We demonstrated here that high-resolution solid-state NMR is a very convenient means to analyze conformation and dynamics of membrane proteins such as bacteriorhodopsin (bR) and their fragments incorporated into bilayers, if an appropriate ¹³C-labelling is feasible either by biosynthesis or chemical synthesis. We assigned regio-specifically resolved ¹³C NMR peaks of [3-¹³C] Ala-bR to the transmembrane helices, N-or C-terminus and loop regions with reference to the conformation-dependent ¹³C chemical shifts so far accumulated. Further assignment of these signals to individual residues has been made on the basis of a variety of experiments, comparison of spectra between wild type and site-directed mutants, enzymatic digestion, pH changes, etc. This approach turned out to be an excellent means to probe conformational changes induced by lipid-protein interaction, cation-binding, etc. We also demonstrated a novel approach to probe the manner of interaction of biologically active peptides with lipids by utilizing magnetically oriented lipid bilayers or bicelles.

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A scanning near-field optical/atomic-force microscope (SNOAM) system was applied to simultaneous topographic and fluorescence imaging of biological samples in air and liquid. The SNOAM uses a bent optical fiber simultaneously as a dynamic mode atomic-force microscopy (AFM) cantilever and a scanning near-field optical microscopy (NSOM) probe. Optical resolution of this system indicated about 50-100 nm in a fluorescence mode for fluorescent latex beads on a quartz glass plate. The SNO-AM system used 458 or 488 nm from Ar ion laser multiline of excitation of green fluorescent protein (GFP), since a native GFP has been known to give a maximum at 395 nm and a broad absorption spectrum until 500 nm. Topographic and fluoresence images of recombinant E. coli were obtained simultaneously with a high spatial resolution which was apparently better than that of a conventional confocal microscope. Nanoscopic GFP fluorescence spectrum was obtained by positioning the optical fiber probe above the bright area of the E. coli cells. Comparing topographic and fluorescence images, individual E. coli cells expressed different fluorescent intensity. Fluorescence obtained by SNOAM indicated GFP oxidation possibly occured near cell surface. SNOAM also provided us with simultaneous topographical and optical images of human chromosomes. Native chromosomes were spread out onto a coverslip using the surface-spreading whole-mount method. Topographic images clearly indicated duplicated structure on metaphase chromosome, while fluorescence images were a different shape probably because it depended on the combination of SYBRTM Green I and chromosome DNA. Atomic force images have some artefacts, however they can be corrected by comparison with the fluorescence image. Topography and fluorescence images of RBL-2H3 mast cells surface were determined with/without DNP-BSA stimulation by SNOAM system. Near-field fluorescence images were obtained from the granules stained with quinacrine. Fluorescence profiles and intensities were largely changed after allergen stimulation. Exocytotic events of granules were specially discussed based on SNOAM.

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This article reviews the development of Fourier transform infrared (FT-IR) and Raman spectroscopy of ultrathin organic films such as Langmuir-Blodgett (LB) films, Langmuir (L) monolayers, and bilayer lipid membranes during 20 years after the establishment of The Membrane Society of Japan. The key factor in the vibrational spectroscopy of ultrathin films is the sensitivity of the measurement technique for an extremely small amount of molecules within the irradiated field of infrared or visible light. In the infrared spectroscopy, the FT-IR technique itself has developed in the computing algorithm and mirror position controls and so forth, combined with various measuring techniques suitable for sensitivity-enhancement. In the Raman spectroscopy, the introduction of low-noise charge coupled device (CCD) detectors or transmission-type gratings has increased the sensitivity applicable to the level of monolayer films.

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An understanding of the functional organization of neural networks and how it generates the organism's repertoire of behaviors, is best achieved using techniques that permit one to monitor the spatio-temporal patterning of its electrical activity. Optical techniques using voltage-sensitive dyes and photodiode arrays have made it possible to monitor electrical/voltage-activity simultaneously from multiple-sites in living systems such as central nervous and other systems. In this article, we review recent advances of optical recording-/imaging-studies of transmembrane voltage activity in neural systems.

