Another Look at Magic-Angle-Detected Fluorescence and Emission Anisotropy Decays in Fluorescence Microscopy

摘要

It is shown that in contrast to a traditional fluorescence spectroscopy with the parallel beams of light, in which the kinetic fluorescence decays are collected at the so-called magic-angle of theta_(mag) = 54.7~(deg), in the fluorescence microscopy, the value of the magic-angle depends on the numerical aperture (NA) of a microscope objective and on the refractive index (n) of an immersion liquid used. Two methods enabling the determination of the magic-angle values corresponding to different values of NA, are discussed. It is shown that theta_(mag) changes from a value of 54.7~(deg) at the NA ->0, to a value of 45~(deg) with NA -> 1. Also in contrast to a traditional fluorescence spectroscopy, in the fluorescence microscopy the term I_(||)(t) + 2I_(perpendicular) (t) does not represent the total fluorescence intensity I_(tot)(t), because the resulting fluorescence decay I_(||)(t) + 2I_(perpendicular)(t) is contributed by the dynamic evolution of excited fluorophores. A correctly defined total fluorescence intensity solely represents the kinetic evolution of excited fluorophores, and in the fluorescence microscopy it equals I_(tot)(t) = 3I_(mag)(t), where I_(mag)(t) represents the fluorescence intensity detected at theta_(mag) corresponding to a particular NA value. If the correct (true) decay of I_(tot)(t) is substituted into the denominator in the expression for the emission anisotropy r(t), r(t) is a (multi)exponential function of time and it accounts for the high-aperture excitation-detection conditions.