Abstract
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Objectives The controlled activation of a luminescent signal can be used to enhance the contrast in fluorescence-based molecular imaging approaches. However, background autofluorescence always remains a problem for imaging. The use of luminescence lifetime imaging can help separate long-living exogenous signals from short-living autofluorescence.
Methods Two dyes were selected based on their photophysical properties. Cy5 is very suitable for diagnostic questions in vitro and in vivo. Ir(ppy)3 has a long luminescence lifetime. By conjugating the two fluorophores through a cleavable disulfide linker, an enzymatically activatable compound was synthesized. The photophysical properties in both ‘conjugated’ and ‘cleaved’ form were studied. Cysteamine was used for activation in solution; this was followed by fluorescence spectrometry. Activation in cells was studied in 4T1 murine breast cancer cells. Confocal microscopy was used to locate the signal of both fluorophores and a dedicated FLIM set-up was used for lifetime measurements.
Results Upon conjugation of the two dyes, the compound becomes optically silent; a combination of energy transfer and spin-orbit coupling leads to signal quenching of both dyes. Cleavage of the disulfide bond leads to activation of luminescent signals, both in solution (>200-fold increase in intensity) and in vitro. This way simultaneous activation of two signals is achieved, one very bright signal (Cy5) and a long luminescence lifetime signal (Ir(ppy)3). Confocal microscopy localized this signal activation in the lysosomes. FLIM showed a clear lifetime dependance of the activatable signal, whereby activation led to a two-fold increase in lifetime, going from 45 ns to 90 ns.
Conclusions This initial feasibility study has shown that integrated fluorescence signal activation with lifetime imaging can help provide more detailed information about local enzymatic activities.
Research Support This research was partially supported by an NWOnano Grant (No. STW 11435).