Masahiro Hibino

Time courses of cell electroporation as revealed by submicrosecond imaging of transmembrane potential

Changes in the membrane conductance of sea urchin eggs, during the course of electroporation, were investigated over the time range of 0.5 microsecond to 1 ms by imaging the transmembrane potential at a submicrosecond resolution with the voltage-sensitive fluorescent dye RH292. When a rectangular electric pulse of moderate intensity was applied across an egg, a position-dependent potential developed synchronously with the pulse, as theory predicts for a cell with an insulating membrane. From the rise and fall times, the membrane capacitance of unfertilized eggs was estimated to be 0.95 microF/cm2 and the intracellular conductance 220 omega.cm. Under an electric pulse of much higher intensity, the rise of the induced potential stopped at a certain level and then slowly decreased on the microsecond time scale. This saturation and subsequent reversal of the potential development was ascribed to the introduction of finite membrane conductance, or permeabilization of the membrane, by the action of the intense pulse (electroporation). Detailed analysis indicated the following: already at 0.5 microsecond in the rectangular electric pulse, the two sides of the egg facing the positive and negative electrodes were porated and gave a high membrane conductance in the order of 1 S/cm2; the conductance on the positive side appeared higher. Thereafter, the conductance increased steadily, reaching the order of 10 S/cm2 by 1 ms. This increase was faster on the negative-electrode side; by 1 ms the conductance on the negative side was more than twice that on the positive side. The recovery of the porated membrane after the pulse treatment was assessed from the membrane conductance estimated in a second electric pulse of a small amplitude. At least two recovery processes were distinguished, one with a time constant of 7 microseconds and the other 0.5 ms, at the end of which the membrane conductance was already < 0.1 S/cm2.

Membrane conductance of an electroporated cell analyzed by submicrosecond imaging of transmembrane potential

Transmembrane potential was induced in a sea urchin egg by applying a microsecond electric pulse across the cell. The potential was imaged at a submicrosecond time resolution by staining the cell membrane with the voltage-sensitive fluorescent dye RH292. Under moderate electric fields, the spatial distribution of the induced potential as well as its time dependence were in accord with the theoretical prediction in which the cell membrane was regarded as an insulator. At higher field intensities, however, the potential apparently did not fully develop and tended to saturate above a certain level. The saturation is ascribed to the introduction of a large electrical conductance, in the form of aqueous openings, in the membrane by the action of the induced potential (electroporation). Comparison of the experimental and theoretical potential profiles indicates that the two regions of the membrane that opposed the electrodes acquired a high membrane conductance of the order of 1 S/cm2 within 2 microseconds from the onset of the external field. The conductance was similar in the two regions, although permeability in the two regions of the membrane long after the pulse treatment appeared quite different.

Atomic Images of Saturated and Unsaturated Fatty Acids at Liquid/Graphite Interface and Difference of Tunneling Currents between Them Observed by Scanning Tunneling Microscopy

Structures of arachidic acid and elaidic acid molecules adsorbed onto a graphite substrate were investigated using a scanning tunneling microscope. The ordered arrangements of the molecules were observed with atomic resolution. The image of an arachidic acid molecule is composed of nineteen bright spots at the individual hydrocarbon groups in the alkyl chain and a dark region at the carboxyl group. The image of an elaidic acid molecule with an unsaturated hydrocarbon chain is similar to that of an arachidic acid molecule except for a much brighter spot at the double bond in the hydrocarbon chain. As a result, functional groups such as hydrocarbon chains, carboxyl groups and double bonds could be distinguished by the tunnel currents.

Molecular Arrangements of Fatty Acids and Cholesterol at Liquid/Graphite Interface Observed by Scanning Tunneling Microscopy

We have directly imaged organic molecules such as fatty acids and cholesterol adsorbed onto a graphite substrate using a scanning tunneling microscope (STM). The arrangements of pure fatty acids such as myristic, palmitic, stearic, arachidic, behenic and elaidic acids are essentially similar except that STM images change systematically with the length of the alkyl chain. The STM images attained atomic resolution and as a result we observed the individual hydrocarbon groups in the alkyl chains. In the direction parallel and perpendicular to the molecular axis, the molecules on the graphite substrate form dimers via hydrogen bonding between carboxyl groups and a superstructure with a period of four or five molecules, respectively. The image of a monolayer of a binary mixture of fatty acids adsorbed onto the substrate reflects two different lengths of alkyl chains. A monolayer of cholesterol molecules was imaged for the first time.

Molecular motion at domain boundaries in fatty acid monolayers on graphite observed by scanning tunneling microscopy

Abstract The domain boundaries in a monolayer of fatty acids absorbed onto a graphite substrate were directly observed using a scanning tunneling microscope (STM). For all the fatty acids observed, the STM images of the monolayers were composed of bright bands corresponding to the alkyl chains and dark regions at the carboxyl groups via hydrogen bonding. There are three configuration of domains in the monolayers according to the three main crystallographic axes of the graphite substrate, i.e. the directions of the bright bands in neighboring domains make an angle of 120 ° with each other at the domain boundaries. A variety of the images was obtained at the domain boundaries, such as bright and noisy regions and dark regions corresponding to free volume. The motion of the domain boundaries were often detected between two successive STM images over several tens of seconds. Molecular rearrangements in small domains were also observed during scanning. These facts indicate the existence of molecular motion on the graphite substrate.

