Recently, photoactivation and photoswitching were used to control single-molecule fluorescent labels and produce
images of cellular structures beyond the optical diffraction limit (e.g., PALM, FPALM, and STORM). While previous
live-cell studies relied on sophisticated photoactivatable fluorescent proteins, we show in the present work that
superresolution imaging can be performed with fusions to the commonly used fluorescent protein EYFP. Rather than
being photoactivated, however, EYFP can be reactivated with violet light after apparent photobleaching. In each cycle
after initial imaging, only a sparse subset fluorophores is reactivated and localized, and the final image is then generated
from the measured single-molecule positions. Because these methods are based on the imaging nanometer-sized single-molecule
emitters and on the use of an active control mechanism to produce sparse sub-ensembles, we suggest the
phrase "Single-Molecule Active-Control Microscopy" (SMACM) as an inclusive term for this general imaging strategy.
In this paper, we address limitations arising from physiologically imposed upper boundaries on the fluorophore
concentration by employing dark time-lapse periods to allow single-molecule motions to fill in filamentous structures,
increasing the effective labeling concentration while localizing each emitter at most once per resolution-limited spot.
We image cell-cycle-dependent superstructures of the bacterial actin protein MreB in live Caulobacter crescentus cells
with sub-40-nm resolution for the first time. Furthermore, we quantify the reactivation quantum yield of EYFP, and find
this to be 1.6 x 10-6, on par with conventional photoswitchable fluorescent proteins like Dronpa. These studies show that
EYFP is a useful emitter for in vivo superresolution imaging of intracellular structures in bacterial cells.