Triplet imaging contrast by modulated excitation

A novel imaging method employing the different kinetics of the energetic states of fluorescent markers is presented, where the contrast depends on the triplet kinetics. An attractive feature of triplet states is their long lifetime, rendering them highly sensitive to the immediate environment of their fluorescent host molecules. They typically have a lifetime of the order of ~10us to 10ms, which is 3-6 orders of magnitude longer than the lifetimes of fluorescent singlet states. The concept exploits characteristic variations of population of these states for different modulated excitations. It is fully compatible with low time- resolution detection by a CCD camera and does not impose any constraints on the concentration or the fluorescence brightness of the sample molecules to be measured. As first proof of principle, we demonstrate how triplet parameters of various fluorophores in different environments can be exploited for imaging. As control, the triplet parameters have been measured by FCS experiments. This concept opens for imaging of fluorophore markers in biological samples, as a novel contrast method for visualizing subtle changes in the immediate microenvironment of the fluorophore markers.