Fluorescence lifetime imaging using a confocal laser scanning microscope

Abstract

Abstract The implementation of a fast fluorescence life‐time imaging method in a confocal laser scanning microscope is described. The set up utilizes a low‐power continuous wave (CW) argon ion laser equipped with an electro‐optic chopper producing nanosecond pulses with a repetition rate up to 25 MHz. A time‐gated detection technique enables the measurement of the lifetime of a pixel in 40 μs. The first confocal fluorescence lifetime contrast images are presented. Application of fluorescence lifetime imaging in multilabelling experiments for discrimination between different labels with overlapping emission bands, for probing the local environment of a fluorescent molecule, and for quantitative fluorescence are discussed.