Triplet State Imaging

We present a functional imaging method based on triplet state kinetics. The triplet state of many known fluorophores shows 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. By means of a modulated excitation scheme of pulses with varying duty cycles, the triplet state population can be controlled: large duty cycles lead to a highly populated triplet state and hence a lower populated excited singlet state. This singlet-triplet interation translates into a diminished fluorescence emission, which can be detected using any standard CCD detector technology. We realised an imaging setup allowing extraction of triplet lifetime maps based on a whole field illumination and detection. 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 rapid 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. These results demonstrate the robustness of the setup to extract triplet lifetimes against high concurrent bleaching components. First imaging of bioassays will be presented indicating the high potential for functional and metabolic imaging in life cell applications.