Quenched coumarin derivatives as fluorescence lifetime phantoms for NADH and FAD

Two-photon fluorescence lifetime imaging is a versatile laboratory technique in the field of biophotonics and its importance is also growing in the field of in vivo diagnostics for medical purposes. After years of experience in dermatology, endoscopic implementations of the technique are now posing new technical challenges. To develop, test, and compare instrumental solutions for this purpose suitable reference samples have been devised and tested. These reference samples can serve as reliable NADH- and FAD-mimicking optical phantoms for 2-photon fluorescence lifetime imaging, as they can be prepared relatively easily with reproducible and stable characteristics for this quite relevant diagnostic technique. The reference samples (mixtures of coumarin 1 and coumarin 6 in ethanol with suitable amounts of 4-hydroxy-TEMPO) have been tuned to exhibit spectral and temporal fluorescence characteristics very similar to those of NADH and FAD, the two molecules most frequently utilized to characterize cell metabolism.