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

Abstract

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 <i>N</i>-hydroxy­succinimide-functionalized derivative enabled covalent labeling of amines on the surface of live <i>Caulobacter crescentus</i> 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 <i>x</i>, <i>y</i>, and <i>z</i>. 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