Department of Biochemistry, School of Medical Sciences, University of Bristol, University Walk, Bristol, BS8 1TD, UK.
Protoplasma. 2010 Aug;244(1-4):91-7. doi: 10.1007/s00709-010-0165-1. Epub 2010 Jun 5.
Bioimaging: the visualisation, localisation and tracking of movement of specific molecules in cells using microscopy has become an increasing field of interest within life science research. For this, the availability of fluorescent and electron-dense markers for light and electron microscopy, respectively, is an essential tool to attach to the molecules of interest. In recent years, there has been an increasing effort to combine light and electron microscopy in a single experiment. Such correlative light electron microscopy (CLEM) experiments thus rely on using markers that are both fluorescent and electron dense. Unfortunately, there are very few markers that possess both these properties. Markers for light microscopy such as green fluorescent protein are generally not directly visible in the electron microscopy and vice versa for gold particles. Hence, there has been an intensive search for markers that are directly visible both in the light microscope and in the electron microscope. Here we discuss some of the strategies and pitfalls that are associated with the use of CLEM markers, which might serve as a "warning" that new probes should be extensively tested before use. We focus on the use of CLEM markers for the study of intracellular transport and specifically endocytosis.
使用显微镜对细胞内特定分子的运动进行可视化、定位和跟踪，已经成为生命科学研究中日益感兴趣的领域。为此，荧光和电子致密标记物分别用于光和电子显微镜，是将其附着到感兴趣的分子上的重要工具。近年来，人们越来越努力地在单个实验中结合使用光和电子显微镜。这种相关的光电子显微镜（CLEM）实验因此依赖于使用既具有荧光性又具有电子密度的标记物。不幸的是，具有这两种特性的标记物非常少。例如，绿色荧光蛋白等用于显微镜的标记物在电子显微镜中通常不可见，反之亦然，金颗粒在光显微镜中也是不可见的。因此，人们一直在积极寻找既能在光显微镜中又能在电子显微镜中直接观察到的标记物。在这里，我们讨论了与使用 CLEM 标记物相关的一些策略和陷阱，这些标记物可能会成为一个“警告”，即在使用新探针之前，应该对其进行广泛的测试。我们重点介绍了 CLEM 标记物在细胞内运输特别是内吞作用研究中的应用。
