Fluorescence lifetime based contrast imaging using variable period excitation pulse trains

The development of an experimental setup capable of contrasting fluorescent materials by their recombinative lifetimes in an imaging mode is discussed. Such materials might include molecular dyes and QDs. The system is comprised of a standard upright microscope fitted with an imaging CCD and a white light laser that illuminates a circular region within the field of view with variable period excitation pulse trains. Different fluorescent species within this region absorb the laser light and fluoresce with a recombination lifetime dependent on material composition and local environment. Species with differing fluorescent lifetimes can be distinguished in an imaging mode by their contrasting intensity response to the pulse train at the range of different pulse frequencies. The technique is discussed and applied to samples containing both CdTe (705 nm) and CdSe (611 nm) QDs showing contrast between long (70-100 ns) and (relatively) short (25-35 ns) lifetime within an image.