Small-Molecule Labeling of Live Cell Surfaces for Three-Dimensional Super-Resolution Microscopy

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• 作者：Marissa K. Lee ; Prabin Rai ; Jarrod Williams ; Robert J. Twieg ; W. E. Moerner
• 刊名：Journal of the American Chemical Society
• 出版年：2014
• 出版时间：October 8, 2014
• 年：2014
• 卷：136
• 期：40
• 页码：14003-14006
• 全文大小：343K
• ISSN：1520-5126

文摘

Precise imaging of the cell surface of fluorescently labeled bacteria requires super-resolution methods because the size-scale of these cells is on the order of the diffraction limit. In this work, we present a photocontrollable small-molecule rhodamine spirolactam emitter suitable for non-toxic and specific labeling of the outer surface of cells for three-dimensional (3D) super-resolution (SR) imaging. Conventional rhodamine spirolactams photoswitch to the emitting form with UV light; however, these wavelengths can damage cells. We extended photoswitching to visible wavelengths >400 nm by iterative synthesis and spectroscopic characterization to optimize the substitution on the spirolactam. Further, an N-hydroxysuccinimide-functionalized derivative enabled covalent labeling of amines on the surface of live Caulobacter crescentus cells. Resulting 3D SR reconstructions of the labeled cell surface reveal uniform and specific sampling with thousands of localizations per cell and excellent localization precision in x, y, and z. The distribution of cell stalk lengths (a sub-diffraction-sized cellular structure) was quantified for a mixed population of cells. Pulse-chase experiments identified sites of cell surface growth. Covalent labeling with the optimized rhodamine spirolactam label provides a general strategy to study the surfaces of living cells with high specificity and resolution down to 10鈥?0 nm.