Elena V. Zagaynova

Optical Coherence Tomography of the Esophagus and Proximal Stomach in Health and Disease

OBJECTIVE: Surveillance of Barrett’s esophagus is problematic, as high-grade dysplasia cannot be recognized endoscopically. Endoscopic ultrasound lacks the resolution to detect high-grade dysplasia. Optical coherence tomography (OCT) employs infrared light reflectance to provide in vivo tissue images at resolution far superior to endoscopic ultrasound, nearly at the level of histology. We have developed a catheter-based system well suited for study of the GI tract. The purpose of this study was to test this catheter-based OCT system and characterize the OCT appearance of normal squamous mucosa, gastric cardia, Barrett’s esophagus, and carcinoma. METHODS: The OCT catheter was passed through the operating channel of the endoscope and placed in contact with the esophageal mucosa. Image acquisition occurred in approximately 3 s. OCT images were correlated with biopsy and/or resection specimens. RESULTS: OCT was used to construct 477 images of the esophagus and stomach in 69 patients. There were unique, distinct OCT appearances of squamous mucosa, gastric cardia, Barrett’s esophagus, and carcinoma. Further, these OCT images were accurately recognized by observers unaware of their site of origin. CONCLUSIONS: OCT provides a highly detailed view of the GI wall, with clear delineation of a multiple layered structure. It is able to distinguish squamous mucosa, gastric cardia, Barrett’s esophagus, and cancer. This technique holds great potential as an adjunct to the surveillance of patients with Barrett’s esophagus, ulcerative pancolitis, and other premalignant conditions.

In vivo optical coherence tomography feasibility for bladder disease

PURPOSE Optical coherence tomography is a new imaging modality capable of imaging luminal surface of biological tissue in the near infrared range with a spatial resolution close to the cellular level. We identified characteristic optical coherence tomography patterns for nonproliferative and proliferative inflammation, and malignant alterations of the bladder. MATERIALS AND METHODS Optical coherence tomography was performed to image the bladder of 66 patients. The probe passed through the operating channel of a cystoscope and was pressed onto the mucosal site of interest. A mucosal biopsy of the image site was obtained. Optical coherence tomography was used to construct 680 images of the bladder and the images were compared with histology slides. RESULTS Optical coherence tomography images of normal bladder showed 3 layers, namely the mucosa or transitional epithelium, submucosa and smooth muscle. In exudative processes there were poor light scattering areas in the connective tissue layer. Images of bladders with proliferative cystitis revealed nonuniform thickening of the epithelium or hyperplasia. Squamous metaplasia appeared as thicker and less transparent epithelium with a jagged boundary. Images of transitional cell carcinoma were characterized by the complete loss of a regular layered structure of the bladder wall and the penetration depth of optical imaging was slight. CONCLUSIONS This study provides the characteristic optical coherence tomography pattern of nonproliferative and proliferative inflammation, and the characteristic appearance of severe dysplasia and transitional cell carcinoma. This technique may be useful as a guide for biopsy and for assisting in establishing resection margins.

Contrasting properties of gold nanoparticles for optical coherence tomography: phantom, in vivo studies and Monte Carlo simulation.

The possibility of using silica-gold nanoshells with 150 nm silica core size and 25 nm thick gold shell as contrasting agents for optical coherence tomography (OCT) is analyzed. Experiments on agar biotissue phantoms showed that the penetration of nanoshells into the phantoms increases the intensity of the optical coherence tomography (OCT) signal and the brightness of the corresponding areas of the OCT image. In vivo experiments on rabbit skin demonstrated that the application of nanoshells onto the skin provides significant contrasting of the borders between the areas containing nanoshells and those without. This effect of nanoshells on skin in vivo is manifested by the increase in intensity of the OCT signal in superficial parts of the skin, boundary contrast between superficial and deep dermis and contrast of hair follicles and glands. The presence of nanoshells in the skin was confirmed by electron microscopy. Monte Carlo simulations of OCT images confirmed the possibility of contrasting skin-layer borders and structures by the application of gold nanoshells. The Monte Carlo simulations were performed for two skin models and exhibit effects of nanoparticles similar to those obtained in the experimental part of the study, thus proving that the effects originate exactly from the presence of nanoparticles.

