Lee Marissa K, Rai Prabin, Williams Jarrod, Twieg Robert J, Moerner W E
Department of Chemistry, Stanford University , Stanford, California 94305, United States.
J Am Chem Soc. 2014 Oct 8;136(40):14003-6. doi: 10.1021/ja508028h. Epub 2014 Sep 24.
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 10-20 nm.
由于荧光标记细菌的细胞表面尺寸在衍射极限范围内，因此对其进行精确成像需要超分辨率方法。在这项工作中，我们展示了一种光控小分子罗丹明螺内酰胺发射体，适用于对细胞外表面进行无毒且特异性标记，以用于三维（3D）超分辨率（SR）成像。传统的罗丹明螺内酰胺在紫外光照射下会光开关转变为发射形式；然而，这些波长会损伤细胞。我们通过迭代合成和光谱表征将光开关扩展到大于400 nm的可见光波长，以优化螺内酰胺上的取代基。此外，一种N-羟基琥珀酰亚胺功能化衍生物能够对活的新月柄杆菌细胞表面的胺进行共价标记。对标记的细胞表面进行3D SR重建，结果显示每个细胞有数千个定位点，具有均匀且特异性的采样，在x、y和z方向上具有出色的定位精度。对混合细胞群体的细胞柄长度（一种亚衍射尺寸的细胞结构）分布进行了量化。脉冲追踪实验确定了细胞表面生长的位点。用优化的罗丹明螺内酰胺标记进行共价标记，为以高特异性和低至10 - 20 nm的分辨率研究活细胞表面提供了一种通用策略。
