fluorescence-recovery-after-merging-droplet-to-measure-two

Fluorescence Recovery after Merging a Droplet to Measure the Two-dimensional Diffusion of a Phospholipid Monolayer

We introduce a new method to measure the lateral diffusivity of a surfactant monolayer at the fluid-fluid interface, called fluorescence recovery after ...

KAIST

In this article we present experimental results demonstrating an approach to controlling the size and spatial patterning of defect domains in a smectic liquid crystal (LC) by geometric confinement in surface-modified microchannels. By confining the LC 4'-octyl-4-cyanobiphenyl in mum-sized rectangular channels with controlled surface polarity, we were able to generate defect domains that are not only nearly uniform in size but also arranged in quasi-2D ordered patterns. Atomic force microscopy measurements revealed that the defects have a toroidal topology, which we argue is dictated by the boundary conditions imposed by the walls of the microchannel. We show that the defects can be considered to be colloidal objects, which interact with each other to form ordered patterns. This method opens the possibility for exploiting the unique optical and rheological properties associated with LC defects to making new materials. For example, the control of the shape, size, and spatial arrangement of the defects at the mesoscale suggests applications in patterning, templating, and when extended to lyotropic LCs, a process leading to uniform-sized spherical particles for chemical encapsulation and delivery.

Ordered patterns of liquid crystal toroidal defects by microchannel confinement.

Liquid-ordered (L(O)) domains reconstituted in model membranes have provided a useful platform for in vitro studies of the lipid-raft model, in which signalling membrane molecules are thought to be compartmentalized in sphingolipid- and cholesterol-rich domains. These in vitro studies, however, have relied on an uncontrolled phase-separation process that gives a random distribution of L(O) domains. Obviously, a precise control of the size and spatial distribution of the L(O) domains would enable a more systematic large-scale in vitro study of the lipid-raft model. The prerequisite for such capability would be the generation of a well-defined energy landscape for reconstituting the L(O) domain without disrupting the two-dimensional (2D) fluidity of the model membrane. Here we report controlling the reconstitution of the L(O) domains in a spatially selective manner by predefining a landscape of energy barriers using topographic surface modifications. We show that the selective reconstitution spontaneously arises from the 2D brownian motion of nanoscale L(O) domains and signalling molecules captured in these nanodomains, which in turn produce a prescribed, concentrated downstream biochemical process. Our approach opens up the possibility of engineering model biological membranes by taking advantage of the intrinsic 2D fluidity. Moreover, our results indicate that the topographic configuration of cellular membranes could be an important machinery for controlling the lipid raft in vivo.

Topographic control of lipid-raft reconstitution in model membranes.

We present persistence length measurements on neurofilaments (NFs), an intermediate filament with protruding side arms, of the neuronal cytoskeleton. Tapping mode atomic force microscopy enabled us to visualize and trace at subpixel resolution photoimmobilized NFs, assembled at various subunit protein ratios, thereby modifying the side-arm length and chain density charge distribution. We show that specific polyampholyte sequences of the side arms can form salt-switchable intrafilament attractions that compete with the net electrostatic and steric repulsion and can reduce the total persistence length by half. The results are in agreement with present X-ray and microscopy data yet present a theoretical challenge for polyampholyte interchain interactions.

Unconventional salt trend from soft to stiff in single neurofilament biopolymers.

The review will describe experiments inspired by the rich variety of bundles and networks of interacting microtubules (MT), neurofilaments, and filamentous-actin in neurons where the nature of the interactions, structures, and structure-function correlations remain poorly understood. We describe how three-dimensional (3D) MT bundles and 2D MT bundles may assemble, in cell free systems in the presence of counter-ions, revealing structures not predicted by polyelectrolyte theories. Interestingly, experiments reveal that the neuronal protein tau, an abundant MT-associated-protein in axons, modulates the MT diameter providing insight for the control of geometric parameters in bio- nanotechnology. In another set of experiments we describe lipid-protein-nanotubes, and lipid nano-tubes and rods, resulting from membrane shape evolution processes involving protein templates and curvature stabilizing lipids. Similar membrane shape changes, occurring in cells for the purpose of specific functions, are induced by interactions between membranes and proteins. The biological materials systems described have applications in bio-nanotechnology.

Nanoscale assembly in biological systems: from neuronal cytoskeletal proteins to curvature stabilizing lipids.

A previous study (Ahmed, A. et al., Anal. Chem. 84, 1146-1151( 2012) reported that toluene used as a solvent was the proton source for polyaromatic hydrocarbon compounds (PAHs) that were subjected to (+)-mode atmospheric-pressure photoionization. In the current study, the exact position of the hydrogen atom in the toluene molecule (either a methyl hydrogen or an aromatic ring hydrogen) involved in the formation of protonated PAH ions was investigated. Experimental analyses of benzene and anisole demonstrated that although the aromatic hydrogen atom of toluene did not contribute to the formation of protonated anthracene, it did contribute to the formation of protonated acridine. Thermochemical data and quantum mechanical calculations showed that the protonation of anthracene by an aromatic ring hydrogen atom of toluene is endothermic, while protonation by a methyl hydrogen atom is exothermic. However, protonation of acridine by either an aromatic ring hydrogen or a methyl hydrogen atom of toluene is exothermic. The different behavior of acridine and anthracene was attributed to differences in gas-phase basicity. It was concluded that both types of hydrogen in toluene can be used for protonation of PAH compounds, but a methyl hydrogen atom is preferred, especially for non-basic compounds.

