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

Detection of In vivo Oral Epithelial Cancer using Fluorescence Lifetime Imaging

Endogenous fluorescence lifetime imaging microscopy (FLIM) provides a nondestructive means to interrogate the biochemical composition of biological tissues. Therefore, it has the potential to identify tissue pre-malignant and malignant transformation. In this study, we evaluate the potential of endogenous FLIM for detecting benign oral lesions from pre-malignant and malignant oral lesions. Using a database of FLIM images (n=20) obtained in vivo from the oral cavity of patients undergoing tissue biopsy, we were able to identify specific features from the characteristic FLIM signal of benign, mild dysplastic, and cancerous oral lesions. These features were used to train statistical classification rules aimed to detect benign lesions from either dysplastic and cancerous lesions. Our results indicated that dysplastic and cancerous lesions could be detected from benign lesions with sensitivity of ~89% and specificity of ~95%. Our future efforts are focused on further developing our classification algorithms with additional FLIM in vivo data

Detection of In vivo Oral Epithelial Cancer using Fluorescence Lifetime Imaging

Endogenous fluorescence lifetime imaging microscopy (FLIM) provides a nondestructive means to interrogate the biochemical composition of biological tissues. Therefore, it has the potential to identify tissue pre-malignant and malignant transformation. In this study, we evaluate the potential of endogenous FLIM for detecting benign oral lesions from pre-malignant and malignant oral lesions. Using a database of FLIM images (n=20) obtained in vivo from the oral cavity of patients undergoing tissue biopsy, we were able to identify specific features from the characteristic FLIM signal of benign, mild dysplastic, and cancerous oral lesions. These features were used to train statistical classification rules aimed to detect benign lesions from either dysplastic and cancerous lesions. Our results indicated that dysplastic and cancerous lesions could be detected from benign lesions with sensitivity of ~89% and specificity of ~95%. Our future efforts are focused on further developing our classification algorithms with additional FLIM in vivo data

Emerging Optical Methods for Endoscopic Barrett’s Surveillance

Barrett’s oesophagus is an acquired metaplastic condition that predisposes patients to the development of oesophageal adenocarcinoma, prompting the use of surveillance regimes to detect early malignancy for endoscopic therapy with curative intent. The currently accepted surveillance regime uses white light endoscopy together with random biopsies, but suffers poor sensitivity and discards information from numerous light-tissue interactions that could be exploited to probe structural, functional and molecular changes in the tissue. Advanced optical methods are now emerging that are exquisitely sensitive to these changes and hold significant potential to improve surveillance of Barrett’s oesophagus if they can be applied endoscopically. The next decade will see some of these exciting new methods applied to Barrett’s surveillance in new device architectures for the first time, potentially leading to a longawaited improvement of the standard of care

Detection of In vivo Oral Epithelial Cancer using Fluorescence Lifetime Imaging

Endogenous fluorescence lifetime imaging microscopy (FLIM) provides a nondestructive means to interrogate the biochemical composition of biological tissues. Therefore, it has the potential to identify tissue pre-malignant and malignant transformation. In this study, we evaluate the potential of endogenous FLIM for detecting benign oral lesions from pre-malignant and malignant oral lesions. Using a database of FLIM images (n=20) obtained in vivo from the oral cavity of patients undergoing tissue biopsy, we were able to identify specific features from the characteristic FLIM signal of benign, mild dysplastic, and cancerous oral lesions. These features were used to train statistical classification rules aimed to detect benign lesions from either dysplastic and cancerous lesions. Our results indicated that dysplastic and cancerous lesions could be detected from benign lesions with sensitivity of ~89% and specificity of ~95%. Our future efforts are focused on further developing our classification algorithms with additional FLIM in vivo data

