Microspectrofluorometry by digital image processing: measurement of cytoplasmic pH.
Microspectrofluorometry by digital image processing: measurement of cytoplasmic pH.
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DOI：
10.1083/jcb.98.2.717
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发表时间：
1984-02
期刊：
影响因子：
--
通讯作者：
Taylor DL
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文献类型：
--
作者：
Tanasugarn L;McNeil P;Reynolds GT;Taylor DL
我们的显微荧光分光光度计与图像处理系统的接口进行显微荧光分光光度测量活细胞的数字图像处理。荧光光谱参数可以通过数字图像处理直接从细胞的显微图像测量，并自动归一化为光程和可达体积。因此，可以产生各种光谱参数的准确的细胞质“图”。通过测量两个连续波长（489和452 nm）激发的荧光素荧光的比率来确定成纤维细胞（3 T3细胞）的静息细胞质pH。微注射到细胞中的黄绿素标记的葡聚糖被用作pH指示剂，因为它被捕获在细胞质中，但被排除在细胞核和其他细胞器之外。平均细胞质pH值为6.83（+/- 0.38）。然而，细胞质pH值呈现非单峰分布，较低的平均pH值为6.74（+/- 0.23）。当3 T3细胞胞饮含有荧光素葡聚糖的培养基时，细胞核周围的胞饮体表现出比靠近细胞皱褶边缘的胞饮体更低的pH。本图像处理系统的线性检测，光散射文物，信噪比，标准曲线，和空间分辨率进行了分析。从数字图像分析得到的结果被证明是可比的标准显微荧光光谱法的结果。我们还讨论了这种比率成像技术在细胞生物学中的其他几个应用。
An interface of our microspectrofluorometer with an image processing system performs microspectrofluorometric measurements in living cells by digital image processing. Fluorescence spectroscopic parameters can be measured by digital image processing directly from microscopic images of cells, and are automatically normalized for pathlength and accessible volume. Thus, an accurate cytoplasmic "map" of various spectroscopic parameters can be produced. The resting cytoplasmic pH of fibroblasts (3T3 cells) has been determined by measuring the ratio of fluorescein fluorescence exited by two successive wavelengths (489 and 452 nm). Fluorescein-labeled dextran microinjected into the cells is used as a pH indicator, since it is trapped in the cytoplasm but is excluded from the nucleus and other organelles. The average cytoplasmic pH is 6.83 (+/- 0.38). However, cytoplasmic pH exhibits a nonunimodal distribution, the lower mean pH being 6.74 (+/- 0.23). When 3T3 cells pinocytose medium containing fluorescein dextran, pinosomes peripheral to the nucleus exhibit a lower pH than those closer to the ruffling edge of the cell. The present image processing system is analyzed for linearity of detection, light scattering artifacts, signal to noise ratio, standard curves, and spatial resolution. The results obtained from digital image analysis are shown to be comparable to the results from standard microspectrofluorometry. We also discuss several other applications of this ratio imaging technique in cell biology.