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The environment-sensitive fluorescent probes, such as prodan, acrylodan and laurdan, change their emission wavelength depending on the polarity of their environment. For example, laurdan changes its emission peak from 440 nm in the gel phase of lipid bilayer to 490 nm in the liquid-crystalline phase. Time-resolved fluorescence spectra of laurdan in liposomes suggested that the 490 nm component of fluorescence was a emission from dipolar-relaxed states of excited fluorophores and that the dipolar relaxation was due to water molecules penetrated into the hydrophobic region of membranes. The generalized polarization (GP), defined by two fluorescence intensities at these characteristic emission wavelengths, is a measure of membrane fluidity and polarity. We have constructed an imaging system which enables us to obtain spatial distribution of the GP. The system is composed of a fluorescence microscope, monochromatic filters, a cooled-CCD camera, and an image-analysis system. It was found that the average GP value in a liposome and its temperature dependence were almost the same as those obtained by a conventional fluorescence spectrometer, by appending only one instrumental constant which described the difference of relative sensitivity between the two wavelength channels. By using this apparatus to the study of membrane heterogeneity of CHO (chinese hamster ovary) cells, it was found that plasma membranes were less polar than the membranes of organelles.

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Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) ethyl esters were effectively separated from oleic acid (OA) and palmitic acid (PA) ethyl esters by oil-in-water (O/W) emulsion liquid membranes containing silver nitrates as a mobile carrier. Since the silver ions in the liquid membrane phase have a high affinity to the carbon-carbon double bonds in polyunsaturated fatty acids (PUFA), DHA and EPA ethyl esters were selectively transported through the complex formation with the silver carrier from the internal material phase to the external recovery phase. The stability of the liquid membranes in the O/W emulsions was the most important factor to achieve a high recovery yield of their PUFA esters with a high purity. Surfactants were limited to nonionic surfactants to obtain a stable emulsion liquid membrane when containing the silver carrier. Concentrations of the surfactant and silver nitrates had a predominant effect to operate the PUFA separation by emulsion liquid membranes effectively. There is an optimal concentration for the silver nitrates and excess dosage of the carrier causes the instability of liquid membranes. Under the best conditions, 86% polyunsaturated fatty acid esters were successfully separated through the silver liquid membrane in a few minutes with a 98% purity.

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By regarding the dialysate flow in a hollow fiber bundle as a flow in a porous medium, the average dialysate pressure of several hollow fibers is numerically determined using the transverse conductivity perpendicular to the hollow fibers and longitudinal conductivity parallel to the hollow fibers. Determination of the dialysate pressure isobars becomes possible if the transverse-to-longitudinal hydraulic conductivity ratio can be obtained experimentally. Since the dialysate fluid flows in the transverse direction at the inlet and outlet, the radial dialysate pressure gradient is enhanced at the inlet and outlet if the transverse conductivity is low. However, even if the transverse conductivity is low, the use of a slender dialyzer suppresses the radial dialysate pressure gradient. If the transverse-to-longitudinal hydraulic conductivity ratio is lower than one-tenth the radius-to-total-length ratio of the cylindrical dialyzer, the radial dialysate pressure gradient is negligible compared to the central axial dialysate pressure gradient.

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Wagner improved the mechanism of magnetic balance which was invented by Clark, and reformed by Gast using the magnetic force. This type of balance is now manufactured and sold by Rubotherm Co. This, compared with conventional balance, has a unique advantage of transmitting the weight change indirectly to a chemical balance which is isolated from the measuring system. This mechanism makes it possible for the materials of the measuring system to have no direct contact with the weighing balance. Therefore, the weight change under even highly corrosive environment can be measured in a wide temperature and pressure range from 77 K to 2, 273 K and from high vacuum to 2, 000 bar.

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