Scanning tunnelling microscopy study on dynamic structural formation in mixed fatty-acid monolayers at liquid/graphite interface

Abstract We directly imaged a monolayer of fatty acids adsorbed onto a graphite substrate from binary mixtures of myristic acid and behenic acid using a scanning tunnelling microscope (STM). The overall STM images of a monolayer of pure fatty acids exhibit an alternate structure composed of bright bands corresponding to the alkyl chains and chained dark regions at the carboxyl groups. In the STM images of a monolayer of binary mixtures, the entire area is filled with 2 kinds of the bright bands, i.e., there are the short and iong bands corresponding to the alkyl chains of 2 kinds of fatty acids. The ratio of the short and long bands in the images suggests that the adsorption of fatty acid with a longer alkyl chain on the graphite substrate is preferential. Moreover, a dynamic exchange of 2 kinds of the bands was often imaged between 2 successive STM images over several tens of seconds. This fact indicates the existence of molecular motion at the interface between a solution and the graphite substrate.

Submicrosecond Imaging Under A Pulsed-Laser Fluorescence Microscope

A microscope system has been constructed that enables digital imaging of a fluorescent cell under pulsed illumination. Each image is produced by a single laser pulse of duration less than 0.3 11 s. With this system, microsecond responses of a single cell to an externally applied electric field have been resolved temporally and spatially. The cell membrane was stained with a voltage-sensitive fluorescent dye. The induction of transsmembrane potential by the applied field, and the perforation (electroporation) of the cell membrane under an intense field, were seen as successive images. The major finding was a transient increase, at the moment of perforation, in the membrane permeability to an enormous level in localized regions of the cell membrane. Possible roles in cell technology, as well as other applications of the microscope system, are discussed.

Ordered Structure Formed by Biologically Related Molecules

The two-dimensional arrangement of biologically related molecules was studied by means of scanning probe microscopy. For monolayers of fatty acid molecules with a saturated hydrocarbon chain adsorbed on a graphite substrate, in the scannings tunneling microscope image, the position associated with the carbon atoms was clearly distinguished. In addition, based on the image for fatty acid molecules with an unsaturated hydrocarbon chain, at the position of a double bond, local electrical conductance was found to increase. Based on the images, it was pointed out that not the position of each carbon but the interaction between it graphite substrate and an alkyl chain plays an important role in imaging. On the other hand, for the surface of Langmuir-Blodgett films composed of phosphatidic acids with cations, the scanning force microscope image shows, for the first time, evidence of the methyl ends in the arrangement of phospholipid molecules.

Point defects in Langmuir–Blodgett films of Cd arachidate

Abstract True molecular resolution with the atomic force microscope (AFM) has been achieved, on Langmuir–Blodgett multilayers, using films of cadmium arachidate on mica. The images were obtained with 12 μm scanner head and commercially purchased cantilevers with spring constant k =0.06 N m −1 under ambient conditions. The presence of vacancies in the Langmuir–Blodgett film was shown for the first time and is the evidence that AFM is a true probe for imaging molecular structure of soft organic materials. Well-preserved raw image of an isolated dislocation was resolved in this film clearly. The probability for existence of vacancies and free dislocations is discussed in the light of KTHNY theory.

Self-Assembled Monolayers of Cholesterol and Cholesteryl Esters on Graphite

The molecular arrangements of self-assembled monolayers (SAMs) of cholesterol, cholesteryl laurate, and cholesteryl stearate adsorbed on a graphite surface were studied using scanning tunneling microscopy (STM) at the liquid-solid interface. The STM images of the SAMs showed two-dimensional periodic arrays of bright regions that corresponded to the sterol rings. However, individual sterol rings could not be observed in the bright regions in the STM images of the cholesterol monolayers. Nevertheless, by comparing the STM images and the crystallographic data, it is concluded that the cholesterol molecules are arranged in pairs oriented head-to-head owing to the hydrogen bonds between the hydroxyl groups. These dimers, in turn, are oriented parallel to each other, owing to the interactions between the sterol rings. The STM images of cholesteryl ester monolayers had molecular resolution and showed pairs of cholesteryl ester molecules oriented in an antiparallel manner, with their fatty acid chains located in the central regions. Furthermore, the fatty acid chains of cholesteryl stearate were observed to be oriented in the (1120) zigzag direction of the graphite lattice, whereas those of cholesteryl laurate were oriented in the (1010) armchair direction. These observations reveal that the interactions between the fatty acid chains affect the structure of the SAMs. The molecular arrangements also depend on the lengths of the fatty acid chains of the cholesterol esters and hence on the interactions between the alkyl chains and the graphite surface. The self-assembly at the liquid-solid interface is therefore controlled by the interactions between sterol rings, between alkyl chains, and between alkyl chains and the substrate.