Dix J A, Verkman A S
Department of Medicine, University of California, San Francisco 94143-0532.
Biophys J. 1990 Feb;57(2):231-40. doi: 10.1016/S0006-3495(90)82526-1.
Steady-state and time-resolved fluorescence properties of probes incorporated into living cells give information about the microenvironment near the probe. We have extended studies of spatially averaged fluorescence anisotropy (r) by using an epifluorescence microscope, equipped with excitation and emission polarizers and an image analysis system, to map r of nonoriented fluorophores incorporated into cultured cells. With this imaging system, r for reflected light or glycogen scattering solutions was greater than 0.98. Measurement of r over the range 0.01-0.35 for fluorophores in bulk solution and in thin capillary tubes placed side-by-side gave values equivalent to r measured by cuvette fluorometry. Cytoplasmic viscosity (eta) in Madin-Darby canine kidney (MDCK) cells and Swiss 3T3 fibroblasts was examined from anisotropy images and time-resolved fluorescence decay of the cytoplasmic probes 2,7-bis-carboxyethyl-5 (and 6)-carboxy-fluorescein (BCECF) and indo-1. Nanosecond lifetimes and anisotropy decay were measured using a pulsed light source and gated detector interfaced to the epifluorescence microscope. Anisotropy images of BCECF in MDCK cells revealed two distinct regions of r: one from the cytoplasm (r = 0.144 +/- 0.008) and a second appearing at late times from the interstitial region (r = 0.08 +/- 0.03), representing BCECF trapped beneath the tight junctions. Anisotropy values, taken together with intracellular life-times and the calibration between r and eta/tau f for water/glycerol mixtures, gave eta values of 10-13 cP at 23 degrees C. These values assume little fluorophore binding to intracellular components and are therefore upper limits to cytoplasmic viscosity. These data establish a new methodology to map anisotropy in intact cells to examine the role of fluidity in cellular physiology.
掺入活细胞中的探针的稳态和时间分辨荧光特性提供了有关探针附近微环境的信息。我们通过使用配备激发和发射偏振器以及图像分析系统的落射荧光显微镜，扩展了对空间平均荧光各向异性（r）的研究，以绘制掺入培养细胞中的非定向荧光团的r值。使用该成像系统，反射光或糖原散射溶液的r大于0.98。对于并排放置的大量溶液和细毛细管中的荧光团，在0.01-0.35范围内测量r，得到的值与通过比色杯荧光测定法测量的r值相当。通过细胞质探针2,7-双羧乙基-5（和6）-羧基荧光素（BCECF）和indo-1的各向异性图像以及时间分辨荧光衰减，研究了马德堡-达比犬肾（MDCK）细胞和瑞士3T3成纤维细胞中的细胞质粘度（eta）。使用与落射荧光显微镜相连的脉冲光源和门控探测器测量纳秒寿命和各向异性衰减。MDCK细胞中BCECF的各向异性图像显示了两个不同的r区域：一个来自细胞质（r = 0.144 +/- 0.008），另一个在后期出现在间质区域（r = 0.08 +/- 0.03），代表被困在紧密连接处下方的BCECF。各向异性值，连同细胞内寿命以及水/甘油混合物的r和eta/tau f之间的校准，在23℃下给出了10-13 cP的eta值。这些值假设荧光团与细胞内成分的结合很少，因此是细胞质粘度的上限。这些数据建立了一种新的方法来绘制完整细胞中的各向异性，以研究流动性在细胞生理学中的作用。
