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Application of the Particular Human Intestinal Microbiota Profile to Disease Precaution
RIKEN Discovery Research Institute |
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| We were only seeing 30%? | |||
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In the mid-19th century, Theodor von Escherich of the University of Vienna found bacteria in feces - the species that would eventually be called Escherichia coli. However, until the mid-20th century, few fecal bacteria other than E. coli had been successfully cultured, leading most to believe that the majority of bacteria existing in the intestines were dead. The dispeller of this long-held belief was Dr. Tomotari Mitsuoka, a RIKEN scientist who, in the 1950s, began to reveal that there are many living bacteria in the intestinal tract through the use of anaerobic culturing. It turned out that most intestinal bacteria are anaerobic bacteria that cannot survive in the presence of oxygen. "I spent my younger days with intestinal microbiota," says Dr. Benno, who joined Dr. Mitsuoka's laboratory at RIKEN approximately 30 years ago. "Different bacterial species require different culturing conditions. Our work can be likened to artisanship - creating agar medium by trial and error, and counting individual bacteria to record bacterial species and numbers. Not only does it require a great deal of perseverance and energy, but the sample feces and media are malodorous and pose a constant risk of bacterial infection. No-one is excited to take on this assignment. 'You have to be crazy to do this kind of work!' as Dr. Mitsuoka used to tell us," laughed Dr. Benno. Dr. Mitsuoka received the Japan Academy Award in 1988 for his work on systematic research on intestinal microbiota utilizing original culturing techniques. "Everyone thought that we knew all there was to know about intestinal microbiota, and that Dr. Mitsuoka's work was complete." However, Dr. Benno could not help but wonder if the culturing methods available at that time had really taught us everything. DNA analysis, which became available for the study of intestinal microbiota in the mid-1980s, enabled researchers to determine the presence of intestinal microbiota without culturing. In 1996, Dr. Benno came across a stunning paper produced by a group of molecular biologists who analyzed DNA extracted from bacteria isolated from human feces, reporting that 10% to 25% of the bacteria can be cultured but the remaining 75% are either extremely difficult or impossible to culture, and that the intestinal microbiota that had been thought to comprise approximately 100 species are in fact 500 species. The intestinal microbiota that seemingly had been almost fully elucidated by the culturing methods available at that time, which were the sum total of the efforts of many researchers, were merely around 30% of the extant bacteria. |
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| Capturing the whole analysis of intestinal microbiota | |||
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In 1998, Dr. Benno shifted his research focus to elucidating the complete picture of intestinal microbiota, including those "difficult or impossible to culture" bacteria, by incorporating DNA analysis with conventional culturing methods. He analyzed 744 DNA clones bacteria isolated from the fecal specimens of three healthy Japanese subjects, and found that 75% were novel intestinal bacteria, and that there are great individual variations in the composition of intestinal microbiota. Subsequently, around 2000, Dr. Benno began to use T-RFLPs (terminal restriction fragment length polymorphisms) to facilitate determination of the compositional patterns of human intestinal microbiota. T-RFLP analysis consists of extraction of genes for 16S ribosomal RNA from various intestinal bacteria in feces, amplification using primers (molecules that provide a starting point for DNA synthesis) labeled with fluorescent dyes, and digestion with two different restriction enzymes. Restriction enzymes cleave DNA, which is composed of four different bases, at specific sequences, generating DNA fragments of various lengths depending on the number of bases. The amount of each DNA fragment is determined based on the intensity of its fluorescence signal, and aligning the DNA fragments by the number of bases provides an intestinal microbiota profile reflecting the composition of intestinal microbiota and their amounts (Figures 1 and 2).
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| Exploring the relationship between intestinal microbiota and disease | |||
Intestinal bacteria live in the colon, which is a "wellspring of disease"; in fact, the colon is associated with the greatest number of different diseases of any human organ. The intestinal tract is also on the "front line of immunity," as it is connected to the outside world.Certain intestinal bacteria generate putrefactive products such as ammonia and hydrogen sulfide, bacteriotoxins, and carcinogens. These toxic substances damage the intestinal tract and induce colon cancer and a variety of other colonic diseases, and some of them are absorbed and circulated throughout the body by the blood, causing damage to various organs. Thus, there is an increasing body of evidence that intestinal bacteria can be the causes of carcinogenesis, aging, and various pathological conditions, including arteriosclerosis resulting from cholesterol deposition, liver damage, dementia, autoimmune diseases, and weakened immunity (Figures 3). For example, Clostridium species are found at high levels in the feces of persons suffering from senile dementia. Once the toxic substances produced by these bacteria are spread throughout the body, the functions of neurotransmitters etc. are inhibited, resulting in impaired brain function. Recent molecular biological studies suggest that toxic substances produced by intestinal bacteria may promote cholesterol deposition in blood vessels, inducing arteriosclerosis, which in turn causes heart and cerebrovascular disease. Intestinal bacteria have also been reported to convert bile acids into secondary bile acids, thereby promoting the development of colon cancer. To date, six species of bacteria that produce secondary bile acids have been identified, three of which were identified by the Molecular Microbial Functions Division. "It is well-known that germ-free animals live 1.5 times longer in aseptic conditions than in normal conditions. Our lifespan is evidently controlled by the endogenous bacteria in our bodies." |
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| Developing an intestinal environment database | |||
