Comparison of Confocal and Super-Resolution Reflectance Imaging of Metal Oxide Nanoparticles.
Comparison of Confocal and Super-Resolution Reflectance Imaging of Metal Oxide Nanoparticles.
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金属氧化物纳米颗粒的共聚焦和超分辨率反射成像的比较。
DOI：
10.1371/journal.pone.0159980
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Rappoport JZ
中科院分区：
文献类型：
--
作者：
Guggenheim EJ;Khan A;Pike J;Chang L;Lynch I;Rappoport JZ
近年来，人类暴露于人造纳米颗粒（NP）的可能性有所增加，部分原因是将工程颗粒纳入了广泛的商业商品和医疗应用中。NP是生物医学中用作治疗和诊断工具的理想候选物，然而存在关于其功效和安全性的担忧。因此，开发用于研究NP摄取到细胞中的技术是至关重要的。目前的细胞内NP研究依赖于使用透射电子显微镜（TEM），它提供了高分辨率，但涉及繁琐的样品制备，使该技术与活细胞成像或荧光标记不兼容，这遭受光漂白，差的生物共轭，并经常改变NP表面性质。反射光成像提供了一种替代的非破坏性无标记技术，其非常适合但不限于模型系统（例如细胞）内NP摄取的可视化。共焦反射显微镜提供光学切片和实时成像功能，样品制备很少。然而，共焦显微镜是衍射受限的，因此X-Y分辨率被限制为~250 nm，基本上大于NP的<100 nm尺寸。超分辨率光学显微镜等技术克服了这一基本限制，提供了更高的X-Y分辨率。反射SIM（R-SIM）用于NP成像以前仅在定制显微镜上进行过演示，限制了广泛使用并限制了NP研究。本文演示了使用商业SIM显微镜的超分辨率反射率数据的收购与X-Y分辨率为115 nm，大于两倍的增长与RCM相比，可达到的。这种分辨率的提高有利于可视化小的紧密间隔的结构，如NP簇，以前无法分辨的RCM。当研究NP在荧光标记的细胞区室内的亚细胞运输时，这是有利的。用RCM、R-SIM和TEM可以观察到NP信号，并进行了直接比较。这些技术中的每一种都有其自身的优点和局限性; RCM和R-SIM提供了新的补充信息，而模式的组合提供了一个独特的机会，以获得有关NP摄取的额外信息。因此，多种成像方法的使用极大地扩大了可以在无标记条件下研究的NP范围。
The potential for human exposure to manufactured nanoparticles (NPs) has increased in recent years, in part through the incorporation of engineered particles into a wide range of commercial goods and medical applications. NP are ideal candidates for use as therapeutic and diagnostic tools within biomedicine, however concern exists regarding their efficacy and safety. Thus, developing techniques for the investigation of NP uptake into cells is critically important. Current intracellular NP investigations rely on the use of either Transmission Electron Microscopy (TEM), which provides ultrahigh resolution, but involves cumbersome sample preparation rendering the technique incompatible with live cell imaging, or fluorescent labelling, which suffers from photobleaching, poor bioconjugation and, often, alteration of NP surface properties. Reflected light imaging provides an alternative non-destructive label free technique well suited, but not limited to, the visualisation of NP uptake within model systems, such as cells. Confocal reflectance microscopy provides optical sectioning and live imaging capabilities, with little sample preparation. However confocal microscopy is diffraction limited, thus the X-Y resolution is restricted to ~250 nm, substantially larger than the <100 nm size of NPs. Techniques such as super-resolution light microscopy overcome this fundamental limitation, providing increased X-Y resolution. The use of Reflectance SIM (R-SIM) for NP imaging has previously only been demonstrated on custom built microscopes, restricting the widespread use and limiting NP investigations. This paper demonstrates the use of a commercial SIM microscope for the acquisition of super-resolution reflectance data with X-Y resolution of 115 nm, a greater than two-fold increase compared to that attainable with RCM. This increase in resolution is advantageous for visualising small closely spaced structures, such as NP clusters, previously unresolvable by RCM. This is advantageous when investigating the subcellular trafficking of NP within fluorescently labelled cellular compartments. NP signal can be observed using RCM, R-SIM and TEM and a direct comparison is presented. Each of these techniques has its own benefits and limitations; RCM and R-SIM provide novel complementary information while the combination of modalities provides a unique opportunity to gain additional information regarding NP uptake. The use of multiple imaging methods therefore greatly enhances the range of NPs that can be studied under label-free conditions.
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影响因子：
2
作者：
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通讯作者：
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