Yuan Lin, Jin Fangping, Zeng Zebing, Liu Chengbin, Luo Shenglian, Wu Jishan
State Key Laboratory of Chemo/Biosensing and Chemometrics , College of Chemistry and Chemical Engineering , Hunan University , Changsha 410082 , P. R. China . Email:
Department of Chemistry , National University of Singapore , 3 Science Drive 3 , 117543 , Singapore.
Chem Sci. 2015 Apr 16;6(4):2360-2365. doi: 10.1039/c4sc03883e. Epub 2015 Jan 28.
Two-photon excitation (TPE) probe-based fluorescence imaging has become one of the most attractive diagnostic techniques to investigate biomolecules and biological events in live cells and tissues. At the current stage most of the TPE-based sensing is reflected by fluorescence intensity changes. Nevertheless the mere altering of intensity could be facilely affected by ambient conditions. On the other hand, TPE probes based on an intramolecular charge transfer (ICT) strategy could solve this problem to some extent with a morphology change-induced emission shift. However their applications are yet constrained due to the inherent limitation of ICT, the high degree of overlap of two emissions bands and shifts of the TPE maxima. To achieve the desired TPE-based sensing and to circumvent the problems stated above, we adapted a Förster resonance energy transfer (FRET) strategy to develop small molecule ratiometric TPE fluorescent probes. Our FRET-based ratiometric TPE fluorescent probe displays a remarkable emission shift (up to 125 nm) with two well-resolved emission bands. Hence the ratio of these two emission bands could enable the measurement of fluorescence changes more accurately, thus further improving imaging in live cells and deep tissues. To the best of our knowledge, the current reported probe has the largest emission shift among all the small molecule ratiometric TPE fluorescent probes while the maximum TPE wavelength remains unchanged. This work has provided a FRET approach to fabricate novel small molecule ratiometric TPE fluorescent probes that improve imaging in deep tissues.
基于双光子激发（TPE）探针的荧光成像已成为研究活细胞和组织中生物分子及生物事件最具吸引力的诊断技术之一。在现阶段，大多数基于TPE的传感是通过荧光强度变化来体现的。然而，强度的单纯改变很容易受到环境条件的影响。另一方面，基于分子内电荷转移（ICT）策略的TPE探针可以通过形态变化引起的发射光谱位移在一定程度上解决这个问题。然而，由于ICT的固有局限性、两个发射带的高度重叠以及TPE最大值的位移，它们的应用仍然受到限制。为了实现基于TPE的理想传感并规避上述问题，我们采用了Förster共振能量转移（FRET）策略来开发小分子比率型TPE荧光探针。我们基于FRET的比率型TPE荧光探针显示出显著的发射光谱位移（高达125 nm），有两个分辨良好的发射带。因此，这两个发射带的比率能够更准确地测量荧光变化，从而进一步改善活细胞和深部组织中的成像。据我们所知，目前报道的探针在所有小分子比率型TPE荧光探针中具有最大的发射光谱位移，而最大TPE波长保持不变。这项工作提供了一种FRET方法来制造新型小分子比率型TPE荧光探针，改善深部组织中的成像。
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