Malignancies of the oral cavity are conventionally diagnosed by white light endoscopy, biopsy, and histopathology. However, it is often difficult to distinguish between benign and premalignant or early lesions. A laser confocal endomicroscope (LCE) offers noninvasive, in vivo surface and subsurface fluorescence imaging of tissue. We investigate the use of an LCE with a rigid probe for diagnostic imaging of the oral cavity. Fluorescein and 5-aminolevulinic acid (ALA) were used to carry out fluorescence imaging in vivo and on resected tissue samples of the oral cavity. In human subjects, ALA-induced protoporphyrin IX (PpIX) fluorescence images from the normal tongue were compared to images obtained from patients with squamous cell carcinoma (SCC) of the tongue. Using rat models, images from normal rat tongues were compared to those from carcinogen-induced models of SCC. Good structural images of the oral cavity were obtained using ALA and fluorescein, and morphological differences between normal and lesion tissue can be distinguished. The use of a pharmaceutical-grade solvent Pharmasolve® enhanced the subsurface depth from which images can be obtained. Our initial results show that laser confocal fluorescence endomicroscopy has potential as a noninvasive optical imaging method for the diagnosis of oral cavity malignancies.
Confocal endomicroscopy is a novel, noninvasive microscopic technique that enables surface and subsurface imaging of living tissues or cells in vivo. This study was to explore the possibility of utilizing a novel rigid confocal endomicroscope (RCE) system for detecting morphological changes in living normal and neoplastic human and murine tongue tissue in combination with different photosensitizers, i.e. hypericin and 5-aminolevulinic acid (ALA) induced endogenous protoporphyrin IX (PPIX) fluorescence. Subjects were topically or systemically applied photosensitizer to the oral mucosa, and then fluorescence confocal endomicroscopy was performed on the tongue using the RCE system with the laser excitation wavelength at 488 nm. The preliminary results showed that confocal fluorescence images of the tongue can be acquired in real-time with well-defined micro-morphological structures, and changes of tissue structures associated with cancer transformation can also be identified. This study suggests that photosensitizer-mediated confocal endomicroscopy have a significant potential for rapid, non-invasive detection of early oral cancers in vivo.
A digitized fluorescence endoscopic imaging (DFEI) system combined with 5-Aminolevulinic Acid (5-ALA) induced protoporphyrin IX (PPIX) was developed for the detection of neoplasms in the oral cavity and uterine-cervix. The system has the capability of producing both the digital and video fluorescence images in real time, and also quantifying the fluorescence images. The results show that using the DFEI system associated with the fluorescence image quantification method, both high sensitivity and specificity can be achieved during the head and neck, and obstetric and gynaecology clinical trials. The red fluorescence intensity distribution in the lesion area can also be obtained after digital image processing to better understand the situation of PPIX accumulation in the tissues. Furthermore, applying the intensity ratio IR/IB at red and blue wavelength regions of the tissue targeted, where IR includes the intensity of PPIX fluorescence and red tissue autofluorescence, and IB is the intensity of diffusely back-scattered excitation blue light, different histopathological grades of lesions can be classified by the DEFI system, suggesting a significant potential of the noninvasive optical biopsy for the early cancer detection.
A digitized fluorescence endoscopy imaging system combined with 5-Aminolevulinic Acid (5-ALA) induced Protoporphyrin IX (PPIX) has been developed for the detection of neoplasms in oral cavity. It mainly consists of the illumination console, fluorescence detection unit, computer system for image acquisition, processing and analysis, and online image display system as well. The developed system can produce both the digital and video fluorescence images in real time, and can be used to quantify fluorescence images acquired. Preliminary results from the Head and Neck clinic show that high sensitivity and high specificity can be achieved. Furthermore, applying the intensity ratios at two different wavelength regions, the developed system shows the capability of differentiating between different histopathological stages of oral lesions, suggesting a significant potential for realizing the non-invasive optical biopsy for early cancer diagnosis.
Fluorescence excitation-emission matrices (EEMs) were measured from normal and tumourous oral tissues in vitro using excitation wavelengths ranging from 220 to 500 nm. The prominent fluorescence excitation-emission peaks: (228 nm; 260/338/475 nm), (282 nm; 337/475/660 nm), (335 nm; 472 nm) and (465 nm, 523 nm) for normal tissues; (228 nm; 335/475/660 nm), (282 nm; 330/660 nm) and (350 nm; 460 nm) for tumour tissues, can be found in EEM maps, indicating the dominant fluorophores responsible for tissue autofluorescence are tryptophan, NADH, FAD, and porphyrin compounds in oral tissues. The optimal excitation wavelengths were found to be 280, 335, and 465 nm. The empirical discrimination algorithms based on I475/I660, and I520/I660 were developed. These algorithms yield an average sensitivity and specificity of 95% and 88%, respectively.
The ratio of autofluorescence intensity at 550+/- 10 nm to that at 630+/- 10 nm (R1=I550/I630) or 680+/- 10 nm (R2=I550/I680) was used as a diagnostic algorithm for identification of malignant tumour tissues. The performance of the ratio diagnostic algorithm was evaluated on more than 100 human colonic specimens under excitation laser light at 457.9 nm. A significant difference of the ratio value R1 and R2 was found between normal and tumour specimens (p<0.001). The diagnostic test has a sensitivity and specificity of 85% and 87% for a threshold value of R1=1.75, and a sensitivity and specificity of 94% and 82% for a threshold of R2=3.5. The experimental results show that the ratio mapping is sensitive to the small changes in the presence of tumour tissues, and the contrast of the contour map for detecting the region of malignancy can also be enhanced significantly if using the diagnostic algorithm R2 (I550/I680).
Based on the microscopic properties of colonic tissues, a five-layer colon optical model was developed to calculate the excitation light distribution in the tissue and the fluorescence escape function from the tissue by Monte Carlo simulations. The theoretically modeled fluorescence spectrum fits well to the experimental results, demonstrating that the microscopic properties of tissue applied in the colon optical model can be quantitatively correlated with the macroscopic autofluorescence measurements.
Based on tissue microscopic fluorescence properties and optical parameters, a three-layer bronchus optical mode was developed to calculate the excitation light distribution in the tissue and the fluorescence escape function from the tissue by Monte Carlo simulations. The contributions of each layer in the tissue to total autofluorescence signal observed at the tissue surface were also estimated. In addition, the fluorescence spectrum of the bronchial tissue was also modeled using the obtained tissue micro- spectroscopic properties. It is found that the theoretically modeled spectrum agrees well with the in vitro tissue fluorescence in the spectral range below 520 nm or above 600 nm.
Using an excitation wavelength of 442 nm from a He-Cd laser, autofluorescence emissions from various layers of human colonic tissues were analyzed by a microspectrophotometric system. The cross section view on the sectioned colonic tissues reveals three distinct tissue layers for fluorescence, i.e., the mucosa, the submucosa and the muscularis propria, with submucosa being the most fluorescent. It was found that the acquired fluorescence imaging from different tissue layers could be used for localizing fluorescence as well as describing tissue architectural details of areas of distinct pathology that are essential for differentiating between normal and abnormal tissues by laser spectroscopic diagnostic approaches.
Early detection of lung cancer has been a significant area of interest due to the large number of cancer-related deaths. The microscopic fluorescence and imaging of excised lung tissue sections were studied using a novel microspectrophotometric system. The intrinsic autofluorescence distributions in different tissue layers of the lung were observed in normal and malignant tissues. The preliminary results show that the microscopic fluorescence analysis on different tissue layers can provide a powerful means to explore the origin of spectral differences between normal and abnormal lung tissues.
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