QUANTITATIVE IMAGING OF GREEN FLUORESCENT PROTEIN IN CULTURED-CELLS - COMPARISON OF MICROSCOPIC TECHNIQUES, USE IN FUSION PROTEINS AND DETECTION LIMITS
QUANTITATIVE IMAGING OF GREEN FLUORESCENT PROTEIN IN CULTURED-CELLS - COMPARISON OF MICROSCOPIC TECHNIQUES, USE IN FUSION PROTEINS AND DETECTION LIMITS
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DOI：
10.1111/j.1365-2818.1995.tb03665.x
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发表时间：
1995-11-01
影响因子：
2
通讯作者：
PISTON, DW
中科院分区：
文献类型：
--
作者：
NISWENDER, KD;BLACKMAN, SM;PISTON, DW
为了确定绿色荧光蛋白（GFP）作为报告基因或蛋白质标签的应用限制，我们表达GFP本身和融合蛋白伴侣，并使用三种不同的成像方法来识别GFP荧光。在常规的落射荧光显微术中，细胞中表达的GFP可以被区分为黄-绿色自体荧光背景上的明亮的绿色信号。然而，在定量荧光显微术中，GFP信号被细胞自体荧光污染。通过使用488-nm激发的共聚焦扫描激光显微术的组合，快速接通分色镜和窄带通发射滤光器。在约390 nm的等效波长处的GFP荧光的双光子激发提供了比488 nm激发更好的吸收，这导致增加的信号/背景，但也产生了不同的自发荧光图案，并且似乎增加了GFP光漂白，当GFP与谷胱甘肽-S-转移酶（GST）或与葡萄糖激酶表达为融合蛋白时，观察到与单独的GFP相似的荧光光谱。此外，纯化的GST。GFP融合蛋白显示出与先前报道的单独GFP的值一致的消光系数和量子产率。在HeLa细胞中，细胞质GFP浓度必须大于约μ M以允许对自发荧光的可定量区分。然而，如果GFP靶向离散的亚细胞区室，例如质膜、细胞器或细胞核，则可以检测到较低的表达水平。
To determine the application limits of green fluorescent protein (GFP) as a reporter gene or protein tag, we expressed GFP by itself and with fusion protein partners, and used three different imaging methods to identify GFP fluorescence. In conventional epifluorescence photomicroscopy, GFP expressed in cells could be distinguished as a bright green signal over a yellow-green autofluorescence background, In quantitative fluorescence microscopy, however, the GFP signal is contaminated by cellular autofluorescence, Improved separation of GFP signal from HeLa cell autofluorescence was achieved by the combination of confocal scanning laser microscopy using 488-nm excitation, a rapid cut-on dichroic mirror and a narrow-bandpass emission filter, Two-photon excitation of GFP fluorescence at the equivalent of approximate to 390 nm provided better absorption than did 488-nm excitation, This resulted in increased signal/background but also generated a different autofluorescence pattern and appeared to increase GFP photobleaching, Fluorescence spectra similar to those of GFP alone were observed when GFP was expressed as a fusion protein either with glutathione-S-transferase (GST) or with glucokinase. Furthermore, purified GST . GFP fusion protein displayed an extinction coefficient and quantum yield consistent with values previously reported for GFP alone, In HeLa cells, the cytoplasmic GFP concentration must be greater than approximate to mu M to allow quantifiable discrimination over autofluorescence, However, lower expression levels may be detectable if GFP is targeted to discrete subcellular compartments, such as the plasma membrane, organelles or nucleus.