Simplification in the Acquisition and Analysis of Fluorescence Decays Acquired with Polarized Emission for Time-Resolved Fluorescence Anisotropy Measurements

摘要

This study introduces a global fluorescence decay analysis that substantially simplifies the acquisition and analysis of time-resolved fluorescence decays acquired with a vertically polarized excitation and vertically (I-VV(t)) and horizontally (I-VH(t)) polarized emission for time-resolved fluorescence anisotropy (TRFA) measurements. TRFA measurements were conducted whereby the I-VV(t) and I-VH(t) fluorescence decays of a series of oligoquinolines labeled at one end with an oligo(phenylenevinylene) dye (OPV-Q(n) with n = 4, 7, 17, 24, 33) were acquired according to the standard protocol that is currently accepted in the scientific literature which involves toggling the emission polarizer before fitting linear combinations of the I-VV(t) and I-VH(t) decays or acquiring the I-VV(t) and I-VH(t) decays with static polarizers before fitting them globally. The rotational time (phi) and initial anisotropy (r(0)) retrieved from these analyses were identical within experimental error regardless of whether the decays were acquired with toggling or static polarizers and fitted according to the standard protocol or globally. These experimental results were further supported by retrieving the parameters used to generate mono-, bi-, and tri-exponential TRFAs from the global analysis of simulated I-VV(t) and I-VH(t) fluorescence decays which were found to match perfectly the values that were inputted. Together, these experiments and simulations demonstrated that the parameters describing any type of TRFA can be extracted directly from the analysis of the I-VV(t) and I-VH(t) fluorescence decays acquired with a standard time-resolved fluorometer, a substantial simplification compared to the protocols currently in place to determine the TRFA.