Double‐pulse fluorescence lifetime measurements

Abstract

It is demonstrated that fluorescence lifetimes in the nanosecond and picosecond time‐scale range can be observed with the recently proposed double‐pulse fluorescence lifetime imaging technique (Müller et al. , 1995, Double‐pulse fluorescence lifetime imaging in confocal microscopy. J. Microsc 177, 171–179). A laser source with an optical parametric amplifier (OPA) system is used to obtain short pulse durations needed for high time resolution, wavelength tunability for selective excitation of specific fluorophores and high pulse energies to obtain (partial) saturation of the optical transition. It is shown that fluorescence lifetimes can be determined correctly also with nonuniform saturation conditions over the observation area. A correction scheme for the effect on the measurements of laser power fluctuations, which are inherently present in OPA systems, is presented. Measurements on bulk solutions of Rhodamine B and Rhodamine 6G in different solvents confirm the experimental feasibility of accessing short fluorescence lifetimes with this technique. Because signal detection does not require fast electronics, the technique can be readily used for fluorescence lifetime imaging in confocal microscopy, especially when using bilateral scanning and cooled CCD detection.