Silver nanostructure sensing platform for maximum-contrast fluorescence cell imaging.

We present herein a silver nanostructure-assisted sensing platform which consists of a combined structure of Ag nanowire (NW) and nanodot (ND) array. Highly enhanced fluorescence from fluorophore is attributed to a strongly coupled optical near-field interaction between proximately located Ag NW and NDs. We obtained enhanced fluorescence intensity with up to 140 folds, as contrasted from background intensity, reaching a theoretical maximum value. On the other hand, fluorescence lifetime was greatly reduced to 0.27 ns (from 2.17 ns for the same fluorophores without nanostructure). This novel platform can be a promising utility for optical imaging and labeling of biological systems with a great sensitivity.

Kinetics of a single cross-bridge in familial hypertrophic cardiomyopathy heart muscle measured by reverse Kretschmann fluorescence.

Familial hypertrophic cardiomyopathy (FHC) is a serious heart disease that often leads to a sudden cardiac death of young athletes. It is believed that the alteration of the kinetics of interaction between actin and myosin causes FHC by making the heart to pump blood inefficiently. We set out to check this hypothesis ex vivo. During contraction of heart muscle, a myosin cross-bridge imparts periodic force impulses to actin. The impulses are analyzed by fluorescence correlation spectroscopy (FCS) of fluorescently labeled actin. To minimize observation volume and background fluorescence, we carry out FCS measurements in surface plasmon coupled emission mode in a reverse Kretschmann configuration. Fluorescence is a result of near-field coupling of fluorophores excited in the vicinity of the metal-coated surface of a coverslip with the surface plasmons propagating in the metal. Surface plasmons decouple on opposite sides of the metal film and emit in a directional manner as far-field p-polarized radiation. We show that the rate of changes of orientation is significantly faster in contracting cardiac myofibrils of transgenic mice than wild type. These results are consistent with the fact that mutated heart muscle myosin translates actin faster in in vitro motility assays.

Single molecule kinetics in the familial hypertrophic cardiomyopathy D166V mutant mouse heart.

One of the sarcomeric mutations associated with a malignant phenotype of familial hypertrophic cardiomyopathy (FHC) is the D166V point mutation in the ventricular myosin regulatory light chain (RLC) encoded by the MYL2 gene. In this report we show that the rates of myosin cross-bridge attachment and dissociation are significantly different in isometrically contracting cardiac myofibrils from right ventricles of transgenic (Tg)-D166V and Tg-WT mice. We have derived the myosin cross-bridge kinetic rates by tracking the orientation of a fluorescently labeled single actin molecule. Orientation (measured by polarized fluorescence) oscillated between two states, corresponding to the actin-bound and actin-free states of the myosin cross-bridge. The rate of cross-bridge attachment during isometric contraction decreased from 3 s(-1) in myofibrils from Tg-WT to 1.4 s(-1) in myofibrils from Tg-D166V. The rate of detachment decreased from 1.3 s(-1) (Tg-WT) to 1.2 s(-1) (Tg-D166V). We also showed that the level of RLC phosphorylation was largely decreased in Tg-D166V myofibrils compared to Tg-WT. Our findings suggest that alterations in the myosin cross-bridge kinetics brought about by the D166V mutation in RLC might be responsible for the compromised function of the mutated hearts and lead to their inability to efficiently pump blood.

Plasmonic platforms of self-assembled silver nanostructures in application to fluorescence.

Fluorescence intensity changes were investigated theoretically and experimentally using self-assembled colloidal structures on silver semitransparent mirrors. Using a simplified quasi-static model and finite element method, we demonstrate that near-field interactions of metallic nanostructures with a continuous metallic surface create conditions that produce enormously enhanced surface plasmon resonances. The results were used to explain the observed enhancements and determine the optimal conditions for the experiment. The theoretical parts of the studies are supported with reports on detailed emission intensity changes which provided multiple fluorescence hot spots with 2-3 orders of enhancements. We study two kinds of the fluorophores: dye molecules and fluorescent nanospheres characterized with similar spectral emission regions. Using a lifetime-resolved fluorescence/reflection confocal microscopy technique, we find that the largest rate for enhancement (~1000-fold) comes from localized areas of silver nanostructures.

