Superresolution imaging techniques based on sequential imaging of sparse subsets of single molecules require fluorophores whose emission can be photoactivated or photoswitched. Because typical organic fluorophores can emit significantly more photons than average fluorescent proteins, organic fluorophores have a potential advantage in superresolution imaging schemes, but targeting to specific cellular proteins must be provided. We report the design and application of HaloTag-based target-specific azido DCDHFs, a class of photoactivatable push-pull fluorogens which produce bright fluorescent labels suitable for single-molecule superresolution imaging in live bacterial and fixed mammalian cells.Recently, sequential imaging of sparse subsets of photoactivatable/photoswitchable singlemolecule fluorophores has enabled optical imaging beyond the diffraction limit (DL), providing insight into the sub-diffraction world (e.g. PALM, FPALM, STORM). 1-3 These single-molecule superresolution (SR) techniques have provided the impetus for development of new controllable fluorophores with large numbers of emitted photons N, because the achievable resolution scales as . 4 Most previous SR experiments in living cells 5 have used photocontrollable fluorescent proteins. 6-9 However, despite having the advantage of being target-specific, fluorescent proteins on average provide 10-fold fewer photons before photobleaching than good organic fluorophores. 10,11 Small organic fluorophores have the additional benefit of synthetic design flexibility for tuning target specificity, spectral wavelength, solubility, and other desired properties. Therefore, targeted bright organic Here we present a target-specific photoactivatable organic fluorophore for use inside living and fixed cells, 3, based on the commercial HaloTag targeting approach. [20][21][22] This method requires a genetic fusion to the HaloEnzyme (HaloEnz), which forms a covalent linkage to the HaloTag substrate, thus labeling the protein of interest (i.e. a protein-HaloEnzHaloTag-fluorophore covalent unit). Specifically, we present: (i) the basic photophysical properties of a new targeted photoactivatable probe; (ii) proof-of-principle labeling of known structures in fixed and living mammalian cells validated by co-staining with antibodies or co-transfection with fluorescent proteins; (iii) specific SR imaging of microtubules in a mammalian cell with quantification of resolution enhancement; (iv) demonstration of targeted labeling in living bacteria with diffraction-limited imaging; and finally, (v) SR imaging of poorly understood structures inside living bacteria.As molecules with bright emission for single-molecule imaging, dicyanomethylenedihydrofuran (DCDHF) push-pull fluorophores emit millions of photons before photobleaching, and can enter living cells. 15,23 Recently, we reported a photoactivatable DCDHF fluorogen based on photocaging the fluorescence by replacing the amine donor with a poorly-donating but photolabile azide, which can then be converted back to an am...

We report the solvatochromic, viscosity-sensitive, and single-molecule photophysics of the fluorophores DCDHF-N-6 and DCDHF-A-6. These molecules are members of the dicyanomethylenedihydrofuran (DCDHF) class of single-molecule emitters that contain an amine electron donor and a DCDHF acceptor linked by a conjugated unit; DCDHF-N-6 and DCDHF-A-6 have naphthalene- and anthracene-conjugated linkers, respectively. These molecules maintain the beneficial photophysics of the phenylene-linked DCDHF (i.e., photostability, emission wavelength dependence on solvent polarity, and quantum yield sensitivity to solvent viscosity), yet offer absorption and emission at longer wavelengths that are more appropriate for cellular imaging. We demonstrate that these new fluorophores are less photolabile in an aqueous environment than several other commonly used dyes (rhodamine 6G, Texas Red, and fluorescein). Finally, we image single copies of the acene DCDHFs diffusing in the plasma membrane of living cells.