绿色荧光蛋白的单分子共聚焦显微镜

G Jung, J Wiehler, W Göhde, J Tittel, Th Basché, B Steipe, C Bräuchle
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引用次数: 43

摘要

单分子检测已通过使用强荧光标记扩展到生命科学。绿色荧光蛋白(GFP)作为一种具有自荧光特性的生物分子受到了广泛的关注。在这里,gfp突变体Glu222Gln的单分子固定在聚乙烯醇基质中,并通过共聚焦荧光显微镜检测。虽然该突变体稳定了野生型GFP的两种构象之一,但对其荧光动力学的研究显示出强烈的信号波动。这种荧光行为——至少部分是由蛋白质框架的可逆光化学变化引起的,这种变化可以在不同的时间尺度上放松到荧光状态。因此,该蛋白似乎特别适合在单分子水平上研究大分子GFP的微观异质性。
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Confocal microscopy of single molecules of the green fluorescent protein

Single molecule detection has been extended into life sciences by use of strongly fluorescent labels. The green fluorescent protein (GFP) as a self-fluorescent biomolecule has attracted considerable attention. Here, single molecules of the GFP-mutant Glu222Gln are immobilized in a polyvinylalcohol matrix and detected by confocal fluorescence microscopy. Although this mutant stabilizes one of both conformers of the wild-type GFP, the investigation of its fluorescence dynamics reveals strong signal fluctuations. This fluorescence behaviour is—at least partly—caused by reversible photochemical changes of the protein framework, that can relax into the fluorescent state on different timescales. Thus, this protein appears particularly appropriate for studying the microheterogeneity of the macromolecule GFP on a single molecule level.

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Magnetic Particle Imaging Magnetic Resonance Imaging Quantitative evaluation of light microscopes based on image processing techniques Confocal microscopy of single molecules of the green fluorescent protein Heavy metal contrast enhancement for the selective detection of gold particles in electron microscopical sections using electron spectroscopic imaging
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