Analyte concentration at the tip of a nanopipette.

Concentration of molecules within the tips of nanopipettes when applying a DC voltage is herein investigated using finite-element simulations. The ion concentrations and fluxes due to diffusion, electro-migration, and electro-osmotic flow, and the electric potential are determined by the simultaneous solution of the Nernst-Planck, Poisson, and Navier-Stokes equations within the water solution containing sodium and chloride ions and negatively charged molecules. The electric potential within the pipette glass wall is at the same time determined by the Poisson equation together with appropriate boundary conditions and accounts for a field effect through the wall. Fixed negative surface charge on both the internal and external glass surfaces of the nanopipette is included together with the field effect through the glass wall to account for the electric double layer and the electro-osmosis. The inclusion of the field effect through the pipette wall is new compared to previous modeling of similar structures and is shown to be crucial for the behavior at the tip. It is demonstrated that the concentration of molecules is a consequence of ionic charge accumulation at the tip screening the electric field, thereby slowing down the electrophoretic motion of the molecules, which is further slowed down or stopped by the oppositely directed electro-osmosis. It is also shown that the trapping is very sensitive to the properties of the molecule, that is, its electrophoretic mobility and diffusion coefficient, the properties of the pipette, the ionic strength of the solution, and the applied electric field.

Reduction of photobleaching and photodamage in single molecule detection: observing single actin monomer in skeletal myofibrils.

Recent advances in detector technology make it possible to achieve single molecule detection (SMD) in a cell. SMD avoids complications associated with averaging signals from large assemblies and with diluting and disorganizing proteins. However, it requires that cells be illuminated with an intense laser beam, which causes photobleaching and cell damage. To reduce these effects, we study cells on coverslips coated with silver nanoparticle monolayers (NML). Muscle is used as an example. Actin is labeled with a low concentration of fluorescent phalloidin to assure that less than a single molecule in a sarcomere is fluorescent. On a glass substrate, the fluorescence of actin decays in a step-wise fashion, establishing a single molecule detection regime. Single molecules of actin in living muscle are visualized for the first time. NML coating decreases the fluorescence lifetime 17 times and enhances intensity ten times. As a result, fluorescence of muscle bleaches four to five times slower than on glass. Monolayers decrease photobleaching because they shorten the fluorescence lifetime, thus decreasing the time that a fluorophore spends in the excited state when it is vulnerable to oxygen attack. They decrease damage to cells because they enhance the electric field near the fluorophore, making it possible to illuminate samples with weaker light.

Focused optical beams obtained at planar structures by an imaginary shift in position.

A theory for focused optical beams at planar structures is described. It is an extension of a previous theory based on summation of plane waves. The focused beam is obtained by an imaginary shift in the position vector of the plane waves. It is well suited for calculations of electromagnetic fields at planar surface plasmon resonance structures excited by a focused optical beam.

Red blood cells do not attenuate the SPCE fluorescence in surface assays.

We describe the positive effect of surface plasmon-coupled fluorescence emission (SPCE) on the detection of a signal from a surface immunoassay in highly absorbing or/and scattering samples. A model immunoassay using fluorescently labeled anti-rabbit antibodies that bind to rabbit immunoglobulin on a silver surface was performed, and the signal was detected in the presence of various highly absorbing and/or scattering solutions or suspensions, such as hemoglobin solution, plastic beads, and red blood cells. The results showed that a highly absorbing solution consisting of small molecules (dye, hemoglobin) attenuates the SPCE signal approximately 2-3-fold. In contrast, suspensions with the same absorption containing large particles (large beads, red blood cell suspension) attenuate the SPCE signal only slightly, approximately 5-10%. Also, a suspension of large undyed, highly scattering beads does not reduce the SPCE signal. The effects on the immunoassay signal of the sample background absorption and scattering, the size of the background particles, and the geometry of the experimental set-up are discussed. We believe that SPCE is a promising technique in the development of biosensors utilized for surface-based assays, as well as any assays performed directly in highly absorbing and/or scattering solutions without washing or separation procedures. Figure Red blood cells (unlike hemoglobin) do not attenuate the SPCE fluorescence in surface assays.

Interference of surface plasmon resonances causes enhanced depolarized light scattering from metal nanoparticles.

