Tetsuichi Wazawa

Biotin-containing phospholipid vesicle layer formed on self-assembled monolayer of a saccharide-terminated alkyl disulfide for surface plasmon resonance biosensing.

We describe a technique to form a biotin-containing phospholipid vesicle layer on a self-assembled monolayer (SAM) deposited on a gold surface to immobilize biotinylated receptor proteins for a surface plasmon resonance (SPR) biosensor. The adsorption of vesicle of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) was examined by SPR on the SAMs of dithiobis(1-deoxy-glucitol-1-carbamoyl pentane) (DDGP), 11-mercaptoundecanoic acid, 11-mercaptoundecanol, 11-amino-1-undecanethiol, and 12-mercaptododecane, and it was found that the DOPC vesicle rapidly adsorbed on the DDGP SAM to achieve the highest coverage of the surface. By quartz crystal microbalance with dissipation monitoring (QCM-D), the DOPC layer formed on the DDGP SAM was shown to be a vesicle layer, in which intact DOPC vesicles physisorbed on the SAM surface. To immobilize a biotinylated receptor protein, one of three biotinylated phospholipids, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(biotinyl) (biotin-DOPE), N-((6-(biotinoyl)amino)hexanoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (biotin-X-DHPE) and N-(biotinoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (biotin-DHPE), was mixed with DOPC to form a biotin-containing vesicle layer on the DDGP SAM. A comparative binding study of NeutrAvidin and the biotin-containing vesicle layers showed that the use of biotin-X-DHPE achieved the most rapid immobilization of NeutrAvidin on the vesicle layer at the highest surface density. Furthermore, biotinylated protein A, as a receptor protein, could be immobilized through NeutrAvidin on the vesicle layer containing DOPC and biotin-X-DHPE, and its reaction with immunoglobulin G, as an analyte, was successfully observed by SPR. The results demonstrate that the biotin-containing vesicle layer on the DDGP SAM must be a useful component for SPR biosensor surfaces.

Imaging And Nano-Manipulation Of Single Actomyosin Motors At Work

1. Muscle contraction is achieved by the sliding movement of myosin and actin using the energy of ATPase.

In vivo brain activity imaging of interactively locomoting mice

Electrophysiological field potential dynamics have been widely used to investigate brain functions and related psychiatric disorders. Conversely, however, various technical limitations of conventional recording methods have limited its applicability to freely moving subjects, especially when they are in a group and socially interacting with each other. Here, we propose a new method to overcome these technical limitations by introducing a bioluminescent voltage indicator called LOTUS-V. Using our simple and fiber-free recording method, named “SNIPA,” we succeeded in capturing brain activity in freely-locomotive mice, without the need for complicated instruments. This novel method further allowed us to simultaneously record from multiple independently-locomotive animals that were interacting with one another. Further, we successfully demonstrated that the primary visual cortex was activated during the interaction. This methodology will further facilitate a wide range of studies in neurobiology and psychiatry.

Highly biocompatible super-resolution fluorescence imaging using the fast photoswitching fluorescent protein Kohinoor and SPoD-ExPAN with Lp-regularized image reconstruction

Far-field super-resolution fluorescence microscopy has enabled us to visualize live cells in great detail and with an unprecedented resolution. However, the techniques developed thus far have required high-power illumination (102-106 W/cm2), which leads to considerable phototoxicity to live cells and hampers time-lapse observation of the cells. In this study we show a highly biocompatible super-resolution microscopy technique that requires a very low-power illumination. The present technique combines a fast photoswitchable fluorescent protein, Kohinoor, with SPoD-ExPAN (super-resolution by polarization demodulation/excitation polarization angle narrowing). With this technique, we successfully observed Kohinoor-fusion proteins involving vimentin, paxillin, histone and clathrin expressed in HeLa cells at a spatial resolution of 70-80 nm with illumination power densities as low as ~1 W/cm2 for both excitation and photoswitching. Furthermore, although the previous SPoD-ExPAN technique used L1-regularized maximum-likelihood calculations to reconstruct super-resolved images, we devised an extension to the Lp-regularization to obtain super-resolved images that more accurately describe objects at the specimen plane. Thus, the present technique would significantly extend the applicability of super-resolution fluorescence microscopy for live-cell imaging.