Fluorescence In Vivo Endomicroscopy
Figure 4: (A) Histoarchitecture of rat lung stained with acridine orange, 0.05% i.v. Ricinus communis lectin in bleomycin induced lung injury model. (B) Enhanced parenchymal thickness (blue and white arrows) together with macrophage and metaplastic epithelial cell “big cell” staining (red arrow). Image published with permis- sion from Dr. Olivier Lesur, University of Sherbrooke, Canada.
patients undergoing brain tumor surgeries was used to evaluate volumetric images of cytoarchitecture and the course of abnor- mal vasculature in 31 human tumors, including meningiomas, gliomas, and pituitary adenomas [50]. Tese findings provided increased spatial understanding of tumor cellular architecture and related structures compared with two-dimensional (2-D) classical procedures. FIVE has an added advantage, as digital biopsy allows real-time transfer of images from the operating room to virtually anywhere in the world. An FDA-approved clinical FIVE neurosurgical system (CONVIVO) has been used intraoperatively to examine brain tumor biopsied ex vivo (Figure 6). Trials currently have been extended in several clinical centers to assess the feasibility of the system for in vivo neurosurgical application, which are showing promising inte- gration into the neurosurgical-pathology workflow. Parallel with their clinical findings, Belykh et al. also
described FIVE imaging in mouse brain and confirmed the histoarchitecture in 3-D volumetric examination [50]. Optical sectioning with FIVE allowed extensive visualization of tumor size, shape, location, and orientation. In a similar glioblastoma study of murine brain cell shape and tissue architecture, San- kar reported well-correlated FIVE and histology images [51]. Peyre also imaged mouse brain meningioma using FIVE, cap- turing accurate imaging of meningothelial and fibroblastic mouse meningiomas, as well as malignant meningiomas that corresponded with pathological findings [52]. Tis study also showed a sharp definition of the brain-tumor interface and enabled identification of embedded nerves, vessels, and an extension of tumor along Virchow–Robin spaces into adjacent tissue. A similar study by Fenton et al. reported Alexa Fluor– labeled, tyrosine-related protein-2 antibodies for imaging
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mouse glioma cells [53]. Martirosyan et al. extended their acri- dine orange histoarchitecture glioma work in rats and normal porcine brains. In a further study, they inoculated 13 rats with either green fluorescent protein-labeled U251 cells or epider- mal growth factor receptor overexpressing F98 cells, and they imaged brain tumors with FIVE, benchtop confocal micros- copy, and standard H&E staining [54]. Fluorescein isothiocya- nate-conjugated EGFR fluorescent antibody (FITC-EGFR) was applied for contrast of F98 cells, while GFP-labeled U251 cells were self-fluorescent. FIVE visualized GFP-labeled tumors as individual satellite cells within peritumoral tissue, a definitive tumor border, and subcellular structures. Both the markers and their tumor regions correlated with standard confocal imaging and histology (Figure 7). In a recent study contributing to FDA approval of a FIVE
system for clinical neurosurgical use, Belykh et al. continued their experiments with the second-generation FIVE imaging and demonstrated that the second-generation FIVE had higher resolution and contrast and offered ease of operation with automated contrast and image metadata analysis [50]. FIVE has also been used in studies of small vascular structures in altered blood flow states, and to assess the characteristics of tumor blood vessels (Figure 8). Liver. FIVE, with a sterile sheath, has been used for imag-
ing surgically exposed human liver. Although basic features of the microvasculature can be imaged with FIVE and fluo- rescein staining, histoarchitecture of liver cannot be visual- ized as effectively due to the thick liver capsule [55]. Hence, a prototype FIVE device with near infrared imaging (780 nm) was used with indocyanine green as a contrast agent [56]. Tis follow-up trial overcame the previous challenges and
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