OPTIMIZATION OF HIGH-SENSITIVITY FLUORESCENCE DETECTION
OPTIMIZATION OF HIGH-SENSITIVITY FLUORESCENCE DETECTION
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DOI：
10.1021/ac00216a012
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发表时间：
1990-09-01
影响因子：
7.4
通讯作者：
STRYER, L
中科院分区：
文献类型：
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作者：
MATHIES, RA;PECK, K;STRYER, L
我们给出了荧光生色团发射的光子数随光照强度和持续时间变化的通用表达式。目的是在基态耗尽和光破坏同时存在的情况下，找到检测荧光分子的最佳条件。关键的分子参数有吸收系数ε、激发单重态寿命τf、激发三重态衰减速率kT、系间窜越速率kI以及固有光破坏时间τd。当只有单重态饱和和光化学起重要作用时，信噪比取决于两个基本变量：k，即吸收速率ka与观测到的荧光衰减速率kf之比，以及τ，即光照持续时间τt与固有光破坏时间τd之比。当三重态形成和光化学起重要作用时，也针对更复杂的情况推导出了方程。通过测量流经聚焦氩离子激光束的B - 藻红蛋白溶液的荧光，对该理论进行了检验。荧光对入射光强度和光照时间的依赖性与单重态饱和和光化学的理论预测吻合良好。当调整光强度和流速使得k和τ都接近1时（5×10²²光子·cm⁻²·s⁻¹以及700 μs的通过时间τt），信噪比达到最佳。该分析对于优化DNA测序、色谱法、荧光显微镜以及单分子荧光检测中的荧光检测应该是有用的。
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.