-
-
-
-
-
Biophysical Journal 12/2009; 98:203. · 3.65 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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 Journal of Physical Chemistry B 10/2009; 114(45):14157-67. · 3.70 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Liquid crystal-based detectors of bio/chemical molecules based on a surface alignment transition of the liquid crystal director have been demonstrated. This type of detector could be made more sensitive if the alignment transition was more abrupt and tunable to be at a ‘trigger’ point for the desired level of concentration of the target molecule. In this study, we investigate the use of ‘double layer alignment films’ to cause a more abrupt change in the bulk surface alignment of a liquid crystal-based sensor of lecithin. We show that the detection level of the percentage of lecithin dissolved in the liquid crystal host can be controlled from greater than 4% for a conventional single layer alignment film, to less than 0.1% using the double layer alignment film method. This result verifies an earlier theory, which predicted that a double layer alignment film should provide an abrupt surface alignment transition. The utility of this controllable surface alignment transition as a sensor lies in the anisotropy of liquid crystal materials and the amplification of a detectable signal arising from this anisotropy that results from the alignment of the bulk liquid crystal material in contact with it. For example, the surface alignment change would be expected to cause a large change in capacitance (×2) of a micro-capacitor on an integrated circuit. This work provides a direction for enhancement of liquid crystal-based sensors of other biomolecules, drugs and chemicals.
Liquid Crystals. 10/2009; 36(10-11):1031-1035.
-
[show abstract]
[hide abstract]
ABSTRACT: We demonstrate single-molecule fluorescence imaging beyond the optical diffraction limit in 3 dimensions with a wide-field microscope that exhibits a double-helix point spread function (DH-PSF). The DH-PSF design features high and uniform Fisher information and has 2 dominant lobes in the image plane whose angular orientation rotates with the axial (z) position of the emitter. Single fluorescent molecules in a thick polymer sample are localized in single 500-ms acquisitions with 10- to 20-nm precision over a large depth of field (2 microm) by finding the center of the 2 DH-PSF lobes. By using a photoactivatable fluorophore, repeated imaging of sparse subsets with a DH-PSF microscope provides superresolution imaging of high concentrations of molecules in all 3 dimensions. The combination of optical PSF design and digital postprocessing with photoactivatable fluorophores opens up avenues for improving 3D imaging resolution beyond the Rayleigh diffraction limit.
Proceedings of the National Academy of Sciences 03/2009; 106(9):2995-9. · 9.68 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: We have designed and studied the photophysics of a class of organic fluorophores termed "DCDHFs," which were originally used as push-pull chromophores for nonlinear optical applications. In this paper, we describe the general photophysics of many realizations of the DCDHF class of single-molecule emitters. Moreover, we have reengineered a red-emitting DCDHF 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 singlemolecule level in living cells. This molecule and its relatives will provide a new class of bright photoactivatable fluorophores, as are needed for super-resolution imaging schemes that require active control of single-molecule emission.© (2009) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.
02/2009;
-
[show abstract]
[hide abstract]
ABSTRACT: A group of new fluorescent dye materials for single-molecule imaging applications comprised of an amine donor, a π-system comprised of phenyl and thiophene rings and a 2-dicyanomethylene-3-cyano-2,5-dihydrofuran acceptor group have been synthesized. Relative to comparable single-ring compounds these doubly aromatic conjugated fluorophores have red-shifted absorption and emission usually accompanied by significantly increased quantum yields. Solvatochromism studies indicate that the photophysical properties of these dyes are sensitive to the solvent polarity and environmental rigidity. Photophysical studies demonstrate that these DCDHF dye materials are strong single-molecule emitters and the total number of detected photons per molecule is among the highest thus far for this family of fluorophores.
Chemistry of Materials 02/2009; 21(5):797. · 7.29 Impact Factor
-
Samuel J Lord,
Nicholas R Conley,
Hsiao-Lu D Lee,
Stefanie Y Nishimura,
Andrea K Pomerantz,
Katherine A Willets,
Zhikuan Lu,
Hui Wang, Na Liu,
Reichel Samuel,
Ryan Weber,
Alexander Semyonov,
Meng He,
Robert J Twieg,
W E Moerner
[show abstract]
[hide abstract]
ABSTRACT: 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.
ChemPhysChem 12/2008; 10(1):55-65. · 3.41 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: A novel double‐layer alignment film (DLAF) was developed to obtain greater control of the alignment characteristics of the liquid crystal director. The DLAF consists of a thin fluorinated polymer layer on the top of a rubbed non‐fluorinated, non‐branched polyimide layer (PI 2555). Two types of fluorinated polymer with different chemical structures and wetting behaviour on PI 2555 were chosen, to provide either continuous or discontinuous top layers. The continuous top layer DLAF (DLAF‐1) exhibits an abrupt pretilt transition from planar to homeotropic as the top layer thickness increases. The discontinuous top layer DLAF (DLAF‐2) exhibits a gradual transition where the pretilt correlates with the coverage of fluorinated top layer. These two types of transitions fit with de Gennes' local Frederick's transition and Kwok's inhomogeneous alignment theories, respectively. The abrupt pretilt transition system may be promising for chemical/biosensor applications, whereas the gradual transition system is suitable for pretilt control in LCD devices.
Liquid Crystals. 10/2008; 35(10):1191-1197.
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
Journal of the American Chemical Society 08/2008; 130(29):9204-5. · 9.91 Impact Factor
