Simple and rapid method for selective enumeration of lactic acid bacteria in commercially prepared yogurt by image analysis and K-means clustering.

We developed a simple and rapid method based on the combination of image analysis and k-means clustering to selectively enumerate cocci and bacilli from among lactic acid bacteria (LAB) in commercially prepared yogurt. We used our previously reported method for recovering only LAB without non-microbial substances from commercial yogurt, and found that the shape and light intensity of LAB cell images taken by optical microscopy were factors that could distinguish cocci and bacilli, allowing the selective enumeration of LAB. Also, k-means clustering was executed on a dataset of the mean light intensity and aspect ratio of each LAB obtained by image analysis, and each LAB in the image could be automatically assigned to either the cocci or bacilli group. The results obtained by this automated method were in good agreement with those obtained by manually counting the LAB under a microscope, with an overall error within 10%. In addition, this method could provide results within a few hours, which is approximately 1/32 of the time required for the conventional colony-counting method.

Chemically Crosslinked Sulfonated Polyphenylsulfone (CSPPSU) Membranes for PEM Fuel Cells.

Sulfonated polyphenylsulfone (SPPSU) with a high ion exchange capacity (IEC) was synthesized using commercially available polyphenylsulfone (PPSU), and a large-area (16 × 18 cm2) crosslinked sulfonated polyphenylsulfone (CSPPSU) membrane was prepared. In addition, we developed an activation process in which the membrane was treated with alkaline and acidic solutions to remove sulfur dioxide (SO2), which forms as a byproduct during heat treatment. CSPPSU membranes obtained using this activation method had high thermal, mechanical and chemical stabilities. In I-ViR free studies for fuel cell evaluation, high performances similar to those using Nafion were obtained. In addition, from the hydrogen (H2) gas crossover characteristics, the durability is much better than that of a Nafion212 membrane. In the studies evaluating the long-term stabilities by using a constant current method, a stability of 4000 h was obtained for the first time. These results indicate that the CSPPSU membrane obtained by using our activation method is promising as a polymer electrolyte membrane.

Simple and Rapid Method for Separating Lactic Acid Bacteria from Commercially Prepared Yogurt.

We have reported a simple method for separating lactic acid bacteria (LAB) from yogurt. This method is based on the process of destructions and denaturation of casein micelle aggregates by vortexing, and can supply samples containing only LAB. Recovered LAB were clearly observable by microscopy, meaning that morphological changes could be directly detected at the single-cell level. This method will be a helpful tool for the analyzing various LAB, including their enzyme activity and protein expression.

Cytosolic Transport of Nanoparticles through Pressurized Plasma Membranes for Molecular Delivery and Amplification of Intracellular Fluorescence.

Transporting nanoparticles into live cells is important for drug delivery and other related applications. We found that cells exposed to hypoosmotic pressures can internalize substantial quantities of gold nanoparticles. Importantly, these nanoparticles can circumvent normal intracellular traffic and be transported directly into the cytosol, without the need for surface functionalization. In contrast, nanoparticles endocytosed at physiological osmolality are segregated inside endocytic organelles and are not able to reach the cytosol. Cytosolic internalization was observed for nanoparticles of various sizes and materials, with minimal short- or long-term damage induced by the internalized particles. Thus, our strategy can be used as a delivery platform for a range of applications from therapeutics to medical imaging. As examples, we demonstrated rapid delivery of membrane-impermeable molecules to the cytosol by using nanoparticles as carriers and the use of nanoparticles assembled within the cytosol as plasmonic nanoantenna to enhance intracellular fluorescence. We propose a model for the mechanisms behind nanoparticle internalization through pressurized plasma membranes via the release of lateral pressures. Such characterizations may constitute a foundation for developing new technologies, including nanoparticle-based drug delivery.

Direct Imaging of Carboxymethyl Cellulose-mediated Aggregation of Lactic Acid Bacteria Using Dark-field Microscopy.

The ecological functions of lactic acid bacteria (LAB) have been utilized in human life for food processing and probiotic therapy. Understanding the interaction mechanisms between LAB and food ingredients may help to clarify the fermentation process and physiological functions of LAB in the production of fermented foods made from plant materials and dairy products. However, the interaction mechanisms have yet to be fully clarified. Although laser diffraction was used for measuring the size changes of aggregates caused by the interaction between LAB and food ingredients, aggregate sizes could not be determined because of the precipitation of aggregates and its disruption from stirring. Therefore, a microscopy-based method for directly visualizing their interactions is required. We directly observed aggregation processes of LAB cells mediated by water-soluble polysaccharides, carboxymethyl cellulose (CMC), by dark-filed microscopy (DFM). DFM could visualize CMC-mediated cell aggregation with high contrast in real time, and revealed that the aggregates were formed by repeated collisions of LAB cells in a suspension. This suggests that our method can be used as a useful assay to directly visualize grain formation caused by interactions between LAB cells and various polysaccharides in food ingredients.

Fluorescence Enhancement of Nanoraspberry Hot-spot Source Composed of Gold Nanoparticles and Aniline Oligomers.

