Xenon gas field ion source from a single-atom tip.

Focused ion beam (FIB) systems have become powerful diagnostic and modification tools for nanoscience and nanotechnology. Gas field ion sources (GFISs) built from atomic-size emitters offer the highest brightness among all ion sources and thus can improve the spatial resolution of FIB systems. Here we show that the Ir/W(111) single-atom tip (SAT) can emit high-brightness Xe+ ion beams with a high current stability. The ion emission current versus extraction voltage was analyzed from 150 K up to 309 K. The optimal emitter temperature for maximum Xe+ ion emission was ∼150 K and the reduced brightness at the Xe gas pressure of 1 × 10-4 torr is two to three orders of magnitude higher than that of a Ga liquid metal ion source, and four to five orders of magnitude higher than that of a Xe inductively coupled plasma ion source. Most surprisingly, the SAT emitter remained stable even when operated at 309 K. Even though the ion current decreased with increasing temperature, the current at room temperature (RT) could still reach over 1 pA when the gas pressure was higher than 1 × 10-3 torr, indicating the feasibility of RT-Xe-GFIS for application to FIB systems. The operation temperature of Xe-SAT-GFIS is considerably higher than the cryogenic temperature required for the helium ion microscope (HIM), which offers great technical advantages because only simple or no cooling schemes can be adopted. Thus, Xe-GFIS-FIB would be easy to implement and may become a powerful tool for nanoscale milling and secondary ion mass spectroscopy.

Two approaches toward a high-efficiency flashing ratchet.

For a flashing ratchet with periodic potentials fluctuating via random shifts of one-half period, a high efficiency is shown to result from two mechanisms. The previously reported one [Yu. A. Makhnovskii, Phys. Rev. E 69, 021102 (2004); V. M. Rozenbaum, JETP Lett. 79, 388 (2004)] is realized in the near-equilibrium region and implies, first, the presence of a high barrier V0 blocking the reverse movement of a Brownian particle and, second, identical, though energy-shifted, portions of the asymmetric flat potential profile on both half periods. We report another mechanism acting far from equilibrium, typical of strongly asymmetric potentials which are shaped identically on both half periods with a large energetic shift DeltaV . The two mechanisms exhibit radically different limiting behavior of the maximum possible efficiency: eta(m) approximately 1-exp (-beta V0 /2) for the former and eta(m) approximately 1-ln (2betaDeltaV) /betaDeltaV for the latter ( beta being the reciprocal temperature in energy units). The flux and the efficiency for a Brownian motor with a piecewise-linear potential are calculated using the transfer matrix method; an exact analytical solution can thus be obtained for an extremely asymmetric sawtooth potential, the simplest example of the second high-efficiency mechanism. As demonstrated, the mechanisms considered are also characteristic of a two-well periodic potential treated in terms of the kinetic approach.

Flashing ratchet model with high efficiency.

As a simple model of the Brownian motor, we consider hopping motion of a particle in a periodic asymmetric double-well potential which randomly switches between two states. The potential profiles of the states are identical but shifted by half a period. The current and the efficiency are explicitly calculated as functions of the parameters of the model, including also a load force. Such a flashing ratchet is shown to be particularly efficient, with the efficiency tending to unity when the highest peak of the potential is high enough to suppress the backward motion.

Vertical friedel oscillations in interface-induced surface charge modulations of ultrathin quantum islands.

Two-dimensional Pb islands of a few atomic layers are grown on the incommensurate Si(111)-Pb surface at low temperatures. Among them, two types of islands having different stacking with the substrate are observed. These islands, respectively, display an alternating image contrast with their thickness. Besides, the contrasts of the islands of different types are complementary to each other layer by layer. These intriguing behaviors do not show significant bias dependence throughout the range from -3 to +3 V and can be explained by the vertical charge oscillation with the growth of a new layer. The charge oscillation in the out-of-plane direction originates from electron scattering by the in-plane potential variation at the Pb/Si interface.

Correlation between quantized electronic states and oscillatory thickness relaxations of 2D Pb islands on Si(111)-(7 x 7) surfaces.

Two-dimensional lead (Pb) islands of varying heights have been grown on the Si(111)-(7 x 7) surface at low temperature. Individual islands are investigated concurrently with real-space and local-probe scanning tunneling microscopy and spectroscopy. Quantum size effects, manifested in the formation of new electronic bound states, redistribution of surface charge density, and oscillatory relaxations in island thickness are found to be perfectly correlated to each other.

