Improved Fluorescent Proteins for Dual-Colour Post-Embedding CLEM.
Improved Fluorescent Proteins for Dual-Colour Post-Embedding CLEM.
复制标题
用于双色包埋后 CLEM 的改进荧光蛋白
作者：
Peng D;Li N;He W;Drasbek KR;Xu T;Zhang M;Xu P
包埋后相关光电子显微镜（CLEM）具有高精度配准的优势，能够对同一切片进行光镜和电镜成像。然而，其广泛应用受到可用荧光蛋白（FP）有限和信号背景比（SBR）低的阻碍。在这里，我们开发了一种绿色光可切换 FP，mEosEM-E，在嵌入 Epon 树脂的 EM 样品中具有相当高的开/关对比度，它最大限度地保留了细胞结构，但淬灭了 FP 的荧光。利用 mEosEM-E 的光开关特性，基于减法的 CLEM (sCLEM) 方法显着降低了树脂的自发荧光背景。同时，我们鉴定了一种红色荧光蛋白（RFP）mScarlet-H，它在树脂中表现出比之前报道的 RFP 更高的亮度和 SBR。使用 mEosEM-E 和 mScarlet-H，成功地获得了具有高 SBR 且无串扰信号的双色 Epon 嵌入后 CLEM 图像，以揭示核仁蛋白的组织。此外，对不同 EM 样品制备步骤对几个 RFP 荧光保存的影响的剖析为进一步的探针开发提供了有用的指导。
Post-embedding correlative light and electron microscopy (CLEM) has the advantage of high-precision registration and enables light and electron microscopy imaging of the same slice. However, its broad application has been hampered by the limited available fluorescent proteins (FPs) and a low signal-to-background ratio (SBR). Here, we developed a green photoswitchable FP, mEosEM-E with substantially high on/off contrast in EM samples embedded in Epon resin, which maximally preserves cellular structures but quenches the fluorescence of FPs. Taking advantage of the photoswitching property of mEosEM-E, the autofluorescence background from the resin was significantly reduced by a subtraction-based CLEM (sCLEM) method. Meanwhile, we identified a red fluorescent protein (RFP) mScarlet-H that exhibited higher brightness and SBR in resin than previously reported RFPs. With mEosEM-E and mScarlet-H, dual-colour post-Epon-embedding CLEM images with high SBR and no cross-talk signal were successfully performed to reveal the organization of nucleolar proteins. Moreover, a dissection of the influences of different EM sample preparation steps on the fluorescence preservation for several RFPs provides useful guidance for further probe development.
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影响因子：
9.8
作者：
Shu X;Lev-Ram V;Deerinck TJ;Qi Y;Ramko EB;Davidson MW;Jin Y;Ellisman MH;Tsien RY
通讯作者：
Tsien RY
一系列独特的可逆可切换荧光蛋白，具有适合各种应用的有益特性
DOI：
10.1073/pnas.1113770109
发表时间：
2012-03-20
影响因子：
11.1
作者：
Chang, Hao;Zhang, Mingshu;Xu, Tao
通讯作者：
Xu, Tao
影响因子：
48
作者：
Helmerich, Dominic A.;Beliu, Gerti;Sauer, Markus
通讯作者：
Sauer, Markus
DOI：
10.1073/pnas.0808882105
发表时间：
2008-11-18
影响因子：
11.1
作者：
Marriott, Gerard;Mao, Shu;Yan, Yuling
通讯作者：
Yan, Yuling
影响因子：
48
作者：
Lam, Stephanie S.;Martell, Jeffrey D.;Ting, Alice Y.
通讯作者：
Ting, Alice Y.