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首页> 美国卫生研究院文献>Biophysical Journal >Cytoplasmic viscosity near the cell plasma membrane: translational diffusion of a small fluorescent solute measured by total internal reflection-fluorescence photobleaching recovery.
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Cytoplasmic viscosity near the cell plasma membrane: translational diffusion of a small fluorescent solute measured by total internal reflection-fluorescence photobleaching recovery.

机译：细胞质膜附近的细胞质粘度：通过全内反射-荧光光漂白恢复测量的一种小的荧光溶质的平移扩散。

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Total internal reflection-fluorescence recovery after photobleaching (TIR-FRAP) was applied to measure solute translational diffusion in the aqueous phase of membrane-adjacent cytoplasm. TIR fluorescence excitation in aqueous solutions and fluorescently labeled cells was produced by laser illumination at a subcritical angle utilizing a quartz prism; microsecond-resolution FRAP was accomplished by acousto-optic modulators and electronic photomultiplier gating. A mathematical model was developed to determine solute diffusion coefficient from the time course of photobleaching recovery, bleach time, bleach intensity, and evanescent field penetration depth; the model included irreversible and reversible photobleaching processes, with triplet state diffusion. The validity and accuracy of TIR-FRAP measurements were first examined in aqueous fluorophore solutions. Diffusion coefficients for fluorescein isothiocyanate-dextrans (10-2000 kDa) determined by TIR-FRAP (recovery t1/2 0.5-2.2 ms) agreed with values measured by conventional spot photobleaching. Model predictions for the dependence of recovery curve shape on solution viscosity, bleach time, and bleach depth were validated experimentally using aqueous fluorescein solutions. To study solute diffusion in cytosol, MDCK epithelial cells were fluorescently labeled with the small solute 2',7'-bis-2-carboxyethyl-5-carboxyfluorescein-acetoxymethyl-ester (BCECF). A reversible photobleaching process (t1/2 approximately 0.5 ms) was identified that involved triplet-state relaxation and could be eliminated by triplet-state quenching with 100% oxygen. TIR-FRAP t1/2 values for irreversible BCECF bleaching, representing BCECF translational diffusion in the evanescent field, were in the range 2.2-4.8 ms (0.2-1 ms bleach times), yielding a BCECF diffusion coefficient 6-10-fold less than that in water. These results establish the theory and the first experimental application of TIR-FRAP to measure aqueous-phase solute diffusion, and indicate slowed translational diffusion of a small solute in membrane-adjacent cytosol.
机译：光漂白后的全内反射-荧光恢复（TIR-FRAP）用于测量溶质在膜邻近细胞质水相中的平移扩散。水溶液和荧光标记细胞中的TIR荧光激发是通过使用石英棱镜在亚临界角下进行激光照射产生的；微秒分辨率FRAP是通过声光调制器和电子光电倍增器选通完成的。建立了一个数学模型，根据光漂白恢复的时间过程，漂白时间，漂白强度和，逝场穿透深度确定溶质扩散系数；该模型包括不可逆和可逆的光漂白过程，以及三重态扩散。首先在荧光团水溶液中检查了TIR-FRAP测量的有效性和准确性。通过TIR-FRAP（回收率t1 / 2 0.5-2.2 ms）测定的异硫氰酸荧光素-右旋糖苷（10-2000 kDa）的扩散系数与常规点光漂白法测得的值相符。使用荧光素水溶液通过实验验证了恢复曲线形状对溶液粘度，漂白时间和漂白深度的依赖性的模型预测。为了研究溶质在细胞质中的扩散，用小的溶质2'，7'-双-2-羧乙基-5-羧基荧光素-乙酰氧基甲基酯（BCECF）对MDCK上皮细胞进行荧光标记。已确定可逆的光漂白过程（t1 / 2约为0.5 ms）涉及三重态弛豫，可以通过用100％氧气进行三重态淬灭来消除。 BIRCF不可逆漂白的TIR-FRAP t1 / 2值代表，逝场中的BCECF平移扩散，范围为2.2-4.8 ms（漂白时间为0.2-1 ms），产生的BCECF扩散系数比BECCF扩散系数低6-10倍在水中。这些结果建立了TIR-FRAP测量水相溶质扩散的理论和首次实验应用，并表明小溶质在膜邻近细胞质中的翻译扩散减慢。

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