Targeting antigen-specific receptors on B lymphocytes to generate high yields of specific monoclonal antibodies directed against biologically active lower antigenic peptides within presenilin 1.

We describe a targeting technique that selects antigen-specific receptors on B lymphocytes using antigen driven selective production of monoclonal antibodies which are directed against functional peptide sequences within the presenilin 1 molecule that is believed to be related to the early-onset of familial Alzheimer's disease. Three different peptide sequences of presenilin 1 were constructed, one including the region around the amino acid position 300, where the putative cleavage site exists and the other two present in the N- and C-terminal regions of that site. The efficiency in production of the desired monoclonal antibodies was at least 5-40-fold that obtained with the poly(ethylene glycol) (PEG)-mediated method. In addition, monoclonal antibodies directed against each of the peptide sequence displayed a high specificity for the corresponding peptide, in contrast to the lack of success using the PEG method. Also, the selection of surface immunoglobulin receptors on B lymphocytes by the peptides of interest was confirmed by immunofluorescent analysis. Here we demonstrate that targeting B lymphocytes results in the successful and efficient production of highly specific monoclonal antibodies against the lower antigenic peptide sequences.

Direct observation of electromigration of Si magic clusters on Si(111) surfaces.

Using scanning tunneling microscopy, we have observed electromigration of Si on Si(111)-(7x7) surfaces and have identified the diffusion species to be Si magic clusters. Effects of the directed motion along the direction of the heating current in electromigration and those in thermal migration are determined separately and quantitatively. We also observe the preferential filling of two-dimensional (2D) Si craters and the preferential detachment of Si magic clusters from the edges of 2D Si islands near the cathode side. The driving force for this anisotropic behavior is much stronger than previously recognized.

Biological effects of electric shock and heat denaturation and oxidation of molecules, membranes, and cellular functions.

Direct exposure of cells in suspension to intense electric pulses is known to produce damages to cell membranes and supramolecular organizations of cells, and denaturation of macromolecules, much like injuries and tears seen in electric trauma patients. Thus, the system has been used as a laboratory model for investigating the biochemical basis of electric injury. An intense electric pulse can produce two major effects on cells--one caused by the field, or the electric potential, and the other by current, or the electric energy. The field-induced transmembrane potential can produce electro-conformational changes of ion channels and ion pumps and, when the potential exceeds the dielectric strength of the cell membrane (approximately 500 mV for a pulse width of a few ms), electro-conformational damages and electroporations of membrane proteins and lipid bilayers. These events lead to passage of electric current through the membrane-porated cells and to heating of cell membranes and cytoplasmic contents. The subsequent denaturation of cell membranes and cytoplasmic macromolecules brings about many complex biochemical reactions, including oxidation of proteins and lipids. The combined effects may cripple the cells beyond repair. This communication will focus on the thermal effects of electric shock. After a brief review of the current state of knowledge on thermal denaturation of soluble enzymes and muscle proteins, this paper will describe experiments on the thermal denaturation of cellular components and functions, such as nucleosomes, and the electron transport chain and ATP synthetic enzymes of the mitochondrial inner membranes. Data will show that lipid peroxidation and the subsequent loss of the energy-transducing ability of the cells may occur even at moderate temperatures between 40 degrees C and 45 degrees C. However, lipid peroxidation may be prevented with reducing reagents such as mercaptoethanol, dithiothreitol, and ascorbic acid. Reactivation of denatured cellular proteins and functions may also be possible and a strategy for doing so is discussed.

Fluctuation-driven directional flow in biochemical cycle: further study of electric activation of Na,K pumps.

