2,2′-thiodiethanol:: A new water soluble mounting medium for high resolution optical microscopy
2,2′-thiodiethanol:: A new water soluble mounting medium for high resolution optical microscopy
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DOI：
10.1002/jemt.20396
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发表时间：
2007-01-01
影响因子：
2.5
通讯作者：
Hell, Stefan W.
中科院分区：
文献类型：
--
作者：
Staudt, Thorsten;Lang, Marion C.;Hell, Stefan W.
在光学显微镜中使用高数值孔径浸没透镜会受到浸没系统和样品嵌入介质之间折射率不匹配引起的球面像差的影响。特别是当成像深度大于 10 μm 的样品时，折射率不匹配会导致图像亮度和分辨率明显损失。该问题的解决方案是使嵌入介质的指数适应浸没系统的指数。不幸的是，目前已知的既可调谐折射率又与荧光成像兼容的封片剂并不多。在这里，我们介绍了一种无毒的包埋介质，2,2'-硫代二乙醇（TDE），它可以与水以任意比例混溶，可以对样品的平均折射率进行微调，范围从水（1.33）到浸油（1.52）。因此，TDE 能够使用具有最高可用孔径角的物镜对固定样本深处进行高分辨率成像，并且有可能使甘油嵌入变得多余。由于较大的细胞结构（例如细胞核）引起的折射率变化在很大程度上得到了补偿。此外，作为一种抗氧化剂，TDE 保留了大多数荧光团的荧光量子产率。我们介绍了这种新介质的光学和化学特性及其在各种不同染色的细胞和细胞亚结构中的应用。
The use of high numerical aperture immersion lenses in optical microscopy is compromised by spherical aberrations induced by the refractive index mismatch between the immersion system and the embedding medium of the sample. Especially when imaging >10 mu m deep into the specimen, the refractive index mismatch results in a noticeable loss of image brightness and resolution. A solution to this problem is to adapt the index of the embedding medium to that of the immersion system. Unfortunately, not many mounting media are known that are both index tunable as well as compatible with fluorescence imaging. Here we introduce a nontoxic embedding medium, 2,2'-thiodiethanol (TDE), which, by being miscible with water at any ratio, allows fine adjustment of the average refractive index of the sample ranging from that of water (1.33) to that of immersion oil (1.52). TDE thus enables high resolution imaging deep inside fixed specimens with objective lenses of the highest available aperture angles and has the potential to render glycerol embedding redundant. The refractive index changes due to larger cellular structures, such as nuclei, are largely compensated. Additionally, as an antioxidant, TDE preserves the fluorescence quantum yield of most of the fluorophores. We present the optical and chemical properties of this new medium as well as its application to a variety of differently stained cells and cellular substructures.