Establishment of rules for interpreting ultraviolet autofluorescence microscopy images for noninvasive detection of Barrett's esophagus and dysplasia.

The diagnostic potential of autofluorescence (AF) microscopy under ultraviolet (UV) excitation is explored using ex vivo human specimens. The aim is to establish optical patterns (the rules for interpretation) that correspond to normal and abnormal histologies of the esophagus, spanning from early benign modifications (Barrett's esophagus) to subsequent dysplastic change and progression toward carcinoma. This was achieved by developing an image library categorized by disease progression. We considered morphological changes of disease as they are compared with histological diagnosis of the pathological specimen, as well as control samples of normal esophagus, proximal stomach, and small intestine tissue. Our experimental results indicate that UV AF microscopy could provide real-time histological information for visualizing changes in tissue microstructure that are currently undetectable using conventional endoscopic methods.

Endomicroscopy imaging of epithelial structures using tissue autofluorescence.

We explore autofluorescence endomicroscopy as a potential tool for real-time visualization of epithelial tissue microstructure and organization in a clinical setting. The design parameters are explored using two experimental systems--an Olympus Medical Systems Corp. stand-alone clinical prototype probe, and a custom built bench-top rigid fiber conduit prototype. Both systems entail ultraviolet excitation at 266 nm and/or 325 nm using compact laser sources. Preliminary results using ex vivo animal and human tissue specimens suggest that this technology can be translated toward in vivo application to address the need for real-time histology.

Characterizing the origin of autofluorescence in human esophageal epithelium under ultraviolet excitation.

The autofluorescence under ultraviolet excitation arising from normal squamous and columnar esophageal mucosa is investigated using multispectral microscopy. The results suggest that the autofluorescence signal arises from the superficial tissue layer due to the short penetration depth of the ultraviolet excitation. As a result, visualization of esophageal epithelial cells and their organization can be attained using wide-field autofluorescence microscopy. Our results show tryptophan to be the dominant source of emission under 266 nm excitation, while emission from NADH and collagen are dominant under 355 nm excitation. The analysis of multispectral microscopy images reveals that tryptophan offers the highest image contrast due to its non-uniform distribution in the sub-cellular matrix. This technique can simultaneously provide functional and structural imaging of the microstructure using only the intrinsic tissue fluorophores.

Real-time microscopic imaging of esophageal epithelial disease with autofluorescence under ultraviolet excitation.

Detection of esophageal disease in current clinical practice is limited to visualization of macroscopic epithelial morphology. In this work, we investigate high resolution autofluorescence imaging under ultra violet excitation to visualize microscopic epithelial changes related to disease progression using a bench top prototype microscope. The approach is based on the hypothesis that UV excitation light can only penetrate the superficial layer of cells resulting in autofluorescence images of the epithelial layer without using an additional image sectioning approach. The experiments were performed using ex vivo human esophagus biopsy specimens. The results indicate that cellular morphology information related to disease progression is attainable without tissue preparation.

Development of a dual-modal tissue diagnostic system combining time-resolved fluorescence spectroscopy and ultrasonic backscatter microscopy.

We report a tissue diagnostic system which combines two complementary techniques of time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) and ultrasonic backscatter microscopy (UBM). TR-LIFS evaluates the biochemical composition of tissue, while UBM provides tissue microanatomy and enables localization of the region of diagnostic interest. The TR-LIFS component consists of an optical fiber-based time-domain apparatus including a spectrometer, gated multichannel plate photomultiplier, and fast digitizer. It records the fluorescence with high sensitivity (nM concentration range) and time resolution as low as 300 ps. The UBM system consists of a transducer, pulser, receiving circuit, and positioning stage. The transducer used here is 45 MHz, unfocused, with axial and lateral resolutions 38 and 200 microm. Validation of the hybrid system and ultrasonic and spectroscopic data coregistration were conducted both in vitro (tissue phantom) and ex vivo (atherosclerotic tissue specimens of human aorta). Standard histopathological analysis of tissue samples was used to validate the UBM-TRLIFS data. Current results have demonstrated that spatially correlated UBM and TR-LIFS data provide complementary characterization of both morphology (necrotic core and calcium deposits) and biochemistry (collagen, elastin, and lipid features) of the atherosclerotic plaques at the same location. Thus, a combination of fluorescence spectroscopy with ultrasound imaging would allow for better identification of features associated with tissue pathologies. Current design and performance of the hybrid system suggests potential applications in clinical diagnosis of atherosclerotic plaque.