Functional Imaging of Colonic Mucosa With a Fibered Confocal Microscope for Real- Time In Vivo Pathology  Thomas D. Wang, Shai Friedland, Peyman Sahbaie, Roy Soetikno, Pei–Lin Hsiung, Jonathan T.C. Liu, James M. Crawford, Christopher H. Contag  Clinical Gastroenterology and Hepatology  Volume 5, Issue 11, Pages 1300-1305 (November 2007) DOI: 10.1016/j.cgh.2007.07.013 Copyright © 2007 AGA Institute Terms and Conditions

Figure 1 (A) The fibered confocal microscope (blue light from laser) passes through the instrument channel of a standard colonoscope, whose objective collects a wide field image under white-light illumination, (B) guiding placement for in vivo imaging of a polyp and normal mucosa. Clinical Gastroenterology and Hepatology 2007 5, 1300-1305DOI: (10.1016/j.cgh.2007.07.013) Copyright © 2007 AGA Institute Terms and Conditions

Figure 2 Movement of fluorescein in colonic mucosa. (A) Initial uptake by crypt (t = 0.08 s); (B) expansion from lumen toward basolateral aspect of epithelium and accumulation into focused points of fluorescence (dotted red circle labeled vesicle) (t = 0.67 s); (C) point of fluorescence enters the lamina propria (t = 0.75 s); (D) concurrent appearance of multiple points in the lamina propria (t = 4.5 s; scale bar, 20 μm). (E) Instantaneous radial speed of en face fluorescein movement through epithelium in the direction of the lumen to the lamina propria (peak occurring at t = 0.5 s). (F) Movement of 2 separate points of fluorescence within the lamina propria at 0.083-second time intervals. Clinical Gastroenterology and Hepatology 2007 5, 1300-1305DOI: (10.1016/j.cgh.2007.07.013) Copyright © 2007 AGA Institute Terms and Conditions

Figure 3 Steady state (t > 5 s) distribution of fluorescence in colon with corresponding histology (H&E) for (A) normal mucosa, (B) hyperplasia, (C) tubular adenoma, and (D) villous adenoma. Scale bar, 40 μm. The crypt lumens (l), apical (ap), and basolateral (bl) borders of epithelial cells (c), and lamina propria (lp) can be distinguished. Clinical Gastroenterology and Hepatology 2007 5, 1300-1305DOI: (10.1016/j.cgh.2007.07.013) Copyright © 2007 AGA Institute Terms and Conditions

Figure 4 Lamina propria:crypt contrast ratios of fluorescence intensities in steady state (t > 5 s) from neighboring sites of normal (n = 54) and lesional (n = 24 hyperplastic, n = 25 tubular adenoma, and n = 5 villous adenomas) mucosa from the same patient are shown in separate columns. ■, Normal; ◊, hyperplastic; ▵, tubular adenoma; ○, villous adenoma. Clinical Gastroenterology and Hepatology 2007 5, 1300-1305DOI: (10.1016/j.cgh.2007.07.013) Copyright © 2007 AGA Institute Terms and Conditions

Figure 5 Quantitative performance of functional parameter (lamina propria:crypt fluorescence contrast ratio) used to assess pathology and receiver-operator characteristic curve of tradeoffs for distinguishing (A and B) normal from lesional, (C and D) hyperplasia from adenoma, (E and F) tubular from villous adenoma. ■, Sensitivity; ⧫, specificity; ▲, positive predictive value; ●, negative predictive value; –, accuracy. Clinical Gastroenterology and Hepatology 2007 5, 1300-1305DOI: (10.1016/j.cgh.2007.07.013) Copyright © 2007 AGA Institute Terms and Conditions