A new dual-channel multifrequency fluorometer is described which employs a mode-locked argon-ion laser as a polarized excitation source. The laser produces high-frequency sinusoidal intensity modulations in the MHz to GHz regime which are used to simultaneously probe the fluorescent molecules. The resulting orthogonally polarized components of the emission are collected by two opposite matched detection channels. Because both the parallel [I∥(ω)] and perpendicular [I⊥(ω)] components of the emission are collected simultaneously and at all modulation frequencies, information about the molecules' rotational rate can be rapidly deduced. With the new instrument, rotational correlation times as short as 15 ps can be routinely determined with data-acquisition times as short as 10 ms. Commonly, however, we employ collection times of several seconds to permit the averaging of between 100 and 500 scans. Examples which demonstrate the utility of the new instrument include the determination of the rotational correlation times for rhodamine 6G, fluorescein, and rubrene as a function of solvent viscosity. In addition, results are presented for the resolution of rhodamine 6G associated with beta-cyclodextrin. In the beta-cyclodextrin studies, little spectral (excitation/emission) or temporal (fluorescence lifetime) change was noted upon association. However, a significant difference exists between rotational correlation times of the free fluorophore and the fluorophore included by beta-cyclodextrin. This difference enables the formation constant for the association to be determined, a measurement which would be impossible with conventional techniques.
Frank V. Bright, Curtis A. Monnig, and Gary M. Hieftje, "A New Dual-Channel Frequency-Domain Fluorometer for the Determination of Picosecond Rotational Correlation Times," Appl. Spectrosc. 42, 272-277 (1988)