Luby-Phelps K, Mujumdar S, Mujumdar R B, Ernst L A, Galbraith W, Waggoner A S
University of Texas Southwestern Medical Center, Dallas 75235-9040.
Biophys J. 1993 Jul;65(1):236-42. doi: 10.1016/S0006-3495(93)81075-0.
Two homologous indocyanine dyes, Cy3.18 and Cy5.18, can be used as a ratio pair for fluorometric determination of solvent viscosity. Succinimidyl ester derivatives of these dyes can be attached to inert carrier macromolecules, such as Ficoll 70, for measurement of intracellular or intravesicular solvent viscosity. When the viscosity of the solvent was varied by various methods, the fluorescence intensity ratio (Cy3/Cy5) in a mixture of Cy3.18-Ficoll 70 (Cy3F70) and Cy5.18-Ficoll 70 (Cy5F70) in solution was found to be solely a function of solvent viscosity and was insensitive to other solvent parameters such as dielectric constant, temperature, and the ability of the solvent to form hydrogen bonds. Most important, it was insensitive to the presence of large macromolecules, such as proteins, which increase the shear viscosity but have little effect on solvent viscosity. Following microinjection into the cytoplasm of living tissue culture cells, no binding of Cy3F70 or Cy5F70 to intracellular components was detected by fluorescence recovery after photobleaching. Fluorescence intensity ratio imaging of Cy3F70 and Cy5F70 in non-motile interphase CV1 and PtK1 cells showed that the solvent viscosity of cytoplasm was not significantly different from water and showed no spatial variation.
两种同源的吲哚菁染料Cy3.18和Cy5.18可作为比率对用于荧光法测定溶剂粘度。这些染料的琥珀酰亚胺酯衍生物可连接到惰性载体大分子上，如聚蔗糖70，用于测量细胞内或囊泡内的溶剂粘度。当通过各种方法改变溶剂粘度时，发现溶液中Cy3.18 - 聚蔗糖70（Cy3F70）和Cy5.18 - 聚蔗糖70（Cy5F70）混合物中的荧光强度比率（Cy3/Cy5）仅为溶剂粘度的函数，对其他溶剂参数如介电常数、温度以及溶剂形成氢键的能力不敏感。最重要的是，它对大分子如蛋白质的存在不敏感，蛋白质会增加剪切粘度但对溶剂粘度影响很小。在显微注射到活组织培养细胞的细胞质中后，通过光漂白后的荧光恢复未检测到Cy3F70或Cy5F70与细胞内成分的结合。在非运动性间期CV1和PtK1细胞中对Cy3F70和Cy5F70进行荧光强度比率成像显示，细胞质的溶剂粘度与水没有显著差异，且没有空间变化。
