Metabolic Imaging of Urothelial Carcinoma by Simultaneous Autofluorescence Lifetime Imaging (FLIM) of NAD(P)H and FAD

Abstract

Abstract Purpose To evaluate a metabolic-imaging system based on the simultaneous recording of NAD(P)H and FAD lifetime images in both normal and urothelial carcinoma cells. Patients and Methods Two lasers with 375nm and 405 nm wavelengths were multiplexed to alternatingly excite NAD(P)H and FAD. One FLIM channel detects the NAD(P)H emission band while the other the FAD emission band. FLIM data were processed by SPCImage software. In both channels, the data analysis delivers images of the amplitude-weighted lifetime (tm), the components lifetimes (t1 and t2), amplitudes (a1 and a2), and the amplitude ratio (a1/a2). Moreover, it provides the fluorescence-lifetime redox ratio (FLIRR), a2NAD(P)H/a1FAD. Results Fifteen patients were prospectively enrolled in this study. All samples were of urinary bladder origin. After histopathological analysis, 9 patients were reported without urothelial malignancy and 6 with urothelial carcinoma. For data analysis, a normal cell sample was compared to a high-grade urothelial carcinoma sample. Overall, tm was shorter in normal cells (1.3 ns) than in tumor cells (1.6 ns). A well-defined difference was detected in a1NAD(P)H of normal (66.6%) vs. tumor cells (76%). a1FAD data showed that a1 was lower in normal cells (68%) as opposed to tumor cells (80%), indicating that they contain less bound FAD. FLIRR values were lower in normal cells (0.3) compared to tumor cells (0.5). Conclusions Normal and urothelial carcinoma cells were discriminated by the NAD(P)H tm and a1 values, a1FAD values, and FLIRR. We demonstrate the accurate performance of the FLIM system for discriminating normal urothelial cells from urothelial carcinoma.