Recent Advances and the Potential for Clinical Use of Autofluorescence Detection of Extra-Ophthalmic Tissues

The autofluorescence (AF) characteristics of endogenous fluorophores allow the label-free assessment and visualization of cells and tissues of the human body. While AF imaging (AFI) is well-established in ophthalmology, its clinical applications are steadily expanding to other disciplines. This review summarizes clinical advances of AF techniques published during the past decade. A systematic search of the MEDLINE database and Cochrane Library databases was performed to identify clinical AF studies in extra-ophthalmic tissues. In total, 1097 articles were identified, of which 113 from internal medicine, surgery, oral medicine, and dermatology were reviewed. While comparable technological standards exist in diabetology and cardiology, in all other disciplines, comparability between studies is limited due to the number of differing AF techniques and non-standardized imaging and data analysis. Clear evidence was found for skin AF as a surrogate for blood glucose homeostasis or cardiovascular risk grading. In thyroid surgery, foremost, less experienced surgeons may benefit from the AF-guided intraoperative separation of parathyroid from thyroid tissue. There is a growing interest in AF techniques in clinical disciplines, and promising advances have been made during the past decade. However, further research and development are mandatory to overcome the existing limitations and to maximize the clinical benefits

Using Fluorescence – Polarization Endoscopy in Detection of Precancerous and Cancerous Lesions in Colon and Pancreatic Cancer

Colitis-associated cancer (CAC) arises from premalignant flat lesions of the colon, which are difficult to detect with current endoscopic screening approaches. We have developed a complementary fluorescence and polarization reporting strategy that combines the unique biochemical and physical properties of dysplasia and cancer for real time detection of these lesions. Utilizing a new thermoresponsive sol-gel formulation with targeted molecular probe allowed topical application and detection of precancerous and cancerous lesions during endoscopy. Incorporation of nanowire-filtered polarization imaging into NIR fluorescence endoscopy served as a validation strategy prior to obtaining biopsies. In order to reduce repeat surgeries arising from incomplete tumor resection, we demonstrated the efficacy of the targeted molecular probe towards margins of sporadic colorectal cancer (SCC). Fluorescence-polarization microscopy using circular polarized (CP) light served as a rapid, supplementary tool for assessment and validation of excised tissue to ensure complete tumor resection for examining tumor margins prior to H&E-based pathological diagnosis. We extended our platform towards non-invasive directed detection of pancreatic cancer utilizing fluorescence molecular tomography (FMT) and NIR laparoscopy using identified targeted molecular probe. We were able to non-invasively distinguished between pancreatitis and pancreatic cancer and guide pancreatic tumor resection using NIR laparoscopy

Seeing the Big Picture: System Architecture Trends in Endoscopy and LED-Based hyperspectral Subsystem Intergration

Early-stage colorectal lesions remain difficult to detect. Early development of neoplasia tends to be small (less than 10 mm) and flat and difficult to distinguish from surrounding mucosa. Additionally, optical diagnosis of neoplasia as benign or malignant is problematic. Low rates of detection of these lesions allow for continued growth in the colorectum and increased risk of cancer formation. Therefore, it is crucial to detect neoplasia and other non-neoplastic lesions to determine risk and guide future treatment. Technology for detection needs to enhance contrast of subtle tissue differences in the colorectum and track multiple biomarkers simultaneously. This work implements one such technology with the potential to achieve the desired multi-contrast outcome for endoscopic screenings: hyperspectral imaging. Traditional endoscopic imaging uses a white light source and a RGB detector to visualize the colorectum using reflected light. Hyperspectral imaging (HSI) acquires an image over a range of individual wavelength bands to create an image hypercube with a wavelength dimension much deeper and more sensitive than that of an RGB image. A hypercube can consist of reflectance or fluorescence (or both) spectra depending on the filtering optics involved. Prior studies using HSI in endoscopy have normally involved ex vivo tissues or xiv optics that created a trade-off between spatial resolution, spectral discrimination and temporal sampling. This dissertation describes the systems design of an alternative HSI endoscopic imaging technology that can provide high spatial resolution, high spectral distinction and video-rate acquisition in vivo. The hyperspectral endoscopic system consists of a novel spectral illumination source for image acquisition dependent on the fluorescence excitation (instead of emission). Therefore, this work represents a novel contribution to the field of endoscopy in combining excitation-scanning hyperspectral imaging and endoscopy. This dissertation describes: 1) systems architecture of the endoscopic system in review of previous iterations and theoretical next-generation options, 2) feasibility testing of a LED-based hyperspectral endoscope system and 3) another LED-based spectral illuminator on a microscope platform to test multi-spectral contrast imaging. The results of the architecture point towards an endoscopic system with more complex imaging and increased computational capabilities. The hyperspectral endoscope platform proved feasibility of a LED-based spectral light source with a multi-furcated solid light guide. Another LED-based design was tested successfully on a microscope platform with a dual mirror array similar to telescope designs. Both feasibility tests emphasized optimization of coupling optics and combining multiple diffuse light sources to a common output. These results should lead to enhanced imagery for endoscopic tissue discrimination and future optical diagnosis for routine colonoscopy