In this study, we examined raspberry-shaped organic/inorganic hybrid structure for potential development of a nanoantenna system capable of detecting and labeling biomolecules. The structure is characterized by a high density of gold nanoparticles (AuNPs) separated by closely packed aniline oligomers that serve as a linkage between adjacent particles. In particular, the structure was based on repeated sequences of AuNP-aniline oligomer-AuNP in a three-dimensional arrangement, which enabled the creation of optical hot spots that can hold multiple molecules. We examine the expression of such features by focusing on the structure and characteristics of the hybrid. We demonstrate that these optical hot spots enhance the dye fluorescence without quenching. As a result, we were able to create a nanoantenna structure enabling the efficient use of light.

Three-dimensional microfabrication using local electrophoresis deposition and a laser trapping technique.

We describe a novel fabrication method of three-dimensional (3D) microstructures using local electrophoresis deposition together with laser trapping. A liquid cell consisting of two-faced conductive substrates was filled with a colloidal solution of Au nanoparticles. The nanoparticles were trapped by a laser spot and positioned on the bottom substrate, then deposited onto the surface by the application of electrical voltage between the two substrates. By moving the liquid cell downward while maintaining the deposition, 3D microstructures were successfully fabricated. The smallest diameter of the fabricated pillar was 500 nm, almost the same as that of the Airy disc. The Young's modulus of the fabricated structure was 1.5 GPa.

Fluorescent carbon nanowires made by pyrolysis of DNA nanofibers and plasmon-assisted emission enhancement of their fluorescence.

We report on a facile method for preparing fluorescent carbon nanowires (CNWs) with pyrolysis of highly aligned DNA nanofibers as carbon sources. Silver nanoparticle (AgNP)-doped CNWs were also produced using pyrolysis of DNA nanofibers with well-attached AgNPs, indicating emission enhancement assisted by localized plasmon resonances.

Construction of nanoantennas on the bacterial outer membrane.

We demonstrate a simple manipulation of gold nanoparticles that creates a structure-dependent nanometer-scale antenna on the surface of bacteria. Our studies illuminate the concept of the "effective use of light" based on the absorption and emission of light by antennas formed on bacteria.

Simple fabrication of one-dimensional metal nanostructures and their application for SERS analysis.

This review highlights work using the author's method for the preparation of highly aligned metallic nanofibers with one-dimensional aggregates of metal nanoparticles (MNPs) and their utilization in surface enhanced Raman scattering (SERS) analysis. The preparation method, which is based on the process of evaporation-induced self-assembly with DNA and a drying front movement, eliminates the need for lithography and an external field; it is also fast, cheap and easy. Dark-field scattering spectroscopy was used to study the strong plasmon coupling of MNPs in metallic nanofibers. Furthermore, Raman spectral imaging of the metallic nanofibers revealed the existence of intense hot spots localized along their axes, which played a significant role in the intensity of SERS signals from DNA bases and rhodamine B in the metallic nanofibers. Our results demonstrate the use of evaporation-induced self-assembly with DNA as a straightforward method to produce the one-dimensional coupling of localized plasmons with a longer scale, and to facilitate the fabrication of optical sensor chips for single-molecule detection via SERS.

Direct observation of one-dimensional plasmon coupling in metallic nanofibers prepared by evaporation-induced self-assembly with DNA.

Here we report a simple method for the preparation of highly aligned metallic nanofibers with anisotropic aggregates of silver nanoparticles (AgNPs) as well as a direct observation of localized plasmon field and its coupling in the prepared metallic nanofibers. Metallic nanofibers of several tens of nanometers wide and millimeters long were prepared. The preparation method, which is based on the process of evaporation-induced self-assembly with DNA and drying front movement, eliminates the need for lithography and an external field, and it is fast, cheap and easy. Dark-field scattering spectroscopy was used to study the strong plasmon coupling of AgNPs in the metallic nanofibers. We observed strong near-field coupling between neighboring nanoparticles, which results in red-shifted multipolar plasmon modes that are highly polarized along the fiber axis. The polarization dependence of plasmon coupling in the metallic nanofibers observed in this study was satisfactorily explained by the morphology of the metallic nanofibers, which was characterized by atomic force microscopy (AFM) and scanning electron microscopy (SEM). Furthermore, Raman spectra imaging of the metallic nanofibers revealed the existence of intense hot spots localized along their axes, which played a significant role in the intensity of surface enhanced Raman scattering (SERS) signals from DNA bases in the metallic nanofiber. Our results demonstrate the use of evaporation-induced self-assembly with DNA as a straightforward method to produce one-dimensional coupling of localized plasmons with a longer scale.

Gold nanoparticles as localization markers for direct and live imaging of particle absorption through a Caco-2 cell monolayer using dark-field microscopy.

Recently, efforts have been made to reduce the size of food particles containing functional ingredients, since reducing the size is expected to improve intestinal absorption. However, the absorption mechanisms have yet to be fully clarified. Therefore, a microscopy-based method for studying interactions between the particles and intestinal cells is required. We optimized the experimental conditions for observing gold nanoparticles (AuNPs) on the surface of an unfixed Caco-2 cell using dark-field microscopy (DFM). Tight junctions were clearly visible with AuNPs on the cells, producing intense scattered light under DFM. This suggests that AuNPs could be used as localization markers to visualize particle absorption through Caco-2 cells.

