Emission by Fluorophores near a Surface

Although the probability of absorption of TIR evanescent energy by a fluorophore of given orientation decreases exponentially with distance z from a dielectric surface, the intensity of the fluorescence actually viewed by a detector varies with z in a much more complicated fashion. Both the angular pattern of the emitted radiation and the fluorescent lifetime are altered as a function of z by the proximity of the surface. 

These effects are not limited to fluorophores excited by TIR, although TIR excitation is necessarily near a surface. The discussion in this section is of relevance to any mode of excitation of surface-proximal fluorescence. In many of the experiments involving fluorescence in cell biology, the fluorophores are located near a surface. Usually, this surface is an aqueous buffer/glass or plastic interface upon which cells grow. Occasionally, the interface may have a thin coating on it, such as a synthetic polymer, a metal, or a lipid bilayer. 

Various aspects of fluorophore emission at surfaces have been investigated, particularly within the past two decades. For nondissipative surfaces (e.g., bare glass), the lifetime(14) and the inversely related total radiated power 15 for a single emission dipole, modeled as a continuous classical oscillator, have been calculated as functions of orientation and distance from the surface. The radiated intensity emitted from a continuous dipole oscillator has been calculated as a function of observation angle, dipole orientation, and distance.(16 21) 

We present here a condensed explanation and summary of the effects. A complete discussion can be found in a paper by Hellen and Axelrod(33) which directly calculates the amount of emission light gathered by a finite-aperture objective from a surface-proximal fluorophore under steady illumination. The effects referred to here are not quantum-chemical, that is, effects upon the orbitals or states of the fluorophore in the presence of any static fields associated with the surface. Rather, the effects are classical-optical, that is, effects upon the electromagnetic field generated by a classical oscillating dipole in the presence of an interface between any media with dissimilar refractive indices. Of course, both types of effects may be present simultaneously in a given system. However, the quantum-chemical effects vary with the detailed chemistry of each system, whereas the classical-optical effects are more universal. Occasionally, a change in the emission properties of a fluorophore at a surface may be attributed to the former when in fact the latter are responsible. 

Another feature of the simplest model that needs modification is the assumption of a fixed dipole amplitude. Because of the efficient capture of nonpropagating near fields by a surface, a fixed-amplitude dipole emits more power, the closer it moves to a surface. However, in steady-state fluorescence, the emitted power can only be as large as the (constant) absorbed power (or less, if the intrinsic quantum yield of the isolated fluorophore is less than 100%). Therefore, the fluorophore must be modeled as a constant -power (and variable-amplitude) dipole. Many of the earlier theoretical references listed above deal only with constant-amplitude dipoles, so their results must be considered to be an approximation. 

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