MAPPING OF FLUORESCENCE ANISOTROPY IN LIVING CELLS BY RATIO IMAGING - APPLICATION TO CYTOPLASMIC VISCOSITY
MAPPING OF FLUORESCENCE ANISOTROPY IN LIVING CELLS BY RATIO IMAGING - APPLICATION TO CYTOPLASMIC VISCOSITY
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DOI：
10.1016/s0006-3495(90)82526-1
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发表时间：
1990-02-01
影响因子：
3.4
通讯作者：
VERKMAN, AS
中科院分区：
文献类型：
--
作者：
DIX, JA;VERKMAN, AS
结合到活细胞中的探针的稳态和时间分辨荧光特性提供了关于探针附近微环境的信息。我们扩展了对空间平均荧光各向异性(R)的研究，使用配备了激发和发射偏振器和图像分析系统的荧光显微镜来绘制掺入培养细胞中的r或无取向的荧光团。在此成像系统中，反射光或糖原散射溶液的r为0.98。在混合溶液和并排放置的细毛细管中，在0.01-0.35范围内测量荧光团的r值与用试管荧光法测得的r值相当。细胞质粘度(eta.)在Madin-Darby犬肾(MDCK)细胞和瑞士3T3成纤维细胞中，观察了胞浆探针2，7-双羧乙基-5(和6)-羧基荧光素(BCECF)和INDO-1的各向异性图像和时间分辨荧光衰减。使用脉冲光源和连接到荧光显微镜的门控探测器测量了纳秒寿命和各向异性衰减。细胞各向异性图像显示了r的两个不同区域：一个来自细胞质(r=0.144.+-。0.008)，第二次出现在间质区的时间较晚(r=0.08+-)。0.03)，代表被困在紧密连接下的BCECF。将各向异性值与细胞内寿命和水/甘油混合物的r和？/？f之间的校准结合在一起，得出？在23℃时10-13Cp的值。这些值假定几乎没有荧光团与细胞内组分结合，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的各向异性图，以检查流动性在细胞生理学中的作用。
Steady-state and time-resolved fluorescence properties of probes incorporated into living cells give information about the microenvironment near the probe. We have extended studied 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 or nonoriented fluorophores incorporated into cultured cells. With this imaging system, r for reflected light or glycogen scattering solutions was >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 anistotropy 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 lifetimes and the calibration between r and .eta./.sigma.f for water/glycerol mixtures, gave .eta. values of 10-13 cP at 23.degree.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.