Triplet Imaging by Modulated Excitation

We present a functional imaging method based on triplet state kinetics. The triplet state of many known fluorophores show a triplet lifetime typically in the order of ~10e-6 – 10e-3 s, 3-6 orders of magnitude longer than the associated fluorescence singlet lifetime. As a consequence triplet state sensing is more exposed to the microenvironment of fluorophores and can be used as a functional molecular antenna. Using an adaptive modulated excitation scheme, these long living triplet state dynamics are accessible by standard CCD detector technology. We built an imaging setup allowing extraction of triplet lifetime maps. Effective correction of the competitive bleaching component is achieved by simultaneous monitoring of bleaching. The signal processing is based on a linear regressor analysis enabling extraction of the triplet state lifetime. As a first proof of principle, we investigated oxygen as a triplet state quencher on a sample of Tetramethylrhodamine (TMR) printed on a glass cover slide by soft lithography. First bioassays will be presented indicating the high potential for functional and metabolic imaging in life cell applications.