Accurate determination of the limiting anisotropy of rhodamine 101. Implications for its use as a fluorescence polarization standard.

The S1-S0 limiting anisotropy of a widely used fluorophore, rhodamine 101, is determined with unprecedented accuracy. From time-resolved and steady-state fluorescence measurements in several solvents, it is shown that the limiting anisotropy of rhodamine 101 is for all practical purposes equal to the theoretical one-photon fundamental anisotropy value of 2/5, both in rigid and in fluid media. This fact, along with the favorable chemical and photophysical properties of rhodamine 101, point to its use as a standard for fluorescence polarization measurements. It is also shown that if the excitation pulse can be considered a delta impulse with respect to the time scale of the anisotropy decay (but not necessarily to the time scale of the intensity decay), then no deconvolution procedure is needed for anisotropy decay analysis.

Fluorescence decays and photon propagation times.

The effect of the time of flight of the exciting and emitted photons on fluorescence decays is studied. Experimental results for a long lifetime molecule (coronene) are analysed according to the fluorescence decay law previously obtained by the authors. The developed model accounts well for the main features of the observations. The inclusion of photon propagation times is essential for a correct description of the fluorescence decays under the described circumstances.