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