Directional flow of information and energies is characteristic of many types of biochemical reactions, for instance, ion transport, energy coupling during ATP synthesis, and muscle contraction. Can a fluctuating force field, or a noise, induce such a directional flux? Previous work has shown that Na,K-ATPase of human erythrocyte can absorb free energy from an externally applied random-telegraph-noise (RTN) electric field to pump Rb+ up its concentration gradient. However, the RTN field used in these experiments was constant in amplitude and would not mimic fluctuating electric fields of a cell membrane. Here we show that electric fields which fluctuate both in life time and in amplitude, and thus, better mimicking the transmembrane electric fields of a cell, can also induce Rb+ pumping by Na,K-ATPase. A Gaussian-RTN-electric field, or a field with amplitude fluctuating according to the Gaussian distribution, with varied standard deviation (sigma), induced active pumping of Rb+ in human erythrocyte, which was completely inhibited by ouabain. Increased values for sigma led to a nonmonotonic reduction in pumping efficiency. A general formula for calculating the ion transport in a biochemical cycle induced by fluctuating electric field has been derived and applied to a simple four-state electroconformational coupling (ECC) model. It was found that the calculated efficiency in the energy coupling decreased with increasing sigma value, and this effect was relatively small and monotonic, whereas experimental data were more complex: monotonic under certain sets of conditions but nonmonotonic under different sets. The agreement in general features but disagreement in some fine features suggest that there are other properties of the electric activation process for Na,K-ATPase that cannot be adequately described by the simple ECC model, and further refinement of the ECC model is required.

Least activation path for protein folding: investigation of staphylococcal nuclease folding by stopped-flow circular dichroism.

Is the pathway of protein folding determined by the relative stability of folding intermediates, or by the relative height of the activation barriers leading to these intermediates? This is a fundamental question for resolving the Levinthal paradox, which stated that protein folding by a random search mechanism would require a time too long to be plausible. To answer this question, we have studied the guanidinium chloride (GdmCl)-induced folding/unfolding of staphylococcal nuclease [(SNase, formerly EC 3.1.4.7; now called microbial nuclease or endonuclease, EC 3.1.31.1] by stopped-flow circular dichroism (CD) and differential scanning microcalorimetry (DSC). The data show that while the equilibrium transition is a quasi-two-state process, kinetics in the 2-ms to 500-s time range are triphasic. Data support the sequential mechanism for SNase folding: U3 <--> U2 <--> U1 <--> N0, where U1, U2, and U3 are substates of the unfolded protein and N0 is the native state. Analysis of the relative population of the U1, U2, and U3 species in 2.0 M GdmCl gives delta-G values for the U3 --> U2 reaction of +0.1 kcal/mol and for the U2 --> U1 reaction of -0.49 kcal/mol. The delta-G value for the U1 --> N0 reaction is calculated to be -4.5 kcal/mol from DSC data. The activation energy, enthalpy, and entropy for each kinetic step are also determined. These results allow us to make the following four conclusions. (i) Although the U1, U2, and U3 states are nearly isoenergetic, no random walk occurs among them during the folding. The pathway of folding is unique and sequential. In other words, the relative stability of the folding intermediates does not dictate the folding pathway. Instead, the folding is a descent toward the global free-energy minimum of the native state via the least activation path in the vast energy landscape. Barrier avoidance leads the way, and barrier height limits the rate. Thus, the Levinthal paradox is not applicable to the protein-folding problem. (ii) The main folding reaction (U1 --> N0), in which the peptide chain acquires most of its free energy (via van der Waals' contacts, hydrogen bonding, and electrostatic interactions), is a highly concerted process. These energy-acquiring events take place in a single kinetic phase. (iii) U1 appears to be a compact unfolded species; the rate of conversion of U2 to U1 depends on the viscosity of solution. (iv) All four relaxation times reported here depend on GdmCl concentrations: it is likely that none involve the cis/trans isomerization of prolines. Finally, a mechanism is presented in which formation of sheet-like chain conformations and a hydrophobic condensation event precede the main-chain folding reaction.

Cell fission and formation of mini cell bodies by high frequency alternating electric field.

We report the use of high frequency alternating electric fields (AC) to induce deformation of sea urchin eggs, leading to budding of membrane vesicles or fission of cells. Several mini cell bodies can be prepared from a single egg by carefully manipulating the frequency and amplitude of the AC field and the ratio between the interelectrode spacing and the cell diameter, alpha. alpha values between 2.2 and 3.5 have been found to be optimal for inducing fission of sea urchin eggs. In a typical experiment, a sea urchin egg (diameter = 75 microns), suspended in a low ionic medium (conductance < 2 mS/m), was located under the microscope between two platinum wire electrodes, separated by a distance of approximately 200 microns. A medium strength AC field (< 100 V/cm at 2 MHz) was applied to attract the egg to one of the two electrodes via dielectrophoresis. This process took place in a few seconds. The voltage was then slowly increased to approximately 1000 V/cm over approximately 30 s. The cell elongated and separated into two fragments, the larger one containing the nucleus. When the field was turned off, the mother cell and the daughter vesicle retracted to form spherical mini cell bodies that appear to be stable as assessed by the absence of swelling for the duration of the experiment (approximately 15 min). This indicates that membranes of these mini cell bodies were not leaky to ions and small molecules. This procedure could be repeated a few times to make several mini cell bodies from a single egg. With practice, several minicell bodies could also be prepared in a single fission experiment by adjusting the field parameters and the a value. Cell fission is a result of the mechanical stress produced by the AC field. These procedures may be used to prepare mini membrane vesicles for voltage clamp experiments or to perform microsurgical manipulation of cells, embryos, or chromosomes.

