Superresolution Imaging of Targeted Proteins in Fixed and Living Cells Using Photoactivatable Organic Fluorophores

Abstract

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...

Chemical tags: Applications in live cell fluorescence imaging

“…Chemical tags have been used to study the localization and dynamics of proteins in living cells, especially in experiments that can not be easily performed with fluorescent proteins. In the last few years, chemical tags have been used to label proteins with fluorophores suited for advanced imaging technologies such as super-resolution (SR) microscopy [57][58][59], Ca 2þ -imaging [60][61][62], pH sensing [63], hydrogen peroxide detection [64], chromophore assisted light inactivation [36,65,66], and multi-photon microscopy [19]. Recently, Kosaka et al demonstrated the use of the Halo-tag to perform in vivo imaging studies in live animals for the first time [67].…”

Expanding the Chemical Space of Biocompatible Fluorophores: Nanohoops in Cells

“…[1][2][3] The power of such imaging methods has led to increased interest in identifying new types of dyes, opticallyactive materials, and nanoparticles that have enhanced photophysical properties suitable for multimodal, multiplexed, and super-resolution imaging. [4][5][6][7][8][9][10][11][12][13][14] Because fluorophores play such a critical role in understanding biological processes, it is somewhat surprising that most advances in small molecule dye technology today rely on structural modifications of scaffolds discovered over a century ago. [15] For example, the robust Janelia FluorÒ and some AlexaFluorÒ dyes are structurally modified versions of rhodamine scaffolds discovered 130 years ago.…”

Super‐Resolution Microscopy: Going Live and Going Fast

“…However, these improvements came at the expense of ease of intracellular labelling. Intracellular labelling with organic fluorophores in living cells can be achieved using genetically encoded tags such as SNAP or HaLo tag as previously demonstrated for live-cell super-resolution imaging of bacterial proteins or histone proteins in mammalian cells [50,51] . These tags react with a small peptide, which contains an organic fluorophore as a label.…”