Stanley Robert J, Hou Zhanjia, Yang Aiping, Hawkins Mary E
Department of Chemistry, Temple University, 201 Beury Hall, Philadelphia, Pennsylvania 19122, USA.
J Phys Chem B. 2005 Mar 3;109(8):3690-5. doi: 10.1021/jp0455982.
6MAP is a fluorescent analogue of adenine that undergoes Watson-Crick base pairing and base stacking in double-stranded DNA. The one-photon absorption spectrum of 6MAP is characterized by a maximum around 330 nm with moderate quantum yield fluorescence centered at about 420 nm. To take advantage of this probe for confocal and single-molecule microscopy, it would be advantageous to be able to excite the analogue via two photons. We report the first determination of the two-photon excitation cross section and spectrum for 6MAP from 614 to 700 nm. The power dependence of the fluorescence indicates that emission results from the absorption of two photons. The one-photon and two-photon emission line shapes are identical within experimental error. A study of the concentration dependence of the fluorescence yield for one-photon excitation shows no measurable quenching up to about 5 microM. The maximum in the two-photon excitation spectrum gives a two-photon cross section, delta(TPE), of 3.4 +/- 0.1 Goeppert-Mayer (G.M.) at 659 nm, which correlates well with the one-photon absorption maximum. This compares quite favorably with cross sections of various naturally fluorescent biological molecules such as flavins and nicotiamide. In addition, we have also obtained the two-photon-induced fluorescence emission spectrum of quinine sulfate. It is approximately the same as that for one-photon excitation, suggesting that two-photon excitation of quinine sulfate may be used for calibration purposes.
6MAP是腺嘌呤的一种荧光类似物，它在双链DNA中进行沃森-克里克碱基配对和碱基堆积。6MAP的单光子吸收光谱的特征是在330nm左右有一个最大值，中等量子产率的荧光集中在约420nm处。为了将这种探针用于共聚焦和单分子显微镜，能够通过双光子激发该类似物将是有利的。我们报道了首次测定6MAP在614至700nm范围内的双光子激发截面和光谱。荧光的功率依赖性表明发射是由两个光子的吸收引起的。在实验误差范围内，单光子和双光子发射线形是相同的。对单光子激发荧光产率的浓度依赖性研究表明，在约5 microM以下没有可测量的猝灭。双光子激发光谱中的最大值给出了在659nm处的双光子截面δ(TPE)为3.4±0.1戈培尔-迈耶(G.M.)，这与单光子吸收最大值很好地相关。这与各种天然荧光生物分子(如黄素和烟酰胺)的截面相比非常有利。此外，我们还获得了硫酸奎宁的双光子诱导荧光发射光谱。它与单光子激发的光谱大致相同，表明硫酸奎宁的双光子激发可用于校准目的。
