Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-171 77, Stockholm, Sweden.
J Fluoresc. 1994 Sep;4(3):259-64. doi: 10.1007/BF01878461.
Using a modified confocal fluorescence microscope and a CW argon laser, we have measured fluorescence bursts from diffusing single Rh6G molecules that clearly exceed the background intensity. The exact average number of molecules in the observable volume elements was measured directly via the fluorescence intensity autocorrelation function. This allowed us to estimate the probability of finding several molecules simultaneously in the volume element. A tradeoff between the number of detected fluorescence photons and the signal-to-background ratio was observed. In a volume element of 0.24 fl, 4 photoelectrons on average were detected from a molecule of Rh6G with a fluorescence-to-background ratio of 1000, while the volume element of 60 fl yielded on average 100 photoelectrons with a background of 25 counts. In fast single-molecule detection the intersystem crossing into the triplet state plays an important role, affecting the maximum emission rate from the molecule.
使用改良的共聚焦荧光显微镜和连续波氩激光器，我们测量了来自扩散的单个 Rh6G 分子的荧光爆发，其强度明显超过背景强度。通过荧光强度自相关函数，我们直接测量了可观察体积元中确切的平均分子数。这使我们能够估计在体积元中同时存在几个分子的概率。我们观察到检测到的荧光光子数量和信号与背景的比率之间存在权衡。在 0.24 fl 的体积元中，平均从 Rh6G 分子检测到 4 个光电子，荧光与背景的比率为 1000，而体积元为 60 fl 时，背景为 25 计数时平均产生 100 个光电子。在快速单分子检测中，系间窜跃进入三重态起着重要作用，影响分子的最大发射速率。
