ABSTRACT
A²er a ¦uorophore has been excited to an upper electronic energy level, it may spontaneously decay back to the ground state either radiatively, with a rate constant, Γ, or non-radiatively, with a rate constant, k, as represented in Figure 28.1. ™e quantum eŸ- ciency, η, of the ¦uorescence process is de›ned as the ratio of the number of ¦uorescent photons emitted compared to the number of excitation photons absorbed and is equal to the radiative decay rate, Γ, divided by the total decay rate, Γ + k, as indicated. In general, the quantum eŸciency is sensitive to the local ¦uorophore environment since it can be ažected by any factors that
change the molecular electronic con›guration or de-excitation pathways and, therefore, the radiative or non-radiative decay rates. Such environmental factors can include the local chemical environment, for example, pH, calcium, or oxygen concentration; the proximity of other ¦uorophores that result in, for example, quenching or FRET; or the local physical environment, for example, refractive index, temperature, viscosity, electric ›eld, etc. Determining the quantum eŸciency requires knowledge of the ¦uorophore concentration and the photon excitation and detection eŸciencies. For many biological samples, however, quantitative measurements of intensity are compromised by optical scattering, internal reabsorption of ¦uorescence (inner ›lter ežect), and background ¦uorescence from other (endogenous) ¦uorophores present in a sample. It can therefore be highly challenging to accurately map variations in quantum eŸciency using ¦uorescence intensity imaging, particularly in biological tissue that is usually highly scattering and heterogeneous, o²en presenting multiple endogenous ¦uorophores.