Morphological Features of Dysplastic Progression in Epithelium: Quantification of Cytological, Microendoscopic, and Second Harmonic Generation Images

Advances in imaging technology have led to a variety of available clinical and investigational systems. In this collection of studies, we tested the relevance of morphological image feature quantification on several imaging systems and epithelial tissues. Quantification carries the benefit of creating numerical baselines and thresholds of healthy and abnormal tissues, to potentially aid clinicians in determining a diagnosis, as well as providing researchers with standardized, unbiased results for future dissemination and comparison. Morphological image features in proflavine stained oral cells were compared qualitatively to traditional Giemsa stained cells, and then we quantified the nuclear to cytoplasm ratio. We determined that quantification of proflavine stained cells matched our hypothesis, as the nuclei in oral carcinoma cells were significantly larger than healthy oral cells. Proflavine has been used in conjunction with translational fluorescence microendoscopy of the gastrointestinal tract, and we demonstrated the ability of our custom algorithm to accurately (up to 85% sensitivity) extract colorectal crypt area and circularity data, which could minimize the burden of training on clinicians. In addition, we proposed fluorescein as an alternative fluorescent dye, providing comparable crypt area and circularity information. In order to investigate the morphological changes of crypts via the supporting collagen structures, we adapted our quantification algorithm to analyze crypt area, circularity, and an additional shape parameter in second harmonic generation images of label-free freshly resected murine epithelium. Murine models of colorectal cancer (CRC) were imaged at early and late stages of tumor progression, and we noted significant differences between the Control groups and the late cancer stages, with some differences between early and late stages of CRC progression

The role of Raman Spectroscopy in the detection of dysplasia in Barrett’s oesophagus

Introduction The incidence of oesophageal adenocarcinoma is increasing. Although improvements have been seen, the overall 5 year survival rate remains poor, at 15.1%. As with other cancers, the survival rate is highest when the disease is confined to the oesophagus. Barrett’s oesophagus is an acquired condition, characterised by the replacement of the normal distal squamous epithelial lining of the oesophagus with columnar epithelium. Oesophageal adenocarcinoma develops, in most instances, along a pathway of increasing dysplasia in the sections of Barrett’s oesophagus. If dysplasia can be diagnosed accurately, then this would permit treatment prior to the development of adenocarcinoma. Methods Samples of Barrett’s oesophagus with varying degrees of dysplasia and adenocarcinoma were measured with Raman point and mapping spectroscopy. Analysis was performed using Matlab®. Results Samples of squamous epithelia, Barrett’s oesophagus without dysplasia, with low-grade dysplasia, with high-grade dysplasia and oesophageal adenocarcinoma were measured and analysed. 2078 point spectra measurements and 117 map regions were analysed. Raman point spectra measurements and Raman mapping differentiated samples without dysplasia from those with dysplasia, and differentiated samples of low-grade dysplasia from those of high-grade dysplasia and adenocarcinoma. The specificity and sensitivity were, however, low. Conclusion This research has illustrated the ability of Raman spectroscopy to discern samples of Barrett’s oesophagus with low-grade dysplasia from those with higher grades of dysplasia. This capability could be utilised clinically with in- vivo measurements to identify the areas requiring detailed surveillance and biopsies. The majority of patients with Barrett’s oesophagus and low-grade dysplasia will never progress to adenocarcinoma. There is currently no means, either via histopathology or via a biomarker, to identify the minority who will develop high- grade dysplasia or adenocarcinoma. Raman spectroscopy may have the ability to do this and I believe this is the path that this technology should pursue

