Poul M. Bendix

Heat Profiling of Three-Dimensionally Optically Trapped Gold Nanoparticles using Vesicle Cargo Release

Irradiated metallic nanoparticles hold great promise as heat transducers in photothermal applications such as drug delivery assays or photothermal therapy. We quantify the temperature increase of individual gold nanoparticles trapped in three dimensions near lipid vesicles exhibiting temperature sensitive permeability. The surface temperature can increase by hundreds of degrees Celsius even at moderate laser powers. Also, there are significant differences of the heat profiles in two-dimensional and three-dimensional trapping assays.

Large-Scale Orientation Dependent Heating from a Single Irradiated Gold Nanorod

We quantify the extreme heating associated with resonant irradiation of individual gold nanorods by using a novel assay based on partitioning of lipophilic dyes between membrane phases. The temperature increase is sensitively dependent on the angle between the laser polarization and the orientation of the nanorod. A dramatic and irreversible decrease in the heating of a nanorod occurs at high-illumination intensities; this effect is attributed to surface melting of the nanorod causing it to restructure into a more spherical shape and lose its extreme photothermal properties.

Quantification of nano-scale intermembrane contact areas by using fluorescence resonance energy transfer

Nanometer-scale intermembrane contact areas (CAs) formed between single small unilamellar lipid vesicles (SUVs) and planar supported lipid bilayers are quantified by measuring fluorescence resonance energy transfer (FRET) between a homogenous layer of donor fluorophores labeling the supported bilayer and acceptor fluorophores labeling the SUVs. The smallest CAs detected in our setup between biotinylated SUVs and dense monolayers of streptavidin were ≈20 nm in radius. Deformation of SUVs is revealed by comparing the quenching of the donors to calculations of FRET between a perfectly spherical shell and a flat surface containing complementary fluorophores. These results confirmed the theoretical prediction that the degree of deformation scales with the SUV diameter. The size of the CA can be controlled experimentally by conjugating polyethylene glycol polymers to the SUV or the surface and thereby modulating the interfacial energy of adhesion. In this manner, we could achieve secure immobilization of SUVs under conditions of minimal deformation. Finally, we demonstrate that kinetic measurements of CA, at constant adhesion, can be used to record in real-time quantitative changes in the bilayer tension of a nano-scale lipid membrane system.

Optical Trapping of Nanoparticles and Quantum Dots

Optical manipulation of nanostructures offers new exciting possibilities for building new nano-architectures and for exploring the fundamental interactions between light and nanoparticles. The optical properties of nanostructures differ substantially from those of similar bulk material and exhibit an exquisite sensitivity on nanoparticle shape and composition. The plethora of particles available today expands the possibilities of optical manipulation to include control over particle temperature, luminescence, orientation, and even over the rotational optical momentum transferred to the nanoparticle. Here, we summarize recent experimental advances within optical manipulation of individual nanoparticles and quantum dots with a focus on resonant versus non-resonant trapping, optically induced heating, spherical aberration, and orientation control. Also, we present novel quantitative data on the photonic interaction between gold nanoshells and a focused laser beam. Lastly, promising applications of the biophotonical properties of nanoparticles within nanoscience and biophysics are pointed out.

FBAR Syndapin 1 recognizes and stabilizes highly curved tubular membranes in a concentration dependent manner

Syndapin 1 FBAR, a member of the Bin-amphiphysin-Rvs (BAR) domain protein family, is known to induce membrane curvature and is an essential component in biological processes like endocytosis and formation and growth of neurites. We quantify the curvature sensing of FBAR on reconstituted porcine brain lipid vesicles and show that it senses membrane curvature at low density whereas it induces and reinforces tube stiffness at higher density. FBAR strongly up-concentrates on the high curvature tubes pulled out of Giant Unilamellar lipid Vesicles (GUVs), this sorting behavior is strongly amplified at low protein densities. Interestingly, FBAR from syndapin 1 has a large affinity for tubular membranes with curvatures larger than its own intrinsic concave curvature. Finally, we studied the effect of FBAR on membrane relaxation kinetics with high temporal resolution and found that the protein increases relaxation time of the tube holding force in a density-dependent fashion.

Expanding the optical trapping range of lipid vesicles to the nanoscale.

Small unilamellar lipid vesicles with diameters down to 50 nm enclosing high refractive index sucrose cores can be optically trapped individually in three dimensions using a focused laser beam. Combined optical trapping and confocal microscopy allows for simultaneous quantitative measurements of the forces exerted on individual vesicles and of their size and shape. The position of individual vesicles in three dimensions is measured with nanometer spatial and ∼10 μs temporal resolution.

Heat Generation by Irradiated Complex Composite Nanostructures

Heating of irradiated metallic e-beam generated nanostructures was quantified through direct measurements paralleled by novel model-based numerical calculations. By comparing discs, triangles, and stars we showed how particle shape and composition determines the heating. Importantly, our results revealed that substantial heat is generated in the titanium adhesive layer between gold and glass. Even when the Ti layer is as thin as 2 nm it absorbs as much as a 30 nm Au layer and hence should not be ignored.

Mapping 3D focal intensity exposes the stable trapping positions of single nanoparticles.

The photonic interactions between a focused Gaussian laser beam and a nanoscopic particle are highly dependent on exact particle location and focal intensity distribution. So far, the 3D focal intensity distribution and the preferred position of a nanoparticle confined within the focal region were only theoretically predicted. Here, we directly map the three-dimensional focal intensity distribution, quantify stable trapping positions, and prove that certain sizes of nanoparticles stably trap in front of the focus.

Helical buckling of actin inside filopodia generates traction.

Significance Filopodia are essential membrane protrusions that facilitate cellular sensing and interaction with the environment. The mechanical properties of filopodia are crucial for their ability to push and pull on external objects and are attributed to actin dynamics. We confirm the presence of F-actin inside extended filopodia and reveal a new mechanism by which actin can exert traction forces on external objects. This mechanism is mediated by rotation and helical buckling, which cause shortening and retraction of the actin shaft. By imaging of F-actin and simultaneous force spectroscopy, we reveal and detail how force propagates through this spiral actin structure and show how torsional twist of the actin shaft is translated into a traction force at the filopodial tip. Cells can interact with their surroundings via filopodia, which are membrane protrusions that extend beyond the cell body. Filopodia are essential during dynamic cellular processes like motility, invasion, and cell–cell communication. Filopodia contain cross-linked actin filaments, attached to the surrounding cell membrane via protein linkers such as integrins. These actin filaments are thought to play a pivotal role in force transduction, bending, and rotation. We investigated whether, and how, actin within filopodia is responsible for filopodia dynamics by conducting simultaneous force spectroscopy and confocal imaging of F-actin in membrane protrusions. The actin shaft was observed to periodically undergo helical coiling and rotational motion, which occurred simultaneously with retrograde movement of actin inside the filopodium. The cells were found to retract beads attached to the filopodial tip, and retraction was found to correlate with rotation and coiling of the actin shaft. These results suggest a previously unidentified mechanism by which a cell can use rotation of the filopodial actin shaft to induce coiling and hence axial shortening of the filopodial actin bundle.

Vesicle Fusion Triggered by Optically Heated Gold Nanoparticles

Membrane fusion can be accelerated by heating that causes membrane melting and expansion. We locally heated the membranes of two adjacent vesicles by laser irradiating gold nanoparticles, thus causing vesicle fusion with associated membrane and cargo mixing. The mixing time scales were consistent with diffusive mixing of the membrane dyes and the aqueous content. This method is useful for nanoscale reactions as demonstrated here by I-BAR protein-mediated membrane tubulation triggered by fusion.