented from both ex vivo and in vivo studies of GI cancer. The second broad section of this thesis focuses more closely on confocal endomicroscopy. Firstly current methods used in this field are discussed and the sources of several drawbacks are explained. A novel approach to laser scanning endomicroscopy is then presented, which requires no moving parts and can be implemented without the need for any distal scanners or optics. This technique is similar in concept to the use of adaptive optics to focus through turbid media: it utilises a proximal spatial light modulator to correct for phase variations across a fibre imaging bundle and then to encode for arbitrary wavefronts at the distal end of that fibre bundle. Thus, it is possible to realise both focussing and beam scanning at the output of the fibre bundle with no distal components, permitting extremely compact endoscopic probes to be developed. Proof-of-principle results are presented illustrating the imaging capabilities of this novel system as well as simulations showing the achievable resolution and field of view in several feasible endoscopic configurations. Overall, this thesis contains work from two quite different projects both aimed at developing novel optical techniques for clinical diagnostic use in endoscopic procedures. The first is aimed at investigating the temporal and spectral properties of the fluorescence and reflectance signatures of cancer, while the goal of the second is to develop improved confocal endomicroscopes

Study of Liver Surface Imaging Marker to Monitor Chronic Liver Disease Progression

Ph.DDOCTOR OF PHILOSOPH

Development of wide-field fluorescence lifetime imaging for biomedical applications

Imperial Users onl

Raman spectroscopy for skin cancer diagnosis and characterisation of thin supported lipid films

Raman spectroscopy is a powerful tool in oncological imaging. Optical biopsies in which an accurate diagnosis of the tumour areas is spectroscopically performed are especially interesting for application to skin cancer treatments. In the first part of this dissertation a study on automated Raman spectral imaging allowed accurate diagnosis and delineation of the borders of a common type of skin cancer, basal cell carcinoma (BCC). Automated detection and imaging of BCC in skin sections excised during surgery was performed by combining Raman micro-spectroscopy with supervised multivariate mathematical algorithms based on linear discriminant analysis (LDA). The model allowed 90±9% sensitivity and 85±9% specificity in BCC detection. Raman spectral images based on the LDA model were created and compared with the gold-standard of the conventional histopathological diagnoses resulting in excellent agreement. Additional studies on the ability of the model in discriminating between BCC and hair follicles produced accurate diagnoses. In this thesis instrumental implementation and design of a Raman spectral imaging prototype aiming to reduce the acquisition time required to build the Raman spectral images was developed. High sensitivity variants of Raman spectroscopy such as surface enhanced Raman spectroscopy (SERS) are known to enable optical detection down to single molecules and can be applied to thin supported lipid research. The combination of SERS with a complementary topographic technique simultaneously synchronised adds to the chemical information the morphology of the sample surface. In the second part of this thesis simultaneous atomic force microscopy (AFM) and SERS characterisation of thin (≈15-20 nm) supported films of arachidic acid and cationic phospholipids on sapphire/silver substrates was successfully achieved. Supports were fabricated with nanosphere lithographic procedures and allowed enhancement of the weak Raman signals from the amphiphilic films by a maximum factor of ×10^8