DIGITAL IMAGING FLUORESCENCE MICROSCOPY - SPATIAL HETEROGENEITY OF PHOTOBLEACHING RATE CONSTANTS IN INDIVIDUAL CELLS
DIGITAL IMAGING FLUORESCENCE MICROSCOPY - SPATIAL HETEROGENEITY OF PHOTOBLEACHING RATE CONSTANTS IN INDIVIDUAL CELLS
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DOI：
10.1083/jcb.100.4.1309
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发表时间：
1985-01-01
影响因子：
7.8
通讯作者：
SMITH, LC
中科院分区：
文献类型：
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作者：
BENSON, DM;BRYAN, J;SMITH, LC
光漂白和相关的光化学过程被认为是显微镜定量荧光的实验障碍。我们测量了活细胞、固定细胞和微乳液中荧光团的光漂白动力学，并证明了这些过程在单个细胞内的空间变异性。倒置荧光显微镜和高灵敏度相机，加上计算机控制的图像处理器的高速数据采集，已经被用来精确控制激发光的曝光时间和记录图像。为了提高信噪比，对32幅数字图像进行了整合。在对相机灵敏度和背景荧光的空间变化进行校正后，得到了物面上0.065~m2区域的相对荧光强度图像，为了客观地评价光漂白，开发了一个算法来拟合从同一显微镜视野记录的20幅图像作为光照时间的函数。分析结果表明，光漂白过程遵循一级反应动力学，速率常数在空间上是不均匀的，并且在同一细胞内根据荧光团的不同在2-65倍之间变化。随着激发强度的增加，光漂白速率常数成比例增加，对于苯并(A)芘，在三个数量级(1.25/~M~1.25 mm)范围内，光漂白速率常数与探针浓度无关。每个荧光团对光漂白的倾向是不同的。在所用的细胞条件下，光漂白的平均速率依次为：N-(7-硝基-2-氧杂-1，3-二唑)-23，24-二羟基-5-氯代-22-胺-3FL-ol>；吖啶橙>；罗丹明-123>；苯并(A)芘>；荧光素>；四甲基罗丹明>；1，1‘-二十八基-3，3，3’，3‘-四甲基亚碳二胺。光漂白是一种氧化反应，在矿物油微乳液中加入饱和的Na2S2O溶液，消除了N-(7-硝基-2-氧杂-1，3-二氮唑)的光漂白。
Photobleaching and related photochemical processes are recognized experimental barriers to quantification of fluorescence by microscopy. We have measured the kinetics of photobleaching of fluorophores in living and fixed cells and in microemulsions, and have demonstrated the spatial variability of these processes within individual cells. An inverted fluorescence microscope and a high-sensitivity camera, together with high-speed data acquisition by a computer-controlled image processor, have been used to control precisely exposure time to excitation light and to record images. To improve the signal-to-noise ratio, 32 digital images were integrated. After correction for spatial variations in camera sensitivity and background fluorescence, the images of the relative fluorescence intensities for 0.065~ m 2 areas in the object plane were obtained.To evaluate photobleaching objectively, an algorithm was developed to fit a three-parameter exponential equation to 20 images recorded from the same microscope field as a function of illumination time. The results of this analysis demonstrated that the photobleaching process followed first-order reaction kinetics with rate constants that were spatially heterogeneous and varied, within the same cell, between 2-and 65-fold, depending on the fluorophore. The photobleaching rate constants increased proportionally with increasing excitation intensity and, for benzo (a) pyrene, were independent of probe concentration over three orders of magnitude (1.25/~ M to 1.25 mM). The propensity to photobleach was different with each fluorophore. Under the cellular conditions used in these studies, the average rates of photobleaching decreased in this order: N-(7-nitrobenz-2-oxa-1, 3-diazole)-23, 24-dinor-5-cholen-22-amine-3fl-ol> acridine orange> rhodamine-123> benzo (a) pyrene> fluorescein> tetramethylrhodamine> 1, 1'dioctadecyl-3, 3, 3', 3'-tetramethylindocarbocyanine. The photobleaching appears to be an oxidation reaction, in that the addition of saturated solutions of Na2S2Os to mineral oil microemulsions eliminated photobleaching of N-(7-nitrobenz-2-oxa-1, 3-diazole)-