Which hydrogen atom of toluene protonates PAH molecules in (+)-mode APPI MS analysis?

Despite the recent development of several super-resolution fluorescence microscopic techniques, there are still few techniques that can be readily employed in conventional imaging systems. We present a very simple, rapid, general and cost-efficient super-resolution imaging method, which can be directly employed in a simple fluorescent imaging system with general fluorophores. Based on diffusion-assisted Förster resonance energy transfer (FRET), fluorescent donor molecules that label specific target structures can be stochastically quenched by diffusing acceptor molecules, thereby temporally separating otherwise spatially overlapped fluorescence signals and allowing super-resolution imaging. The proposed method provides two- to three-fold-enhancement in spatial resolution, a significant optical sectioning property, and favorable temporal resolution in live-cell imaging. We demonstrate super-resolution live-cell dynamic imaging using general fluorophores in a standard epi-fluorescence microscope with light-emitting diode (LED) illumination. Due to the simplicity of this approach, we expect that the proposed method will prove an attractive option for super-resolution imaging.

Simple super-resolution live-cell imaging based on diffusion-assisted Förster resonance energy transfer.

Understanding the distinctive phase behaviors in random packing due to particle shapes is an important issue in condensed matter physics. In this paper, we investigate the random packing structure of two-dimensional (2D) snowmen via wax-snowman packing experiments and Brownian dynamics simulations. Both experiments and simulations reveal that neighboring snowmen have a strong short-range orientational correlation and consequently locally form particular conformations. A chiral conformation is dominant for high area fractions near the jamming condition (φ>0.8), and the proportion of the chiral conformation increases with γ. We also found that the attractive interaction does not have a significant impact on the results. The geometry of chirally ordered snowmen causes a mismatch with 2D crystalline symmetries and thus inhibits the development of long-range spatial order, despite the strong orientational correlation between neighbors.

Shape-induced chiral ordering in two-dimensional packing of snowmanlike dimeric particles.

Bundles of taxol-stabilized microtubules (MTs)--hollow tubules comprised of assembled αβ-tubulin heterodimers--spontaneously assemble above a critical concentration of tetravalent spermine and are stable over long times at room temperature. Here we report that at concentrations of spermine several-fold higher the MT bundles (B(MT)) quickly become unstable and undergo a shape transformation to bundles of inverted tubulin tubules (B(ITT)), the outside surface of which corresponds to the inner surface of the B(MT) tubules. Using transmission electron microscopy and synchrotron small-angle X-ray scattering, we quantitatively determined both the nature of the B(MT)-to-B(ITT) transformation pathway, which results from a spermine-triggered conformation switch from straight to curved in the constituent taxol-stabilized tubulin oligomers, and the structure of the B(ITT) phase, which is formed of tubules of helical tubulin oligomers. Inverted tubulin tubules provide a platform for studies requiring exposure and availability of the inside, luminal surface of MTs to MT-targeted drugs and MT-associated proteins.

Transformation of taxol-stabilized microtubules into inverted tubulin tubules triggered by a tubulin conformation switch.

Microtubules (MTs) are nanometer scale hollow cylindrical biological polyelectrolytes. They are assembled from alpha/beta-tubulin dimers, which stack to form protofilaments (PFs) with lateral interactions between PFs resulting in the curved MT. In cells, MTs and their assemblies are critical components in a range of functions from providing tracks for the transport of cargo to forming the spindle structure during mitosis. Previous studies have, shown that while cations with valence equal to or larger than 3+ tend to assemble tight 3D bundles of taxol-stabilized MTs, certain divalent cations induce relatively loose 2D bundles of different symmetry (D. J. Needleman et al., Proc. Natl. Acad. Sci. U. S. A., 2004, 101, 16099). Similarly, divalent cations form 2D bundles of DNA adsorbed on cationic membranes (I. Koltover et al., Proc. Natl. Acad. Sci. U. S. A., 2000, 97, 14046). The bundling behavior for these biological polyelectrolyte systems is qualitatively in agreement with current theory. Here, we present results which show that, unlike the case for DNA adsorbed on cationic membranes, bundling of taxol-stabilized MTs occurs only for certain divalent cations above a critical ion concentration (e.g. Ca2+, Sr2+, Ba2+). Instead, many divalent cations pre-empt the bundling transition and depolymerize taxol-stabilized MTs at a lower counterion concentration. Although previous cryogenic TEM has shown that, in the absence of taxol, Ca2+ depolymerizes MTs assembling in buffers containing GTP (guanosine triphosphate), our finding is surprising given the know stabilizing effects of taxol on GDP (guanosine diphosphate)-MTs. The ion concentration required for MT depolymerization decreases with increasing atomic number for the divalents Mg2+, Mn2+, Co2+, and Zn2+. GdCl3 (3+) is found to be extremely efficient at MT depolymerization requiring ion concentrations of about 1 mM, while oligolysine(2+), is observed not to depolymerize MTs at concentrations as high as 144 mM. The surprising MT depolymerization results are discussed in the context of divalents either disrupting lateral interactions between PFs (which are strengthened for taxol containing beta-tubulin) or interfering with taxol's ability to induce flexibility at the interface between two tubulin dimers in the same PF (which has been recently suggested as a mechanism by which taxol stabilizes MTs post-hydrolysis with the induced flexibility counteracting the kink between GDP-tublin dimers in a PF).

