Using POPOP's viscosity-dependent lifetime as a picosecond resolution standard in near-UV fluorescence lifetime imaging microscopy

Abstract

Fluorescence lifetime imaging microscopy (FLIM) using near-UV excitation is being developed to probe endogenous fluorophores in cells and tissues. Here, we describe such a UV-FLIM system and present a useful method for measuring the fluorescence lifetime discrimination of the system using the viscosity dependent lifetime of 1,4-Bis(5-phenyloxazol-2-yl)benzene (POPOP). The time-domain UV-FLIM system employed a nitrogen laser (337.1 nm) fiber-optic coupled to an inverted microscope. Wide-field fluorescence images were obtained at controlled time delays with a 200 ps gated, intensified-CCD camera. Lifetimes were calculated from the intensity decay on a pixel-by-pixel basis. The system was capable of imaging endogenous fluorescence in living cells using UV excitation. POPOP is a nanosecond lifetime standard suitable for UV excitation (325-375 nm). Its single-exponential lifetime (1.4 ns in ethanol) is comparable to endogenous lifetime values measured in living cells. Increasing solvent viscosity via the incremental addition of glycerol produced a series of POPOP lifetime standards having a range of 1 ns. The FLIM system’s ability to discriminate lifetime differences of 50 ps was demonstrated using the POPOP series. Thus, POPOP’s viscosity dependent lifetime represents a useful and convenient resolution standard for UV-FLIM calibration.