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Multi-Photon Microscopy: A Potential Application for Future Multi-Photon Endoscopy and Non-Invasive Biopsy.
Michael J. Schwartz, David Hwang, Warren R. Zipfel, Rebecca M. Williams, Marcus Loo, Watt W. Webb, Douglas S. Scherr
Department of Urology, New York Presbyterian Hospital, Weill Medical College of Cornell University
Department of Applied and Engineering Physics Cornell University
Introduction and objective: Two and three photon fluorescence imaging allows for real time observation of cellular events in live tissue. In particular, it has been used in animal models to image gene expression, track cell populations and patterns of cellular migration, monitor molecular therapeutics, and quantify tumor angiogenesis. We show efficacy of multi-photon microscopy (MPM) for imaging of murine tissue and for diagnosis of malignancy, for the first time, in human tissue.
Methods: Using MPM, we assessed mouse bladders and human bladder tumors in order to identify intrinsic fluorophores that would allow for accurate in vivo imaging. By performing ex vivo MPM of previously excised bladder tumors we developed sufficient imaging criteria for differentiation of malignant from normal urothelium.
Results: Using similar criteria developed for evaluation of Hematoxylin & Eosin stained tissue—degree of cellular atypia, mitotic figures, nuclear to cytoplasmic ratio, alteration of polarity—the application of MPM to human bladder tissue allows the operator to distinguish malignant from atypical from normal urothelium. (Figures 1,2)
Fig. 1 Multiphoton Images of a Mouse Urinary Bladder (ex vivo) (A) 20 microns deep. Blue pseudocolor is collagen second harmonic signal; yellow/white pseudocolor represents epithelial tissue of the bladder wall. (B) 120 microns deep. Epithelial cells can be clearly seen in the upper left-hand side. (C) Higher zoom image showing the epithelia and umbrella cells (large yellow cells on the lower edge).
Fig 2. Human Bladder Tumor Biopsy (ex vivo) Blue autofluorescent cells of low-grade transitional cell carcinoma stage Ta, grade 1, showing rounded homogeneous cell array of low nuclear/cytoplasmic ratio and some disorganization of cell layer structure. Images comparable to Hematoxylin & Eosin pathologist’s stained images. (All scale bars, 20 mm)
Conclusions: MPM is a viable technology for the imaging and diagnosis of urothelial carcinoma of the bladder. Currently it is necessary to perform an invasive biopsy of the bladder to make the diagnosis of TCC. Potential complications include bladder perforation, gross hematuria with clot retention, and need for reoperation. Using photonic crystal fibers for development of an endoscopic probe to image the bladder in vivo, the potential exists for the diagnosis and staging of urothelial carcinoma without the need for biopsy.