Two-photon excitation of the fluorescent nucleobase analogues 2-AP and tC
Two-photon excitation of the fluorescent nucleobase analogues 2-AP and tC
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DOI：
10.1039/c2ra21881j
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发表时间：
2012-01-01
期刊：
影响因子：
3.9
通讯作者：
Magennis, S. W.
中科院分区：
文献类型：
--
作者：
Lane, R. S. K.;Magennis, S. W.
我们提出了双光子（2 P）激发测量两种常用的和市售的荧光核碱基类似物在水溶液中：环境敏感的腺嘌呤类似物2-氨基嘌呤（2-AP），它在UV区域发射，和环境不敏感的胞嘧啶类似物1，3-二氮杂-2-氧代吩噻嗪（TC），它在绿色区域发射。我们研究了自由2-AP的2 P激发，发现通过单光子（1 P）和三光子（3 P）激发获得相同的发射态。2-AP的2 P截面为0.2GM，与相关分子如NADH的2 P截面相似。我们通过1 P和2 P光谱和显微镜研究了三种单链DNA寡核苷酸中的tC。我们发现，相同的发射状态通过共振或非共振路径填充，2 P截面为1.5 GM。时间分辨荧光研究表明，TC存在于溶液中的两个物种，我们指定为氨基和亚氨基互变异构体。使用共聚焦显微镜，我们发现，可以实现类似的发射率为2 P激发，与1 P激发相比，但没有强烈的光漂白观察到在后者的情况下。虽然我们无法在单分子水平上检测到tC荧光，但这些结果表明核碱基类似物的多光子激发是成像应用的一种有前途的方法。
We present two-photon (2P) excitation measurements of two commonly-used and commercially available fluorescent nucleobase analogues in aqueous solution: the environment-sensitive adenine analogue 2-aminopurine (2-AP), which emits in the UV region, and the environment-insensitive cytosine analogue 1,3-diaza-2-oxophenothiazine (tC), which emits in the green region. We studied the 2P excitation of free 2-AP and found that the same emissive states are accessed as via one-photon (1P) and three-photon (3P) excitation. The 2P cross section of 2-AP was 0.2 GM, which is similar to that of related molecules such as NADH. We studied tC incorporated in three single-stranded DNA oligonucleotides by 1P and 2P spectroscopy and microscopy. We found that the same emissive states were populated via either the resonant or non-resonant pathway with a 2P cross section of 1.5 GM. Time-resolved fluorescence studies suggested that tC exists in solution as two species, which we assigned as the amino and imino tautomers. Using confocal microscopy, we found that similar emission rates could be achieved for 2P excitation, as compared with 1P excitation, but without the strong photobleaching observed in the latter case. Although we were unable to detect tC fluorescence at the single-molecule level, these results indicate that multiphoton excitation of nucleobase analogues is a promising approach for imaging applications.