Optical Biopsy of the Upper GI Tract Using Fluorescence Lifetime and Spectra.

Screening and surveillance for gastrointestinal (GI) cancers by endoscope guided biopsy is invasive, time consuming, and has the potential for sampling error. Tissue endogenous fluorescence spectra contain biochemical and physiological information, which may enable real-time, objective diagnosis. We first briefly reviewed optical biopsy modalities for GI cancer diagnosis with a focus on fluorescence-based techniques. In an ex vivo pilot clinical study, we measured fluorescence spectra and lifetime on fresh biopsy specimens obtained during routine upper GI screening procedures. Our results demonstrated the feasibility of rapid acquisition of time-resolved fluorescence (TRF) spectra from fresh GI mucosal specimens. We also identified spectroscopic signatures that can differentiate between normal mucosal samples obtained from the esophagus, stomach, and duodenum.

5-Aminolevulinic acid induced protoporphyrin IX as a fluorescence marker for quantitative image analysis of high-grade dysplasia in Barrett's esophagus cellular models.

Early detection and treatment of high-grade dysplasia (HGD) in Barrett's esophagus may reduce the risk of developing esophageal adenocarcinoma. Confocal endomicroscopy (CLE) has shown advantages over routine white-light endoscopic surveillance with biopsy for histological examination; however, CLE is compromised by insufficient contrast and by intra- and interobserver variation. An FDA-approved PDT photosensitizer was used here to reveal morphological and textural features similar to those found in histological analysis. Support vector machines were trained using the aforementioned features to obtain an automatic and robust detection of HGD. Our results showed 95% sensitivity and 87% specificity using the optimal feature combination and demonstrated the potential for extension to a three-dimensional cell model.

5-aminolevulinic acid for quantitative seek-and-treat of high-grade dysplasia in Barrett's esophagus cellular models.

High-grade dysplasia (HGD) in Barrett's esophagus (BE) poses increased risk for developing esophageal adenocarcinoma. To date, early detection and treatment of HGD regions are still challenging due to the sampling error from tissue biopsy and relocation error during the treatment after histopathological analysis. In this study, CP-A (metaplasia) and CP-B (HGD) cell lines were used to investigate the "seek-and-treat" potential using 5-aminolevulinic acid-induced protoporphyrin IX (PpIX). The photodynamic therapy photosensitizer then provides both a phototoxic effect and additional image contrast for automatic detection and real-time laser treatment. Complementary to our studies on automatic classification, this work focused on characterizing subcellular irradiation and the potential phototoxicity on both metaplasia and HGD. The treatment results showed that the HGD cells are less viable than metaplastic cells due to more PpIX production at earlier times. Also, due to mitochondrial localization of PpIX, a better killing effect was achieved by involving mitochondria or whole cells compared with just nucleus irradiation in the detected region. With the additional toxicity given by PpIX and potential morphological/textural differences for pattern recognition, this cellular platform serves as a platform to further investigate real-time "seek-and-treat" strategies in three-dimensional models for improving early detection and treatment of BE.

Monitoring photosensitizer uptake using two photon fluorescence lifetime imaging microscopy.

Photodynamic Therapy (PDT) provides an opportunity for treatment of various invasive tumors by the use of a cancer targeting photosensitizing agent and light of specific wavelengths. However, real-time monitoring of drug localization is desirable because the induction of the phototoxic effect relies on interplay between the dosage of localized drug and light. Fluorescence emission in PDT may be used to monitor the uptake process but fluorescence intensity is subject to variability due to scattering and absorption; the addition of fluorescence lifetime may be beneficial to probe site-specific drug-molecular interactions and cell damage. We investigated the fluorescence lifetime changes of Photofrin(®) at various intracellular components in the Mat-LyLu (MLL) cell line. The fluorescence decays were analyzed using a bi-exponential model, followed by segmentation analysis of lifetime parameters. When Photofrin(®) was localized at the cell membrane, the slow lifetime component was found to be significantly shorter (4.3 ± 0.5 ns) compared to those at other locations (cytoplasm: 7.3 ± 0.3 ns; mitochondria: 7.0 ± 0.2 ns, p < 0.05).

Effects of incomplete decay in fluorescence lifetime estimation.

Fluorescence lifetime imaging has emerged as an important microscopy technique, where high repetition rate lasers are the primary light sources. As fluorescence lifetime becomes comparable to intervals between consecutive excitation pulses, incomplete fluorescence decay from previous pulses can superimpose onto the subsequent decay measurements. Using a mathematical model, the incomplete decay effect has been shown to lead to overestimation of the amplitude average lifetime except in mono-exponential decays. An inverse model is then developed to correct the error from this effect and the theoretical simulations are tested by experimental results.