FLIPPER, a combinatorial probe for correlated live imaging and electron microscopy, allows identification and quantitative analysis of various cells and organelles.
FLIPPER, a combinatorial probe for correlated live imaging and electron microscopy, allows identification and quantitative analysis of various cells and organelles.
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DOI：
10.1007/s00441-015-2142-7
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发表时间：
2015-04
影响因子：
3.6
通讯作者：
Giepmans, Ben N. G.
中科院分区：
文献类型：
--
作者：
Kuipers, Jeroen;van Ham, Tjakko J.;Kalicharan, Ruby D.;Veenstra-Algra, Anneke;Sjollema, Klaas A.;Dijk, Freark;Schnell, Ulrike;Giepmans, Ben N. G.
通过电子显微镜 (EM) 对细胞和组织进行超微结构检查，可得出有关亚细胞结构的详细信息。然而，电子显微镜通常仅限于高放大倍率下的小视场；这使得量化多个大面积样本部分中的事件变得极其困难。即使将光学显微镜 (LM) 与 EM（相关 LM 和 EM：CLEM）结合起来寻找感兴趣的区域，分子标记仍然是一个挑战。我们提出了一种新的 CLEM 基因编码探针，名为“FLIPPER”，它有助于对细胞超微结构特征进行定量分析。 FLIPPER 由用于 LM 可视化的荧光蛋白（青色、绿色、橙色或红色）组成，与过氧化物酶融合，允许在 EM 水平上可视化目标。 FLIPPER 的使用非常简单，并且由于该模块是完全基因编码的，因此可以为细胞做好最佳的电镜检查准备。我们使用 FLIPPER 在 EM 水平上量化表达正常和致病点突变细胞表面蛋白（称为 EpCAM（上皮细胞粘附分子））的细胞的细胞形态。突变蛋白保留在内质网 (ER) 中，因此可以改变 ER 功能和形态。为了揭示可能的 ER 改变，将细胞与颜色编码的全长或突变 EpCAM 和针对 ER 的 FLIPPER 共转染。混合细胞群的 CLEM 检查允许基于颜色的细胞识别，然后通过 EM 对 ER 超微结构进行公正的定量分析。因此，FLIPPER 结合了针对实时成像优化的明亮荧光蛋白和针对 EM 标记的高灵敏度，从而成为 CLEM 的一种有前途的工具。本文的在线版本 (doi:10.1007/s00441-015-2142-7) 包含补充材料，可供授权用户使用。
Ultrastructural examination of cells and tissues by electron microscopy (EM) yields detailed information on subcellular structures. However, EM is typically restricted to small fields of view at high magnification; this makes quantifying events in multiple large-area sample sections extremely difficult. Even when combining light microscopy (LM) with EM (correlated LM and EM: CLEM) to find areas of interest, the labeling of molecules is still a challenge. We present a new genetically encoded probe for CLEM, named “FLIPPER”, which facilitates quantitative analysis of ultrastructural features in cells. FLIPPER consists of a fluorescent protein (cyan, green, orange, or red) for LM visualization, fused to a peroxidase allowing visualization of targets at the EM level. The use of FLIPPER is straightforward and because the module is completely genetically encoded, cells can be optimally prepared for EM examination. We use FLIPPER to quantify cellular morphology at the EM level in cells expressing a normal and disease-causing point-mutant cell-surface protein called EpCAM (epithelial cell adhesion molecule). The mutant protein is retained in the endoplasmic reticulum (ER) and could therefore alter ER function and morphology. To reveal possible ER alterations, cells were co-transfected with color-coded full-length or mutant EpCAM and a FLIPPER targeted to the ER. CLEM examination of the mixed cell population allowed color-based cell identification, followed by an unbiased quantitative analysis of the ER ultrastructure by EM. Thus, FLIPPER combines bright fluorescent proteins optimized for live imaging with high sensitivity for EM labeling, thereby representing a promising tool for CLEM. The online version of this article (doi:10.1007/s00441-015-2142-7) contains supplementary material, which is available to authorized users.
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影响因子：
16.6
作者：
通讯作者：
--
DOI：
10.1038/nsb1297-1032
发表时间：
1997-12-01
期刊：
NATURE STRUCTURAL BIOLOGY
影响因子：
--
作者：
Gajhede, M;Schuller, DJ;Poulos, TL
通讯作者：
Poulos, TL
影响因子：
4.6
作者：
Ravelli, Raimond B. G.;Kalicharan, Ruby D.;Giepmans, Ben N. G.
通讯作者：
Giepmans, Ben N. G.
影响因子：
48
作者：
Rothbauer, Ulrich;Zolghadr, Kourosh;Leonhardt, Heinrich
通讯作者：
Leonhardt, Heinrich
影响因子：
48
作者：
Lam, Stephanie S.;Martell, Jeffrey D.;Ting, Alice Y.
通讯作者：
Ting, Alice Y.