Tanasugarn L, McNeil P, Reynolds G T, Taylor D L
J Cell Biol. 1984 Feb;98(2):717-24. doi: 10.1083/jcb.98.2.717.
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.
我们的显微分光荧光计与图像处理系统相结合，通过数字图像处理对活细胞进行显微分光荧光测量。荧光光谱参数可通过数字图像处理直接从细胞的显微图像中测量得到，并会自动针对光程和可及体积进行归一化处理。因此，可以生成各种光谱参数的精确细胞质“图谱”。通过测量由两个连续波长（489和452纳米）激发的荧光素荧光比率，已确定了成纤维细胞（3T3细胞）的静息细胞质pH值。微注射到细胞中的荧光素标记葡聚糖用作pH指示剂，因为它被困在细胞质中，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3T3细胞胞饮含有荧光素葡聚糖的培养基时，细胞核周围的胞饮小体的pH值低于那些靠近细胞边缘的胞饮小体。对当前的图像处理系统进行了检测线性、光散射伪影、信噪比、标准曲线和空间分辨率的分析。数字图像分析得到的结果与标准显微分光荧光测量的结果相当。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。