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

Endoscopic Optical Coherence Tomography for Clinical Gastroenterology

Optical coherence tomography (OCT) is a real-time optical imaging technique that is similar in principle to ultrasonography, but employs light instead of sound waves and allows depth-resolved images with near-microscopic resolution. Endoscopic OCT allows the evaluation of broad-field and subsurface areas and can be used ancillary to standard endoscopy, narrow band imaging, chromoendoscopy, magnification endoscopy, and confocal endomicroscopy. This review article will provide an overview of the clinical utility of endoscopic OCT in the gastrointestinal tract and of recent achievements using state-of-the-art endoscopic 3D-OCT imaging systems. Keywords: optical coherence tomography; optical biopsy; endoscopic imaging; Barrett’s esophagus; inflammatory bowel diseaseNational Institutes of Health (U.S.) (Grant R01-CA75289-17)National Institutes of Health (U.S.) (Grant R44-CA101067-06)National Institutes of Health (U.S.) (Grant R01-CA178636-01)National Institutes of Health (U.S.) (Grant R44EY022864-01)National Institutes of Health (U.S.) (Grant R01-EY011289-27)National Institutes of Health (U.S.) (Grant R01-NS057476-05)United States. Air Force Office of Scientific Research (Grant FA9550-12-1-0499)United States. Air Force Office of Scientific Research (Grant FA9550-10-1-0551

Examining lipid metabolism of colorectal adenomas and carcinomas using Rapid Evaporative Ionisation Mass Spectrometry (REIMS)

Background There is an unmet need for real-time intraoperative colorectal tissue recognition, which would promote personalised oncologic decision making. Rapid Evaporative Ionization Mass Spectrometry (REIMS) analyses the composition of cellular lipids through the aerosol generated from electrosurgical instruments, providing a novel diagnostic platform and surgeon feedback. Thesis Hypothesis Colorectal lipid metabolism and cellular lipid composition are associated with the phenotype of colorectal adenomas and carcinomas, which can be leveraged for tissue recognition in vivo. Methods This thesis contains three work packages. First, a method for REIMS spectral quality control was developed based on a human dataset and analysis of a porcine model assessed the spectral impact of technical and environmental factors. Second, an ex vivo spectral reference database was constructed from analysis of human colorectal tissues, assessing the ability of REIMS for tissue recognition. Finally, REIMS was translated into the operating theatre, for proof-of-principle application of during transanal minimally invasive surgery (TAMIS). Results Sensitivity analyses revealed seven minimum quality criteria for REIMS spectra to be included in all future statistical analyses, with quality also impacted by low diathermy power, coagulation mode and tissue contamination. Based on tissue of 161 patients, REIMS could differentiate colorectal normal, adenoma and cancer tissue with 91.1% accuracy, and disease from normal with 93.5% accuracy. REIMS could risk-stratify adenomas by predicting grade of dysplasia, however not histological features of poor prognosis in cancers. 61 pertinent lipid metabolites were structurally identified. REIMS was coupled to TAMIS in seven patients. Optimisation of the workflow successfully increased signal intensity, with tissue recognition showing high accuracy in vivo and identification of a cancer-involved margin. Discussion This thesis demonstrates that REIMS can be optimised and applied for accurate real-time colorectal tissue recognition based on cellular lipid composition. This can be translated in vivo, with promising results during first-in-man mass spectrometry-coupled TAMIS.Open Acces

A roadmap for the clinical implementation of optical-imaging biomarkers

Clinical workflows for the non-invasive detection and characterization of disease states could benefit from optical-imaging biomarkers. In this Perspective, we discuss opportunities and challenges towards the clinical implementation of optical-imaging biomarkers for the early detection of cancer by analysing two case studies: the assessment of skin lesions in primary care, and the surveillance of patients with Barrett’s oesophagus in specialist care. We stress the importance of technical and biological validations and clinical-utility assessments, and the need to address implementation bottlenecks. In addition, we define a translational roadmap for the widespread clinical implementation of optical imaging-technologies