Influence of Fluorescence Anisotropy on Fluorescence Intensity and Lifetime Measurement: Theory, Simulations and Experiments

Abstract

The significance of fluorescence anisotropy in fluorescence intensity and lifetime measurements, and erroneous measurements and interpretations resulting from its disregard, are thoroughly discussed, formulated and quantified. In all fluorescence-related measurements--including excitation and emission spectra, relative fluorescence intensity (FI), fluorescenc life time (FLT), fluorescence resonance energy transfer (FRET), etc., with the exception of fluorescence polarization and anisotropy--it is generally true that the higher the fluorescence anisotropy, the greater the distortion of fluorescence measurements. Quantifiable distortions occur when fluorescence measurements are conducted without considering the influence of fluorescence anisotropy. Here, this influence is described by numerous newly developed mathematical expressions which are simulated and experimentally confirmed utilizing single and binary fluorescent solutions of fluorophores with different spectroscopic characteristics. A marked agreement is shown between the theory and experimental data, clearly indicating the legitimacy of the physical suppositions and the mathematical expressions presented in this paper. Practical and instructive implications are discussed. The following findings are of special applicative importance: 1) the existence of an infinite number of couples of Magic Angles; 2) the deviation between two equally fluorescing particles having different fluorescence anisotropies; 3) the relation between the detected fluorescence intensity and anisotropy when measured under various setups of emission and excitation polarizers; 4) the dependence of the artificial normalized steady-state weight of a single-exponentially decaying fluorophore on its fluorescence anisotropy.

Time-averaged fluorescence intensity analysis in fluorescence fluctuation polarization sensitive experiments

“…The PBS buffer was used in order to maintain a constant pH in all measurements. Glycerol-concentration is known to change the fluorescein characterizations like FLT and quantum yield [40] but not change the absorbance and emission spectrum [30,53]. Viscosities of these solutions at 20 ° C were estimated at 1, 4, 10.8, 59.6 cP respectively, by a four-parameter correlation function [54].…”

“…That is:$I(t)=I_{\perp }(t)+I_{\left|\right|}(t)=2I_e(A+B{\sin }^2\phi (t))$whereas the total emission intensity measured from one channel, i.e. 2I || + 4I ⊥ or more simply I || + 2I ⊥ [30,40,41] is also θ dependent. However, in single fluorophore experiments where a large numerical aperture (NA) must be used, the total emission intensity is not always equivalent to I || + 2I ⊥ , as it is for narrow beam angles [42,43].…”

Fluorescence lifetime imaging (FLIM): Basic concepts and some recent developments

“…The value of z depends on the NA of the microscope objective, where 1 ≤ z ≤ 2 (z ≈ 1 for a high NA objective, z = 2 for a collimated beam) (Axelrod, 1979;Fisz, 2007aFisz, ,b, 2009Fixler et al, 2006;Ha et al, 1999;Koshioka et al, 1995;Yan and Marriott, 2003). Although a rigorous treatment of the effect of high NA objectives to ''see around'' the fluorophore and therefore collect all three emission components F x , F y , F z leads to a slightly more complex description than Eq.…”

Whole-Object Fluorescence Lifetime Setup for Efficient Non-Imaging Quantitative Intracellular Fluorophore Measurements

“…The FDTRM comprises two main stand-alone components: the optical system-an upright epi-fluorescent Zeiss microscope Z-1, and a modified frequency domain FLT and FAD ISS K-2 spectrofluorimeter. For FLT measurements, the excitation and emission polarizers of the FDTRM were configured to measure at a set of magic angles [18]. As shown in Fig.…”

“…In order to minimize inner filtration influence upon FLT [33], front face fluorescence measurements were performed. In order to simulate the viscosity conditions that exist inside the cell, measurements were performed in viscous media (90% glycerol in PBS v/v) that are known to reduce the FLT from water media [18,31].…”