The three-dimensional cell surface provides context for biological processes, defines the cell shape, and serves as a gateway for material to enter or exit the cell. Precisely studying 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. Single-molecule-based super-resolution imaging requires bright fluorophores with photocontrollable emission. Though fluorescent proteins are commonly used for super-resolution experiments, small-molecule organic fluorophores can emit on average of ten-fold more photons. Since the resolution enhancement scales as 1/vN (where N is the number of detected photons), developing suitable organic fluorophores is desirable. Additionally, the photophysical properties of small-molecule fluorophores can be readily modified using organic synthesis. In this work, we present a small-molecule photocontrollable rhodamine spirolactam emitter suitable for non-toxic and specific labeling of the outer surface of cells for three-dimensional super-resolution imaging. Rhodamine spirolactams photoswitch with UV light; however, these wavelengths can damage cells. We optimized the substitution on the spirolactam by iterative synthesis and spectroscopic characterization to lower the energy required for photoswitching to visible wavelengths at 400 nm, and used N-hydroxy-succinimide chemistry for attachment to amines. Three-dimensional super-resolution images of covalently labeled live Caulobacter crescentus cell surfaces revealed uniform sampling specific to the exterior of the cell. The distribution of cell stalk lengths (a sub-diffraction-sized cellular structure) was quantified for a mixed population of cells. Pulse-chase experiments revealed the sites of new membrane incorporation. Covalent labeling with the optimized rhodamine spirolactam label is a general strategy to study the membranes of living cells with high specificity and resolution down to 10-20 nm, which also can be applied to other surfaces.

Small-molecule labeling of live cell surfaces for 3D super-resolution microscopy

248th National Meeting (2014)

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