Fluorescence-lifetime imaging of multiple fluorophores implemented in confocal microscopy

There exists a number of fluorophores whose lifetimes that vary with the properties of the surrounding medium, such as pH and calcium ion concentration. Lifetime information can be gathered in the form of lifetime images where each pixel value represents the fluorophore lifetime. By using confocal microscopy it is possible to record such lifetime information in three dimensions, thus recording 3-D lifetime images. We have built a confocal microscope that allows simultaneous lifetime imaging of two fluorophores. Two intensity-modulated laser beams are used as excitation sources, and fluorescence is detected using phase-sensitive lock-in technique. We have previously verified that this instrument can simultaneously record lifetime images of two fluorophores with good channel separation. Excitation wavelengths of 488 and 568 nm were used, and the fluorophores tested had lifetimes in the range 2.3 - 6.5 nanoseconds. With the present study we have started work on biomedical applications of lifetime imaging with the IMS technique. In this first stage we have concentrated on one parameter, namely pH. Using the pH sensitive fluorophore SNAFL-2, we found that the lifetime varied between 1 and 3.7 ns for a pH range of 5.0 - 10.0. Experiments with SNAFL-2 were also carried out on living cells. The signal quality in confocal lifetime imaging is also discussed.