We show that the strongly depolarized light scattering from noble metal particles is a result of interference of two surface plasmon resonances on the same particle. The maximum depolarization occurs between two resonances. Under favorable conditions the anisotropy of the scattering light can be much lower than what is possible for dielectric particles. This explanation is discussed in relation to earlier published experimental measurements. Comparison of experimental results with theoretical calculations provides information on the shape distribution of metallic particles in the suspension.

Propensity of a circadian clock to adjust to the 24h day-night light cycle and its sensitivity to molecular noise.

The circadian clock of Drosophila melanogaster and its tendency to adjust to the day-night light cycle is simulated by deterministic and stochastic methods. The robustness of the locking to the light-cycle with respect to molecular noise is studied. It is found that within the model studied, the molecular noise in the stochastic simulation erases the finer injection-locking structures, stronger injection signals are needed and the locking has the character of prolonged locked time intervals with cycle slips in between. The simulations are compared to a simple injection-locking model with noise that seems to describe the overall behavior well.

Trapping single molecules by dielectrophoresis.

We have trapped single protein molecules of R-phycoerythrin in an aqueous solution by an alternating electric field. A radio frequency voltage is applied to sharp nanoelectrodes and hence produces a strong electric field gradient. The resulting dielectrophoretic forces attract freely diffusing protein molecules. Trapping takes place at the electrode tips. Switching off the field immediately releases the molecules. The electric field distribution is computed, and from this the dielectrophoretic response of the molecules is calculated using a standard polarization model. The resulting forces are compared to the impact of Brownian motion. Finally, we discuss the experimental observations on the basis of the model calculations.

Directional two-photon induced surface plasmon-coupled emission.

We measured a directional surface plasmon-coupled emission (SPCE) induced by a two-photon absorption. A 60 nm thick layer of poly(vinyl alcohol) film doped with rhodamine 123 was deposited on a silvered (50 nm Ag) glass slide, which was attached to a hemicylindrical glass prism. The 820 nm excitation from a femtosecond Ti:Sapphire laser was used either in reverse Kretschmann or Kretschmann configuration. The angular distribution of two-photon induced SPCE does not depend on the used configuration. The two-photon induced SPCE can be applied to improve immunoassays and deoxyribonucleic acid detection.

Surface plasmon-coupled emission and Fabry-Perot resonance in the sample layer: A theoretical approach.

A theoretical approach is used to investigate the coupling of surface plasmon-coupled emission to Fabry-Perot resonance in the sample layer. Quantities investigated are emission angles, polarization, power levels, and fluorescence lifetimes. The results are compared to experimental findings. For comparison a layered structure without surface plasmons, possessing only dielectric Fabry-Perot resonances, is explored. This structure seems to be amenable to s-polarization only but is in principle loss-less and has more degrees of freedom for design and optimization.

Theory and simulation of surface plasmon-coupled directional emission from fluorophores at planar structures.

A theoretical approach to surface plasmon-coupled emission (SPCE) from planar structures is developed. It is used for simulations. The results are compared to experimental findings. The match is almost perfect concerning emission angles. Power relations and decay times are reproduced qualitatively. The theory is based on Fresnel plane wave refraction at planar multilayered structures and the Weyl identity for expressing the dipolar radiation in terms of plane waves. One-dimensional integrals, used for the numerical computations, are derived for the fields, powers, and decay enhancements. This theoretical approach is shown to be well suited for design of SPCE setups and for prediction and explanation of experimental results. It also shows promise for refinement and optimization of SPCE, concerning enhancement of weak fluorophores, and usage of decay times.

Optical trapping of single fluorescent molecules at the detection spots of nanoprobes.

We propose a scheme of optical trapping of fluorescent molecules, based on the strongly enhanced optical field due to surface plasmon resonances at laser illuminated metal tips or particles. A semiclassical approach is compared to a quantum-mechanical one. Attractive as well as repulsive forces are possible depending on the wavelength of the optical field. The trapping potential is shown to be strong enough to overcome the Brownian motion in water solution for common optical tweezer light inten-sities. Single molecule resonance Raman spectroscopy probes are particularly well suited for the trap-ping scheme. Finally we propose intracellular probing of the function of biomolecules as an application.