Facile method for constructing metallic nanoarrays on a solid surface.

This review highlights work using the author's method to construct metallic nanoarrays. This method is a DNA-templated assembly, to assemble metal nanoparticles (MNPs) in which DNA molecules are initially stretched and aligned on surfaces. Simple interface (air-liquid) moving and surface coating with polymers containing pi-conjugation units, lead to the controlled formation of one-dimensional (1D) nanoarrays of DNA without special equipment. The assembly of MNPs onto DNA can be driven by electrostatic binding of newly prepared gold nanoparticles with positive charges, leading the formation of 1D metallic nanoarrays. Dark-field microscopy observations indicate that MNPs on DNA are uniaxially aligned. Finally, the fabrication and patterning of metallic nanoarrays achieved with DNA nanofibers and transfer printing techniques are discussed.

Highly localized light field on metallic nanoarrays prepared with DNA nanofibers.

We have reported a one-step method for creating one-dimensional metallic nanoarrays on surfaces. This method is based on the processes of solvent vapor-induced buildup and controlled drying front movement, and forms parallelly aligned metallic nanoarrays exceeding several hundred micrometers in length on a poly(dimethylsiloxane) (PDMS) sheet. Strong light scattering originating from highly localized electromagnetic (light) fields was observed on prepared metallic nanoarrays. Furthermore, the enhancement of electromagnetic fields localized on prepared metallic nanoarrays strongly depended on the incident-light polarization.

Self-organization of an organic-inorganic hybrid nanomushroom by a simple synthetic route at the organic/water interface.

A new strategy for the synthesis of a mushroom-like aggregate, based on repeated sequences of a 3D gold nanoparticle-conducting polymer-gold nanoparticle arrangement, by a single-step process at the organic/water interface has been described.

Simple one-step growth and parallel alignment of DNA nanofibers via solvent vapor-induced buildup.

This communication describes the growth and parallel patterning of 1D DNA nanofibers, exceeding several hundred micrometers in length and 40 nm in diameter, induced by solvent evaporation.

One-step preparation of positively-charged gold nanoraspberry.

Self-assembling particles were prepared by a new synthetic strategy for a raspberry-like aggregate, based on three-dimensional particle-aniline oligomer-particle repeated sequences; this one-step process, without the need for extra control, organic solvents, or ligand exchange, could further help in the realization of nanoscale electronics and molecular devices.

Fabricating and aligning pi-conjugated polymer-functionalized DNA nanowires: atomic force microscopic and scanning near-field optical microscopic studies.

We report a simple method to functionalize DNA with pi-conjugated polymer, forming highly aligned and integrated arrays of pi-conjugated polymer nanowires of a few nanometers diameter. pi-conjugated polymer, polyphenazasiline, having alkylammonium salts on the N atom (PPhenaz-TMA), synthesized in this study can be directly attached to DNA, which can be organized along stretched and aligned DNA molecules on surfaces as a template. Furthermore, PPhenaz-TMA/DNA nanowires were stretched and aligned on surfaces, even when PPhenaz-TMA/DNA complexes formed in solutions. The resulting PPhenaz-TMA/DNA nanowires could be easily converted to oxidized states or metallic nanowires by using adequate oxidant or metal salts. The direct visualization of PPhenaz-TMA/DNA nanowires and its structural changes have been studied by atomic force microscopy and scanning near-field optical microscopy.

Transfer-printing of highly aligned DNA nanowires.

We developed a simple method of reproducibly creating highly aligned DNA nanowires without any surface modifications or special equipment. Stretched DNA molecules initially present on the PDMS sheet were transferred onto another surface using transfer-printing (TP). Fluorescent microscopic and atomic force microscopic images revealed that many DNA molecules were highly aligned on surfaces after TP. Furthermore, it was also possible to realize the two-dimensional assembly of DNA nanowires by repeating TP.

Nano-scale imaging of chromosomes and DNA by scanning near-field optical/atomic force microscopy.

Nano-scale structures of the YOYO-1-stained barley chromosomes and lambda-phage DNA were investigated by scanning near-field optical/atomic force microscopy (SNOM/AFM). This technique enabled precise analysis of fluorescence structural images in relation to the morphology of the biomaterials. The results suggested that the fluorescence intensity does not always correspond to topographic height of the chromosomes, but roughly reflects the local amount and/or density of DNA. Various sizes of the bright fluorescence spots were clearly observed in fluorescence banding-treated chromosomes. Furthermore, fluorescence-stained lambda-phage DNA analysis by SNOM/AFM demonstrated the possibility of nanometer-scale imaging for a novel technique termed nano-fluorescence in situ hybridization (nano-FISH). Thus, SNOM/AFM is a powerful tool for analyzing the structure and the function of biomaterials with higher resolution than conventional optical microscopes.