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

Abstract

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.

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

“…We provided a quantitative assessment of this difference in a previous publication. 32 Our hypothesis that the origin of these images was due to the spectral contribution from different fluorophores including tryptophan, NADH, flavoproteins, and/or lipopigments was supported by a calculated 30% intensity variation. The H&E staining of the non-dysplastic esophageal columnar mucosa section also demonstrates the typical honeycomb pattern observed in this type of tissue.…”

Endomicroscopy imaging of epithelial structures using tissue autofluorescence

“…This fiber conduit only transmitted the emission at wavelengths longer than about 400 nm. Therefore, as the AF signal under 266-nm excitation is mostly due to tryptophan emission centered in the 320 to 350 nm spectral region, 25 only a small portion of this signal (on the order of 10%) was transmitted through the fiber conduit. Using a UV compatible image transporting element would increase the signal available to be detected by the CCD detector by about 1 order of magnitude.…”

“…4. Experimental results of human esophageal mucosal biopsy specimens 25 suggested that the mechanism behind the high contrast imaging of the epithelial layer using wide-field microscopy designs was due to the selected short excitation wavelengths, which only propagated through the tissue to about 50 μm, the approximate penetration depth of UV excitation light. As a result, a sufficient amount of the AF signal produced in the superficial tissue layer could be contained within the microscope's depth of field, sufficiently reducing the out-of-focus signal to allow the formation of high contrast images without the need to use spatial filtering.…”

“…23,24 It was also found using ex vivo human esophageal tissue biopsy specimens under 266-nm excitation resulted in the best image contrast arising mainly from tryptophan emission and its nonuniform distribution within the cells. 25 It was postulated that imaging the epithelial layer with this method would provide more specific histopathologic information related to the disease. Since imaging could be achieved without the use of a signal intensive optical sectioning method (such as confocal or nonlinear excitation), the implementation of high signal throughput image acquisition designs would enable AF microscopic imaging at clinically relevant image acquisition times.…”

Ratio images and ultraviolet C excitation in autofluorescence imaging of neoplasms of the human colon

“…24 Ratio images in the ultraviolet were found to enhance structure visibility in microscopic images of human esophagus. 25 A third study used AF ratio images to highlight adenomatous regions in cross sections of excised colonic polyps. 26 Still others have demonstrated the use of AF ratio images microscopically for monitoring cell metabolism and differentiating cancerous tissue.…”