The rapid development of super-resolution microscopy has been driven by improvements in imaging hardware, sophisticated image acquisition and processing algorithms, and most importantly, through the use of robust fluorophores for biological imaging. While super-resolution methods have been applied widely in eukaryotic cells, their applications in bacteria remain limited due to challenges in targeting superior fluorophores to intracellular proteins. Further, high photostability of fluorescent labels is the most important requirement for achieving the ultimate spatio-temporal resolution through super-resolution microscopy. Here, we report the first use of a highly photostable fluoromodule, dL5, to label proteins in the Gram negative bacterium Caulobacter crescentus, enabling long time scale single protein tracking and super-resolution microscopy. dL5 is a 28 kDa scFv that specifically binds to the fluorogen Malachite Green (MG) and turns on its fluorescence. Chemical modification of MG with an ester group enables the delivery of the fluorogen beyond the complex bacterial membranes and peptidoglycan layer. The fluorescent dL5-MG complex can be used to label proteins sparsely in live bacteria without the need for initial bleaching, enabling the acquisition of non-overlapping protein tracks for extended time scales, although washing is required. We have characterized the dL5-MG complex and report that these complexes emit two-fold more photons than organic dyes such as Cy5 and Alexa 647 in vitro; and five-fold more photons compared to eYFP in live bacteria. The high number of emitted photons makes dL5-MG a robust fluoromodule and enables acquisition of single protein tracks lasting over 10 seconds without the use of antifade agents. Furthermore, we demonstrate the application of the dL5 fluoromodule to image three functionally different proteins in live Caulobacter cells using STED microscopy. STED microscopy of dL5 labeled proteins enabled a four-fold resolution enhancement compared to diffraction-limited imaging in live Caulobacter cells. This fluoromodule opens up many opportunities for a systematic study of poorly understood bacterial protein dynamics through single-protein tracking and/or superstructure measurement through STED microscopy, with a single label. The labeling and imaging methods thus developed are general, and can also be applied to other Gram negative bacteria and possibly Gram positive bacteria for super-resolution imaging.

Ultra-photostable, genetically directed fluoromodule enables STED nanoscopy and long time scale single protein tracks in live bacteria

253rd National Meeting (2017)

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