Recognition and processing of randomly fluctuating electric signals by Na,K-ATPase.

Previous work has shown that Na,K-ATPase of human erythrocytes can extract free energy from sinusoidal electric fields to pump cations up their respective concentration gradients. Because regularly oscillating waveform is not a feature of the transmembrane electric potential of cells, questions have been raised whether these observed effects are biologically relevant. Here we show that a random-telegraph fluctuating electric field (RTF) consisting of alternating square electric pulses with random lifetimes can also stimulate the Rb(+)-pumping mode of the Na,K-ATPase. The net RTF-stimulated, ouabain-sensitive Rb+ pumping was monitored with 86Rb+. The tracer-measured, Rb+ influx exhibited frequency and amplitude dependencies that peaked at the mean frequency of 1.0 kHz and amplitude of 20 V/cm. At 4 degrees C, the maximal pumping activity under these optimal conditions was 28 Rb+/RBC-hr, which is approximately 50% higher than that obtained with the sinusoidal electric field. These findings indicate that Na,K-ATPase can recognize an electric signal, either regularly oscillatory or randomly fluctuating, for energy coupling, with high fidelity. The use of RTF for activation also allowed a quantitative theoretical analysis of kinetics of a membrane transport model of any complexity according to the theory of electroconformational coupling (ECC) by the diagram methods. A four-state ECC model was shown to produce the amplitude and the frequency windows of the Rb(+)-pumping if the free energy of interaction of the transporter with the membrane potential was to include a nonlinear quadratic term. Kinetic constants for the ECC model have been derived. These results indicate that the ECC is a plausible mechanism for the recognition and processing of electric signals by proteins of the cell membrane.

On the efficiency and reversibility of active ligand transport induced by alternating rectangular electric pulses.

The stationary-state kinetic properties of a simplified two-state electro-conformational coupling model (ECC) in the presence of alternating rectangular electric potential pulses are derived analytically. Analytic expressions for the transport flux, the rate of electric energy dissipation, and the efficiency of the transducing system are obtained as a function of the amplitude and frequency of the oscillation. These formulas clarify some fundamental concept of the ECC model and are directly applicable to the interpretation and design of experiments. Based on these formulas, the reversibility and the degree of coupling of the system can be studied quantitatively. It is found that the oscillation-induced free energy transduction is reversible and tight-coupled only when the amplitude of the oscillating electric field is infinitely large. In general, the coupling is not tight when the amplitude of the electric field is finite. Furthermore, depending on the kinetic parameters of the model, there may exist a "critical" electric field amplitude, below which free energy transduction is not reversible. That is, energy may be transduced from the electric to the chemical, but not from the chemical to the electric.

Chain condensation in protein folding.

Three reactions (relaxation times: tau 1 = 140 +/- 8 ms, tau 2 = 840 +/- 30 ms and tau 3 = 30 + 3 s, at pH 7.0 at 25 degrees C) have been resolved by the stopped-flow kinetic method for the folding of the acid denatured staphylococcal nuclease (Chen et al (1991) J Mol Biol 220, 771; Biochemistry (1992) 31, 1483). Of the three reactions only the tau 2 reaction was dependent on the viscosity of the solution in a manner consistent with the diffusion/collision/coalescence model. Experiments with site-directed mutagenesis suggest that the forming of local structures by the electrostatic interactions between Glu75 and His121 and Lys9 may induce the chain condensation.

Comparison of kinetics of formation of helices and hydrophobic core during the folding of staphylococcal nuclease from acid.

