Hsiao-lu D. Lee

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.

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.

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).

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.

Single-Molecule Photocontrol and Nanoscopy

Fluorescence microscopy is a ubiquitous tool in biological studies, but fundamental diffraction limits its resolution to ~200 nm for visible light. To overcome this physical limit, but still retain the advantages of far-field noninvasive fluorescence imaging, single-molecule photocontrol has been utilized to achieve optical nanoscopy. Superlocalization, combined with photocontrol of single molecules, allows individual molecules to be localized to precisions of tens of nanometers as part of a larger biological structure, thereby achieving super-resolution. Photoactivation, photoswitching, and photoinduced blinking are all methods of photocontrol, and critical characterization and performance parameters of photocontrollable fluorophores are discussed. We describe two classes of small molecules for use in photoactivation (azido-dicyanomethylenedihydrofuran molecules) and photoswitching (Cy3–Cy5 covalent heterodimer) studies. Furthermore, the use of the first-reported photoswitchable fluorescent protein, enhanced yellow fluorescent protein (eYFP), is also discussed for photoswitching and for photoinduced blinking experiments. Importantly, all of these methods of photocontrol have demonstrated remarkable usefulness in super-resolution studies of structures in living cells.

Photoactivatable Push-Pull Fluorophores for Single-Molecule Imaging in and out of Cells

We have designed a series of photoactivatable push-pull organic fluorophores, single molecules of which can be imaged in living cells. Photoactivatable probes are needed for superresolution imaging schemes that require active control of single-molecule emission.