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 .tau.t 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 B-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 .times. 1022 photons cm-2 s-1 and a transit time .tau.t of 700 .mu.s). This analysis should be useful for optimizing fluorescence detection in DNA sequencing, chromatography, fluorescence microscopy, and single-molecule fluorescence detection.