Our previous kinetic study of the acid and base-induced folding/unfolding transitions of staphylococcal nuclease (SNase) has monitored Trp-140 fluorescence. Trp-140 is located near the flexible COOH terminus and whether or not its fluorescence reflects the overall conformation of the protein has yet to be established. Here we show that the fluorescence intensity of Try-140 correlated closely with the thermal stability (i.e., the calorimetric enthalpy, delta Hcal, of unfolding) of the protein in the pH range 7 to 2.5, confirming that it is a good measure of the overall protein structure. Circular dichroism (CD) at 222 nm, which reflects the helical content of the protein molecule, was used to follow the same folding/unfolding transition in order to compare kinetics of the helix formation and of the appearance of the hydrophobic core. In addition to the three kinetic phases reported earlier with the fluorescence detection, there were a rapid reaction (completed within the 25 ms mixing time of the instrument), which comprised 15% of the signal, and a very slow reaction (time constant > 300 s), which comprised 19% of the signal. With the fluorescence detection for the folding from acid, only 5% of the signal occurred in the rapid phase and there was no reaction slower than 300 s. By comparing kinetics of folding at pH 7 by the CD and fluorescence detection methods, we concluded that: (a) Roughly 15% of the helix content of SNase accumulated before significant changes in the hydrophobic environment (< 5%) of Trp-140 could be detected. The rapid appearance of CD signal reminiscent of helix formation within 25ms would be consistent with the framework model of protein folding. Note, however, that, 15% of the 22% helix content of the protein amounts to an equivalent of fewer than 5 amino acid residues. (b) For the time-resolved signal between 2 ms and 300s, kinetics measured by both properties are consistent with the sequential model, D4 = D3 = D2 = D1 = No (the four Ds are the four substates of the denatured protein and No is the native state). The major folding step by both signals is the D1 to No transition, which gave approximately a 50% change in fluorescence and CD and had a time constant of 160 ms at 25 degree C, pH7.0. (c) The slow phase with the CD signal (>300 s), which is insensitive to Trp-140 fluorescence, has been assigned to be the cis/trans isomerization of Pro-1 17 by other studies. (d) Kinetics in the unfolding direction are consistent with the above interpretation.

Study of mechanisms of electric field-induced DNA transfection. V. Effects of DNA topology on surface binding, cell uptake, expression, and integration into host chromosomes of DNA in the mammalian cell.

Neumann and coworkers (Neumann, E., M. Schaefer-Ridder, Y. Wang, and P. H. Hofschneider. 1982. EMBO J. 1:841-845) have shown that the efficiency of pulsed electric field (PEF)-induced DNA transfection of mouse L-cells by the thymidine kinase gene is several times higher for the linear DNA than for the closed circular DNA. Transfection of Escherichia coli bacteria by several plasmids indicates that the transfection efficiency was much higher for the closed circular/supercoiled (sc-) and circular/relaxed (cr-) DNA than for the linearized (In-) DNA (Xie, T. D., L. Sun, H. G. Zhao, J. A. Fuchs, and T. Y. Tsong. 1992. Biophys. J. 63:1026-1031). To resolve these conflicting observations, we have systematically examined electrotransfection of NIH3T3 mouse fibroblast by the plasmids, pRSVcat, pRSVneo, and pRSVgpt. Mg(2+)-facilitated surface binding of DNA before, and DNA uptake by 3T3 cells after treatment with PEF, were monitored by 3H-labeled plasmids. Transfection efficiency was evaluated by both the transient expression of chloramphenicol acetyltransferase (cat) activity 2-3 days after, and the permanent expression of neomycin phosphotransferase (neo) and xanthine-guanine phosphoribosyltransferase (gpt) genes in the transformants 2 weeks after the PEF treatment. Our results indicate that cell surface binding and PEF-induced cell uptake of DNA did not depend on the topology of DNA. However, both the transient and the permanent expression of the plasmids were three to five times more efficient for the cr-DNA and the sc-DNA than for the in-DNA. These results indicate that electrotransfection of cells involves several steps: the cation-dependent binding of DNA to the cell surface, the electric field-driven DNA entry into the cells, the transient expression of DNA, and the integration of DNA into the host chromosomes. For understanding mechanisms of electrotransfection, only the DNA binding to the cell surface and the electric field assisted membrane-crossing of DNA are relevant. Both the expression of the loaded DNA and the DNA integration into the host chromosomes depend more on the properties of the cell and its interactions with a foreign gene. Since these properties and interactions will be similar irrespective of the method chosen to facilitate DNA transfer, they are not relevant for the study of mechanisms of electrotransfection. Our results also support the idea that the PEF-induced cellular uptake of DNA is mainly by the electrophoresis of the surface bound DNA across the plasma membrane.