DOUBLE-PULSE FLUORESCENCE LIFETIME MEASUREMENTS

It is demonstrated that fluorescence lifetimes in the nanosecond and p icosecond time-scale range can be observed with the recently proposed double-pulse fluorescence lifetime imaging technique (Muller et al., 1 995, 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 excitat ion of specific fluorophores and high pulse energies to obtain (partia l) 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 eff ect on the measurements of laser power fluctuations, which are inheren tly present in OPA systems, is presented. Measurements on bull; soluti ons of Rhodamine B and Rhodamine 6G in different solvents confirm the experimental feasibility of accessing short fluorescence lifetimes wit h this technique. Because signal detection does not require fast elect ronics, the technique can be readily used for fluorescence lifetime im aging in confocal microscopy, especially when using bilateral scanning and cooled CCD detection.