Contrasting properties of gold nanoshells and titanium dioxide nanoparticles for optical coherence tomography imaging of skin: Monte Carlo simulations and in vivo study

The effect of silica/gold nanoshells and titanium dioxide nanoparticles on the optical properties of skin is studied. By implementing in vivo measurements and Monte Carlo simulations, we analyze the efficiency of using these nanoparticles as contrasting agents for optical coherence tomography (OCT) imaging of skin. In vivo measurements are performed on pig skin, where nanoparticle suspension drops have been applied. The identification of skin layers is performed by comparison with corresponding histology images. Experimental results exhibit an increase in contrast of the obtained OCT images after a single nanoparticles application. Multiple applications do not lead to increase in the obtained contrast. To interpret the obtained experimental OCT images of skin and understand the mechanisms of contrasting, a set of Monte Carlo calculations is performed. The results of the simulations exhibit good qualitative agreement with the experimental images, and prove that the contrasting originates from the nanoparticles added, while the contrast of inclusion originates from the absence of nanoparticles within it and their presence in the surrounding area.

Endoscopic OCT with forward-looking probe: clinical studies in urology and gastroenterology.

In the current paper we present results of application of endoscopic time-domain OCT (EOCT) with lateral scanning by forward looking miniprobe. We analysed material of clinical studies of 554 patients: 164 patients with urinary bladder pathology, and 390 with gastrointestinal tract pathology. We reviewed the materials obtained in different clinics using the OCT device elaborated at the Institute of Applied Physics. We demonstrate results of EOCT application in detection of early cancer and surgery guidance, examples of combined use of OCT and fluorescence imaging. As a result, we show the diagnostic accuracy of EOCT in specific clinical tasks. The sensitivity of EOCT cancer determination in Barretts esophagus is from 71% to 85% at different stages of neoplasia with specificity 68% for all stages. As for bladder carcinoma, the sensitivity and specificity are 85% and 68%, respectively. In colon dysplasia EOST demonstrates high efficacy: sensitivity 92% and specificity 84%.

Phototoxic effects of fluorescent protein KillerRed on tumor cells in mice

KillerRed is known to be a unique red fluorescent protein displaying strong phototoxic properties. Its effectiveness has been shown previously for killing bacterial and cancer cells in vitro. Here, we investigated the photototoxicity of the protein on tumor xenografts in mice. HeLa Kyoto cell line stably expressing KillerRed in mitochondria and in fusion with histone H2B was used. Irradiation of the tumors with 593 nm laser led to photobleaching of KillerRed indicating photosensitization reaction and caused significant destruction of the cells and activation of apoptosis. The portion of the dystrophically changed cells increased from 9.9% to 63.7%, and the cells with apoptosis hallmarks from 6.3% to 14%. The results of this study suggest KillerRed as a potential genetically encoded photosensitizer for photodynamic therapy of cancer.

Flavoprotein miniSOG as a genetically encoded photosensitizer for cancer cells

BACKGROUND Genetically encoded photosensitizers are a promising optogenetic instrument for light-induced production of reactive oxygen species in desired locations within cells in vitro or whole body in vivo. Only two such photosensitizers are currently known, GFP-like protein KillerRed and FMN-binding protein miniSOG. In this work we studied phototoxic effects of miniSOG in cancer cells. METHODS HeLa Kyoto cell lines stably expressing miniSOG in different localizations, namely, plasma membrane, mitochondria or chromatin (fused with histone H2B) were created. Phototoxicity of miniSOG was tested on the cells in vitro and tumor xenografts in vivo. RESULTS Blue light induced pronounced cell death in all three cell lines in a dose-dependent manner. Caspase 3 activation was characteristic of illuminated cells with mitochondria- and chromatin-localized miniSOG, but not with miniSOG in the plasma membrane. In addition, H2B-miniSOG-expressing cells demonstrated light-induced activation of DNA repair machinery, which indicates massive damage of genomic DNA. In contrast to these in vitro data, no detectable phototoxicity was observed on tumor xenografts with HeLa Kyoto cell lines expressing mitochondria- or chromatin-localized miniSOG. CONCLUSIONS miniSOG is an excellent genetically encoded photosensitizer for mammalian cells in vitro, but it is inferior to KillerRed in the HeLa tumor. GENERAL SIGNIFICANCE This is the first study to assess phototoxicity of miniSOG in cancer cells. The results suggest an effective ontogenetic tool and may be of interest for molecular and cell biology and biomedical applications.

