Fluorescence Ratiometry and Fluorescence Lifetime Imaging: Using a Single Molecular Sensor for Dual Mode Imaging of Cellular Viscosity
Fluorescence Ratiometry and Fluorescence Lifetime Imaging: Using a Single Molecular Sensor for Dual Mode Imaging of Cellular Viscosity
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荧光比率测定和荧光寿命成像：使用单分子传感器进行细胞粘度的双模式成像
DOI：
10.1021/ja1104014
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发表时间：
2011-05-04
影响因子：
15
通讯作者：
Yang, Meng
中科院分区：
文献类型：
--
作者：
Peng, Xiaojun;Yang, Zhigang;Yang, Meng
细胞内黏度强烈影响质量和信号的运输、生物分子之间的相互作用以及活细胞中活性代谢物的扩散。荧光分子转子是近年来发展起来的用于测定溶液或生物流体粘度的试剂。由于活细胞的复杂性，为保证高可靠性和准确性，进行多模态粘度测定非常重要。报道了第一个能够双模式荧光成像（比率成像和荧光寿命成像）细胞内粘度的分子转子（RY3）。RY3是一种在中心（中位）被醛基（CHO）取代的五胺菁染料。在非粘性介质中，CHO基团的旋转通过非辐射过程引起内部转化。在粘稠或低温介质中抑制旋转导致强荧光（增加6倍）并延长荧光寿命（从200到1450 ps）。这种特殊设计的分子传感器具有两个吸收最大值（λ (abs)在乙醇中为400和613 nm）和两个发射最大值（蓝色，λ (em)为456nm，红色，在乙醇中为650 nm）。然而，只有红色发射对粘度或温度变化明显敏感，提供比率响应（12倍）以及大的伪斯托克斯位移（250 nm）。基于量子化学计算和低温H-1核磁共振谱，提出了一种机理。通过比值成像和荧光寿命成像（FLIM）可以清楚地观察到细胞内粘度的变化，并表现出一定的区域差异。虽然活细胞是复杂的，但观察到的两种成像程序之间的相关性为确定细胞内粘度提供了以前不可用的可靠性和准确性。
Intracellular viscosity strongly influences transportation of mass and signal, interactions between the bioma-cromolecules, and diffusion of reactive metabolites in live cells. Fluorescent molecular rotors are recently developed reagents used to determine the viscosity in solutions or biological fluid. Due to the complexity of live cells, it is important to carry out the viscosity determinations in multimode for high reliability and accuracy. The first molecular rotor (RY3) capable of dual mode fluorescence imaging (ratiometry imaging and fluorescence lifetime imaging) of intracellular viscosity is reported. RY3 is a pentamethine cyanine dye substituted at the central (meso-) position with an aldehyde group (CHO). In nonviscous media, rotation of the CHO group gives rise to internal conversion by a nonradiative process. The restraining of rotation in viscous or low-temperature media results in strong fluorescence (6-fold increase) and lengthens the fluorescence lifetime (from 200 to 1450 ps). The specially designed molecular sensor has two absorption maxima (lambda(abs) 400 and 613 nm in ethanol) and two emission maxima (in blue, lambda(em) 456 nm and red, 650 nm in ethanol). However it is only the red emission which is markedly sensitive to viscosity or temperature changes, providing a ratiometric response (12-fold) as well as a large pseudo-Stokes shift (250 nm). A mechanism is proposed, based on quantum chemical calculations and H-1 NMR spectra at low-temperature. Inside cells the viscosity changes, showing some regional differences, can be clearly observed by both ratiometry imaging and fluorescence lifetime imaging (FLIM). Although living cells are complex the correlation observed between the two imaging procedures offers the possibility of previously unavailable reliability and accuracy when determining intracellular viscosity.