Superresolution imaging of targeted proteins in fixed and living cells using photoactivatable organic fluorophores.

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 super-resolution 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.

Single-molecule spectroscopy and imaging of biomolecules in living cells.

The number of reports per year on single-molecule imaging experiments has grown roughly exponentially since the first successful efforts to optically detect a single molecule were completed over two decades ago. Single-molecule spectroscopy has developed into a field that includes a wealth of experiments at room temperature and inside living cells. The fast growth of single-molecule biophysics has resulted from its benefits in probing heterogeneous populations, one molecule at a time, as well as from advances in microscopes and detectors. This Perspective summarizes the field of live-cell imaging of single biomolecules.

DCDHF fluorophores for single-molecule imaging in cells.

There is a persistent need for small-molecule fluorescent labels optimized for single-molecule imaging in the cellular environment. Application of these labels comes with a set of strict requirements: strong absorption, efficient and stable emission, water solubility and membrane permeability, low background emission, and red-shifted absorption to avoid cell autofluorescence. We have designed and characterized several fluorophores, termed "DCDHF" fluorophores, for use in live-cell imaging based on the push-pull design: an amine donor group and a 2-dicyanomethylene-3-cyano-2,5-dihydrofuran (DCDHF) acceptor group, separated by a pi-rich conjugated network. In general, the DCDHF fluorophores are comparatively photostable, sensitive to local environment, and their chemistries and photophysics are tunable to optimize absorption wavelength, membrane affinity, and solubility. Especially valuable are fluorophores with sophisticated photophysics for applications requiring additional facets of control, such as photoactivation. For example, we have reengineered a red-emitting DCDHF fluorophore so that it is dark until photoactivated with a short burst of low-intensity violet light. This molecule and its relatives provide a new class of bright photoactivatable small-molecule fluorophores, which are needed for super-resolution imaging schemes that require active control (here turning-on) of single-molecule emission.

A photoactivatable push-pull fluorophore for single-molecule imaging in live cells.

We have reengineered a red-emitting dicyanomethylenedihydrofuran push-pull fluorophore so that it is dark until photoactivated with a short burst of low-intensity violet light. Photoactivation of the dark fluorogen leads to conversion of an azide to an amine, which shifts the absorption to long wavelengths. After photoactivation, the fluorophore is bright and photostable enough to be imaged on the single-molecule level in living cells. This proof-of-principle demonstration provides a new class of bright photoactivatable fluorophores, as are needed for super-resolution imaging schemes that require active control of single molecule emission.

The double-helix microscope super-resolves extended biological structures by localizing single blinking molecules in three dimensions with nanoscale precision.

The double-helix point spread function microscope encodes the axial (z) position information of single emitters in wide-field (x,y) images, thus enabling localization in three dimensions (3D) inside extended volumes. We experimentally determine the statistical localization precision σ of this approach using single emitters in a cell under typical background conditions, demonstrating σ < 20 nm laterally and <30 nm axially for N ≈ 1180 photons per localization. Combined with light-induced blinking of single-molecule labels, we present proof-of-concept imaging beyond the optical diffraction limit of microtubule network structures in fixed mammalian cells over a large axial range in three dimensions.

Fluorescent saxitoxins for live cell imaging of single voltage-gated sodium ion channels beyond the optical diffraction limit.

A desire to better understand the role of voltage-gated sodium channels (Na(V)s) in signal conduction and their dysregulation in specific disease states motivates the development of high precision tools for their study. Nature has evolved a collection of small molecule agents, including the shellfish poison (+)-saxitoxin, that bind to the extracellular pore of select Na(V) isoforms. As described in this report, de novo chemical synthesis has enabled the preparation of fluorescently labeled derivatives of (+)-saxitoxin, STX-Cy5, and STX-DCDHF, which display reversible binding to Na(V)s in live cells. Electrophysiology and confocal fluorescence microscopy studies confirm that these STX-based dyes function as potent and selective Na(V) labels. The utility of these probes is underscored in single-molecule and super-resolution imaging experiments, which reveal Na(V) distributions well beyond the optical diffraction limit in subcellular features such as neuritic spines and filopodia.

Azido push-pull fluorogens photoactivate to produce bright fluorescent labels.

Dark azido push-pull chromophores have the ability to be photoactivated to produce bright fluorescent labels suitable for single-molecule imaging. Upon illumination, the aryl azide functionality in the fluorogens participates in a photochemical conversion to an aryl amine, thus restoring charge-transfer absorption and fluorescence. Previously, we reported that one compound, DCDHF-V-P-azide, was photoactivatable. Here, we demonstrate that the azide-to-amine photoactivation process is generally applicable to a variety of push-pull chromophores, and we characterize the photophysical parameters including photoconversion quantum yield, photostability, and turn-on ratio. Azido push-pull fluorogens provide a new class of photoactivatable single-molecule probes for fluorescent labeling and super-resolution microscopy. Lastly, we demonstrate that photoactivated push-pull dyes can insert into bonds of nearby biomolecules, simultaneously forming a covalent bond and becoming fluorescent (fluorogenic photoaffinity labeling).

Photophysical properties of acene DCDHF fluorophores: long-wavelength single-molecule emitters designed for cellular imaging.

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.