Optimization of high-sensitivity fluorescence detection.

Abstract

We present general expressions for the number of photons emitted by a fluorescent chromophore as a function of the intensity and the duration of illumination. The aim is to find optimal conditions for detecting fluorescent molecules in the presence of both ground-state depletion and photodestruction. The key molecular parameters are the absorption coefficient epsilon, the excited singlet-state lifetime tau f, the excited triplet-state decay rate kT, the intersystem crossing rate kI, and the intrinsic photodestruction time tau d. When only singlet saturation and photochemistry are important, the signal-to-noise ratio depends on two fundamental variables: k, the ratio of the absorption rate ka to the observed fluorescence decay rate kf, and tau, the ratio of the duration of illumination taut to the intrinsic photodestruction time tau d. Equations are also developed for the more complicated cases when triplet formation and photochemistry are important. This theory was tested by measuring the fluorescence from a solution of beta-phycoerythrin flowed through a focused argon ion laser beam. The dependence of the fluorescence on the incident light intensity and the illumination time agrees well with the theoretical prediction for singlet saturation and photochemistry. The signal-to-noise ratio is optimal when the light intensity and the flow rate are adjusted so that both K and tau are close to unity (5 X 10(22) photons cm-2 s-1 and a transit time tau t of 700 mus). This analysis should be useful for optimizing fluorescence detection in DNA sequencing, chromatography, fluorescence microscopy, and single-molecular fluorescence detection.