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

Detection of In vivo Oral Epithelial Cancer using Fluorescence Lifetime Imaging

Endogenous fluorescence lifetime imaging microscopy (FLIM) provides a nondestructive means to interrogate the biochemical composition of biological tissues. Therefore, it has the potential to identify tissue pre-malignant and malignant transformation. In this study, we evaluate the potential of endogenous FLIM for detecting benign oral lesions from pre-malignant and malignant oral lesions. Using a database of FLIM images (n=20) obtained in vivo from the oral cavity of patients undergoing tissue biopsy, we were able to identify specific features from the characteristic FLIM signal of benign, mild dysplastic, and cancerous oral lesions. These features were used to train statistical classification rules aimed to detect benign lesions from either dysplastic and cancerous lesions. Our results indicated that dysplastic and cancerous lesions could be detected from benign lesions with sensitivity of ~89% and specificity of ~95%. Our future efforts are focused on further developing our classification algorithms with additional FLIM in vivo data

Wide-field fluorescence lifetime imaging of cancer

Optical imaging of tissue autofluorescence has the potential to provide rapid label-free screening and detection of surface tumors for clinical applications, including when combined with endoscopy. Quantitative imaging of intensity-based contrast is notoriously difficult and spectrally resolved imaging does not always provide sufficient contrast. We demonstrate that fluorescence lifetime imaging (FLIM) applied to intrinsic tissue autofluorescence can directly contrast a range of surface tissue tumors, including in gastrointestinal tissues, using compact, clinically deployable instrumentation achieving wide-field fluorescence lifetime images of unprecedented clarity. Statistically significant contrast is observed between cancerous and healthy colon tissue for FLIM with excitation at 355 nm. To illustrate the clinical potential, wide-field fluorescence lifetime images of unstained ex vivo tissue have been acquired at near video rate, which is an important step towards real-time FLIM for diagnostic and interoperative imaging, including for screening and image-guided biopsy applications

Fluorescence lifetime spectroscopy of tissue autofluorescence in normal and diseased colon measured ex vivo using a fiber-optic probe

We present an ex vivo study of temporally and spectrally resolved autofluorescence in a total of 47 endoscopic excision biopsy/resection specimens from colon, using pulsed excitation laser sources operating at wavelengths of 375 nm and 435 nm. A paired analysis of normal and neoplastic (adenomatous polyp) tissue specimens obtained from the same patient yielded a significant difference in the mean spectrally averaged autofluorescence lifetime −570 ± 740 ps (p = 0.021, n = 12). We also investigated the fluorescence signature of non-neoplastic polyps (n = 6) and inflammatory bowel disease (n = 4) compared to normal tissue in a small number of specimens

Fluorescence Lifetime Imaging Microscopy (FLIM) System for Imaging of Oral Cancer and Precancer

Standard diagnosis of oral cancer is based on visual inspection and palpation by a clinician followed by histological examination of one or more tissue biopsies. Choosing the right location for biopsies, which represents the most severe lesion, is difficult and subjective to each clinician’s experience, especially for precancer lesions which are often diffuse, multifocal, and clinically indistinguishable from benign lesions. This may lead to low diagnosis sensitivity. The aim of this dissertation is to design a more sensitive and objective screening tool to guide the biopsy of oral cancer and precancer. Fluorescence lifetime imaging microscopy (FLIM) is a noninvasive optical technique which is able to detect the information of tissue metabolism and biochemistry based on fluorescence as a source of contrast. Recently, there is increasing interest in the application of multispectral FLIM for medical diagnosis. Central to the clinical translation of FLIM technology is the development of compact and high-speed clinically compatible systems. In this dissertation, four multispectral FLIM systems were designed and built. A bench-top multispectral FLIM system was first built and combined with reflectance confocal microscopy (RCM) for the preclinical validation by imaging a hamster cheek pouch model of oral carcinogenesis. After that, in order to facilitate in vivo imaging of human oral mucosa, three different multispectral FLIM endoscopes were designed. The first FLIM endoscope was built based on a fiber bundle and the time-gated implementation by an intensified charged-coupled device (ICCD). The system was validated by imaging a hamster cheek pouch model of oral carcinogenesis. To achieve faster imaging speed and more accurate lifetime estimation, two rigid handheld FLIM endoscopes were built based on a pulse sampling implementation. These two handheld endoscopes were different in weight and size. The more compact one might serve as the clinical prototype for oral cancer and precancer detection. Both systems were validated by imaging the human oral biopsy ex vivo and human oral mucosa in vivo. The development of these systems will facilitate the evaluation of multispectral FLIM for oral cancer and precancer detection