Measurements of excited state lifetimes are instrumental to differentiate fluorophores located in different environments in a compartmentalized system. The lifetime of excited pyrene is 130 ns in water, and it is lengthened when pyrene is incorporated into different supramolecular host systems. The fits of the decays of the fluorescence of pyrene in the NaDC/pyrene (Figure ) and OTPh/NaDC/pyrene gels recovered one lifetime in each gel of 381 ± 1 ns and 380 ± 2 ns, respectively. These lifetimes are similar to values observed when pyrene is bound to the primary aggregates of bile salts (320-400 ns). The presence of only one lifetime for the pyrene emission indicates that pyrene is solely located in the NaDC primary aggregates of the gel, since a shorter lived component would have been detected if some pyrene molecules were in the aqueous phase of the gel. These results indicate that the local environment in the binding sites for pyrene in the NaDC gels is not affected by the addition of OTPh. The measurement of one lifetime for pyrene also suggests that no detectable excimer emission is present since a fast decay for the formation of excimers would have been observed (see below). However, the measurements of the I/III ratios and lifetimes cannot eliminate the possibility that OTPh interacts with the primary aggregates containing pyrene by forming structures where pyrene has a different accessibility to molecules residing in solution. Quenching studies are required to probe for the presence of such structures because these quenching experiments are a measure of the accessibility of the quencher to excited pyrene bound to NaDC aggregates. The iodide anion and nitromethane quenchers primarily reside in the aqueous phase of the NaDC and OTPh/NaDC gels. These molecules are known quenchers of the singlet excited state of pyrene in water ((1.1 ± 0.1) × 10 9 M -1 s -1 for iodide anions 57 and (7.1 ± 0.2) × 10 9 M -1 s -1 for nitromethane ) with rate constants close to the diffusion limit. Therefore, a lower quenching rate constant observed when pyrene is bound to bile salt aggregates is a measure of the association rate constant of the quencher to the aggregate containing pyrene. Nitromethane has an easier access to pyrene in sodium cholate aggregates than the iodide anions, because the iodide anions are repelled by the negative charge of the bile salt aggregates. An increase in ionic strength was shown to increase the accessibility of iodide anions to the primary aggregates of sodium cholate which was attributed to charge screening at the higher ionic strength. A similar behaviour is expected for NaDC. The quenching of the singlet excited state of pyrene in OTPh/NaDC hydrogels led to a decrease of the pyrene fluorescence intensity and occurred at lower concentrations of nitromethane than iodide anions (Figure ), showing that in the gel nitromethane has a better access to pyrene bound to the NaDC aggregates than iodide anions. Fluorescence spectra (λex = 335 nm) of pyrene (10 µM) in the OTPh (0.006 mg mL -1 )/NaDC (90 mM) hydrogel with increasing NaI concentrations (0-91 mM).