A Photoactivatable Push−Pull Fluorophore for Single-Molecule Imaging in Live Cells

Abstract

Recent advances in optical imaging with single molecules beyond the diffraction limit (e.g., PALM, FPALM, STORM) 1-3 have introduced a new requirement for fluorescent labels: fluorophores must be actively controlled (usually via photoswitching or photoactivation) to ensure that only a single emitter is switched on at a time in a diffraction-limited region. The location of each of these sparse molecules is precisely determined, and a super-resolution image is obtained from the summation of many successive rounds of photoactivation. The ultimate spatial resolution is determined by a number of factors, most importantly the total number of photons detected from each individual molecule. 4 Super-resolution imaging by these methods, and cell imaging in general, often use photoswitchable fluorescent proteins, which have the advantage of being genetically targeted; 5,6 however, they provide 10-fold fewer photons before photobleaching than good small-molecule emitters. 7 Bright organic fluorophores that can be turned on and/or off are therefore attractive; also smaller labels might be less perturbative than fluorescent proteins, their chemistries and photophysics can be more readily tailored, and they can be targeted using schemes actively under development. 8-12Here, we report a new, bright photoactivatable organic fluorophore that can be imaged at the single-molecule level in living cells. We use the DCDHF class of single-molecule cellular labels, 13-15 in which an amine donor is connected to a dicyanomethylenedihydrofuran acceptor via a conjugated π-bonded network. We have reengineered a red-emitting DCDHF to produce the fluorogen 1, a molecule that is dark until photoactivated with a short burst of low-intensity violet light. Photoactivation leads to conversion of the azide moiety to an amine, which shifts the absorption to long wavelengths and creates a bright, red emitter that is photostable enough to be imaged on the single-molecule level in living cells. Our proof-ofprinciple demonstration shows that photoactivation of an azide-based DCDHF fluorogen provides a new class of labels that would be useful for super-resolution imaging schemes that require active control of single molecules.A fluorescent label useful for live-cell imaging must be pumped at wavelengths >500 nm to avoid cellular autofluorescence. Figure 1A shows that activating the fluorogen 1 at 407 nm leads to loss of the azido-DCDHF absorption at 424 nm and the formation of long-wavelength absorption at 570 nm from 2. The fluorogen does not absorb at 594 nm, but this wavelength does strongly pump the emissive photoproduct 2. Thus, imaging with 594 nm produces no emission until 407 nm is used to turn-on of fluorescence.Drawing from the extensive work on the photochemistry of aryl azides, 16 we expected that photoelimination of N 2 would produce a highly reactive nitrene, which could subsequently react either by inserting into bonds of surrounding molecules (2 and Supporting Information (SI)) or intramolecularly via a ring-expansion to an azepine. Produc...

Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread function

“…The molecule is a derivative of the previously described class of photoswitchable fluorogenic azido-DCDHF molecules, specifically (E)-2-(4-(4-azido-2,3,5,6-tetrafluorostyryl)-3-cyano-5,5-dimethylfuran-2(5H)-ylidene)malononitrile, abbreviated as DCDHF-V-PF 4 -azide (see SI Appendix for structure). Although a full photophysical characterization of this molecule is needed, it was chosen for this study because (i) the azido-DCDHF class of fluorophores emit on the order of 10 6 photons before photobleaching (27) (an order of magnitude more than photoswitchable fluorescent proteins), (ii) the molecule has a suitable emission wavelength for our SLM, and (iii) a relatively small amount of blue light irradiance is necessary for photoactivation. For the data shown, the fluorogenic azide functionalized molecule was previously irradiated with 407-nm light to generate the amine functionalized emissive form (27).…”

“…Although a full photophysical characterization of this molecule is needed, it was chosen for this study because (i) the azido-DCDHF class of fluorophores emit on the order of 10 6 photons before photobleaching (27) (an order of magnitude more than photoswitchable fluorescent proteins), (ii) the molecule has a suitable emission wavelength for our SLM, and (iii) a relatively small amount of blue light irradiance is necessary for photoactivation. For the data shown, the fluorogenic azide functionalized molecule was previously irradiated with 407-nm light to generate the amine functionalized emissive form (27). For imaging, the molecule was pumped with 514 nm and the fluorescence peaking at 580 nm was recorded for 500 ms per frame to yield images similar to Fig.…”

“…photoactivatable 2-dicyanomethylene-3-cyano-2,5-dihydrofuran (DCDHF) fluorophore, a modification of the recently reported azido-DCDHF (27). Two molecules as close as 14 nm (x), 26 nm (y), and 21 nm (z) are resolved by this technique.…”

Photoswitches: Key molecules for subdiffraction‐resolution fluorescence imaging and molecular quantification

“…Recent examples of photoactivatable fluorophores are "push-pull fluorophores" [114] that are activated through a conversion of an azide moiety into an amine, and photochromic rhodamines (rhodamine amides) that can be photoactivated upon excitation in the UV at ∼ 375 or via two-photon excitation at ∼ 750 nm, and as such, even allow for three-dimensional high-resolution microscopy ( Fig. 9b) [50].…”

Photoswitchable Turn-on Mode Fluorescent Diarylethenes: Strategies for Controlling the Switching Response

“…Although several turn-on mode photoactivatable fluorophores, such as dihydrofuran, 41 coumarine, 42 fluorescein, 43 rhodamine 44 and anthracene, 45 have been reported, their photoactivation quantum yields are relatively low and the reactions are irreversible. Recently, a new type of turn-on mode photoswitchable fluorescent molecules has been developed.…”