Light-induced blockage of cell division with a chromatin-targeted phototoxic fluorescent protein

Proteins of the GFP (green fluorescent protein) family are widely used as passive reporters for live cell imaging. In the present study we used H2B (histone H2B)-tKR (tandem KillerRed) as an active tool to affect cell division with light. We demonstrated that H2B-tKR-expressing cells behave normally in the dark, but transiently cease proliferation following green-light illumination. Complete light-induced blockage of cell division for approx. 24 h was observed in cultured mammalian cells that were either transiently or stably transfected with H2B-tKR. Illuminated cells then returned to normal division rate. XRCC1 (X-ray cross complementing factor 1) showed immediate redistribution in the illuminated nuclei of H2B-tKR-expressing cells, indicating massive light-induced damage of genomic DNA. Notably, nondisjunction of chromosomes was observed for cells that were illuminated during metaphase. In transgenic Xenopus embryos expressing H2B-tKR under the control of tissue-specific promoters, we observed clear retardation of the development of these tissues in green-light-illuminated tadpoles. We believe that H2B-tKR represents a novel optogenetic tool, which can be used to study mitosis and meiosis progression per se, as well as to investigate the roles of specific cell populations in development, regeneration and carcinogenesis in vivo.

Intracellular pH imaging in cancer cells in vitro and tumors in vivo using the new genetically encoded sensor SypHer2.

BACKGROUND Measuring intracellular pH (pHi) in tumors is essential for the monitoring of cancer progression and the response of cancer cells to various treatments. The purpose of the study was to develop a method for pHi mapping in living cancer cells in vitro and in tumors in vivo, using the novel genetically encoded indicator, SypHer2. METHODS A HeLa Kyoto cell line stably expressing SypHer2 in the cytoplasm was used, to perform ratiometric (dual excitation) imaging of the probe in cell culture, in 3D tumor spheroids and in tumor xenografts in living mice. RESULTS Using SypHer2, pHi was demonstrated to be 7.34±0.11 in monolayer HeLa cells in vitro under standard cultivation conditions. An increasing pHi gradient from the center to the periphery of the spheroids was displayed. We obtained fluorescence ratio maps for HeLa tumors in vivo and ex vivo. Comparison of the map with the pathomorphology and with hypoxia staining of the tumors revealed a correspondence of the zones with higher pHi to the necrotic and hypoxic areas. CONCLUSIONS Our results demonstrate that pHi imaging with the genetically encoded pHi indicator, SypHer2, can be a valuable tool for evaluating tumor progression in xenograft models. GENERAL SIGNIFICANCE We have demonstrated, for the first time, the possibility of using the genetically encoded sensor SypHer2 for ratiometric pH imaging in cancer cells in vitro and in tumors in vivo. SypHer2 shows great promise as an instrument for pHi monitoring able to provide high accuracy and spatiotemporal resolution.

Cross-polarization optical coherence tomography for early bladder-cancer detection: statistical study.

The capabilities of cross-polarization optical coherence tomography (CP OCT) for early bladder-cancer detection are assessed in statistical study and compared with the traditional OCT. Unlike the traditional OCT that demonstrates images only in copolarization, CP OCT acquires images in cross-polarization and copolarization simultaneously. 116 patients with localized flat suspicious lesions in the bladder were enrolled, 360 CP OCT images were obtained and analyzed. CP OCT demonstrated sensitivity 93.7% (vs. 81.2%, <0.0001), specificity 84% (vs. 70.0%, <0.001) and accuracy 85.3% (vs. 71.5%, <0.001) in detecting flat malignant bladder lesions, which is significantly better than with the traditional OCT. Higher diagnostic efficacy of CP OCT in detecting early bladder cancer is associated with the ability to detect changes in epithelium and connective tissues.