Ion specific effects in bundling and depolymerization of taxol-stabilized microtubules.

A unilamellar polymeric vesicle is a self-assembled structure of a block copolymer that forms a spherical single bilayer structure with a hydrophobic interlayer and a hydrophilic surface. Due to their enhanced colloidal stability and mechanical property, controllable surface functionality, or tunable membrane thickness, polymeric vesicles are useful in nano and bio-science, providing potential applications as nanosized carriers for catalysts, drugs, and enzymes. For fabrication of a unilamellar vesicle, however, preparative procedures with a few steps are inherently required. Herein, without complicated preparative procedures, we report spontaneous unilamellar polymeric vesicles with nanometer sizes (<100 nm), which are prepared by simply mixing a triblock copolymer, Pluronic P85 (PEO26PPO40PEO26), and an organic derivative, 5-methyl salicylic acid (5mS), in aqueous solution. Depending on the 5mS concentration and the temperature, the P85-5mS mixtures presented various self-assembled nanostructures such as spherical and cylindrical micelles or vesicles, which were characterized by small angle neutron scattering and cryo-TEM, resulting in a phase diagram drawn as a function of temperature and the 5mS concentration. Interestingly the critical temperature for the micelle-to-vesicle phase transition was easily controlled by varying the 5mS concentration, i.e. it was decreased with increasing the 5mS concentration.

Spontaneous unilamellar polymer vesicles in aqueous solution.

Optical trapping of nanometer-sized lipid vesicles has been challenging due to the low refractive index contrast of the thin lipid bilayer to the aqueous medium. Using an "optical bottle", a recently developed technique to measure interactions of nanoparticles trapped by an infrared laser, we report, for the first time, quantitative measurements of the trapping energy of charged lipid vesicles. We found that the trapping energy increases with the relative amount of anionic lipids (DOPG) to neutral lipids (DOPC) in vesicles. Moreover, as monovalent salt is added into the exterior solution of vesicles, the trapping energy rapidly approaches zero, and this decrease in trapping energy strongly depends on the amount of anionic lipids in vesicles. A simple model with our experimental observations explains that the trapping energy of charged lipid vesicles is highly correlated with the surface charge density and electric double layer. In addition, we demonstrated selective trapping of a binary mixture of vesicles in different mole fractions of charged lipids, a strategy that has potential implications on charge selective vesicle sorting for engineering applications.

Surface charge effects on optical trapping of nanometer-sized lipid vesicles.

We study electrostatic interactions of polystyrene particles at an oil/water interface controlled by a chemical reaction of carboxylate surface functional groups. By replacing the carboxyl functional groups with hydrocarbon chains using the well-known EDC (1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide) coupling reaction, the surface charge density decreases while the hydrophobicity of the colloid surface increases. Direct visualization of the particle-laden interface reveals that, depending on the extent of hydrocarbon coupling, the strength of the electrostatic repulsion can be regulated: the repulsive interaction increases with the reaction, removing aggregates, but rapidly decreases if the reaction proceeds too much, forming a large aggregation. This simple reaction, thus, dramatically changes the structures of the colloidal monolayers at the oil/water interface. We conclude that such structural change is the result of change of the repulsive interactions from the oil phase, although interactions in the water phase are also changed slightly.

Surface charge regulation of carboxyl terminated polystyrene latex particles and their interactions at the oil/water interface.

We present a novel technique to measure diffusion coefficients of insoluble surfactant monolayers. We merge a surfactant-coated droplet with a fluorescently labeled planar monolayer. During the merging process, a monolayer on a droplet displaces the existing planar monolayer, leaving a dark area when viewed under a fluorescence microscope. We measure fractional intensities as the dyes recover, which allows diffusion coefficients to be computed. We validate this technique with the two most common phospholipid monolayers (DPPC and DOPC) and study the diffusion of their mixtures. The proposed technique has several advantages over the FRAP technique and is potentially capable of measuring the diffusion of any soluble/insoluble surfactant monolayers.

Fluorescence recovery after merging a surfactant-covered droplet: a novel technique to measure the diffusion of phospholipid monolayers at fluid/fluid interfaces.

Myung Chul Choi

Department of Bio and Brain Engineering

KAIST

Fluorescence Recovery after Merging a Droplet to Measure the Two-dimensional Diffusion of a Phospholipid Monolayer

Myung Chul Choi

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Bio and Brain Engineering

KAIST

Fluorescence Recovery after Merging a Droplet to Measure the Two-dimensional Diffusion of a Phospholipid Monolayer

Department of Bio and Brain Engineering