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While the relationship between intestinal microbiota and disease is becoming clearer, Dr. Benno strongly feels that conventional research methods require significant modification if we are to further advance intestinal microbiota research and to apply the knowledge gained to actual preventive medicine and other fields. "Previously, researchers primarily targeted bacteria that can be cultured, and studied bacterial dynamics in patients with particular diseases, which did not produce the intended results. Now we need to focus on studying the interactions between bacteria as a whole and our bodies and food, and identify the substances they produce. The first step is to develop an intestinal environment database integrating intestinal microbiota profiles that reflect the composition of intestinal microbiota as a whole, with intestinal metabolic profiles that reflect the composition of the products they generate, namely metabolites." The accumulation of lifestyle-related data on diet, medical condition, and so on, and a study of the correlations present provide information about healthy intestinal environmental states and intestinal environments associated with particular diseases. We can then utilize this intestinal environment data for prevention and early detection of diseases. The U.S. scientist Dr. Craig Venter, one of the leaders of the Human Genome Project, and his colleagues undertook the development of an intestinal microbiota database as well. However, the group only targeted culturable bacteria, and the group of molecular biologists who published that "stunning paper" in 1996 were unable to further advance their intestinal microbiota research. The Molecular Microbial Functions Division led by Dr. Benno is leading the way in this area through research aimed at elucidating the whole analysis of intestinal microbiota and linking the data obtained to preventive medicine. The intestinal environment is unique to each individual, and changes depending on lifestyle factors such as diet and age. Therefore, the development of an intestinal environment database requires both samples from a broad range of areas and age groups and quick analysis of large numbers of samples. Dr. Benno is attempting to expand the routes to acquire large numbers of samples (100,000+) from throughout Japan. "With conventional culturing methods, one researcher can test 200 individuals per year at most. Molecular biological analysis methods like T-RFLP enable automation of the entire process, and with further advances in instrumentation and taking proper advantage of the technological capabilities of RIKEN, we will probably be able to analyze hundreds of thousands of samples in just a few years. The use of T-RFLP has greatly enhanced our opportunities for collaborative research with medical schools, universities, and hospitals." A focus on specific geographic regions is also an important consideration for elucidating the relationships among lifestyle, intestinal microbiota and diseases. As an example, the group is planning to perform annual testing, in collaboration with Hirosaki University, of the intestinal microbiota of 3,000 residents in specific regions of Aomori prefecture, and to study their association with the health of those individuals in five or 10 years. Aomori prefecture is ranked lowest in longevity for both men and women and is a high-risk region for colon cancer. "Recently, the number of colon cancer patients, including young people, has been increasing nationwide. If we could use intestinal microbiota patterns associated with high susceptibility to colon cancer to screen for individuals at high risk, we could implement prevention and early detection at the national level and greatly reduce medical costs. Colon cancer has some of the highest associated medical costs among diseases." According to Dr. Benno, intestinal microbiota research will bring us closer to order-made medicine, which allows selection of drugs suitable for an individual. "For example, if we could examine a patient's intestinal microbiota, we could solve one of the major issues with antibiotic administration, the adverse reaction of diarrhea, by prescribing antibiotics with a low risk of causing diarrhea for that particular patient. Examination of intestinal microbiota will also allow the most effective prescription of Chinese herbal medicines for specific individuals as well, since the enzymes released by specific intestinal bacteria often activate the medicinal properties of Chinese herbal medicines." In addition to the intestine, the oral cavity and the vagina are home to an estimated approximately 500 microbial species each. A large number live on the skin as well. "Only ten percent of oral bacteria have been isolated to date. We are also conducting collaborative research using T-RFLP to obtain the complete picture of oral bacteria and to apply the information for the prevention and treatment of periodontal disease." Dr. Benno indicated his interest in expanding partnerships with other groups within RIKEN as well. "For example, I am interested in studying the relationships between intestinal microbiota and allergies through collaborative research with the RIKEN Research Center for Allergy and Immunology of the RIKEN Yokohama Institute." |
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| Connecting medicine and diet with intestinal microbiota | |||
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Some individuals when infected with enterohemorrhagic Escherichia coli O-157 or Salmonella develop symptoms of food poisoning while others do not. Thus, there are obviously individual variations in the response to infection. "The explanation for this phenomenon lies in whether individuals have intestinal microbiota resistant to pathogenic bacteria. What is the ideal intestinal microbiota pattern and how should we change our diet to achieve it? The day on which individuals are able to undergo intestinal microbiota testing at local hospitals and receive such advice is fast approaching," stated Dr. Benno. "The Japanese like to say 'Food is medicine,' but the door between food and medicine can only be opened with intestinal microbiota." Development of intestinal microbiota research has brought with it progress in our understanding of the mechanisms of various bacteria of the genus Lactobacillus, which confer beneficial effects on our health. These advances have triggered a boom in probiotics - functional foods incorporating these bacteria. "The concept of 'probiotics' was developed as a concept standing in opposition to 'antibiotics,' which kill not only pathogenic bacteria but beneficial bacteria as well. Probiotics utilize the functions of 'friendly' bacteria to promote human health." The best-known examples of probiotics are fermenteded milk and yogurt, which are both listed as "food for specified health uses (FOSHU)." The "FOSHU" designation is permitted by the government on labels for foods expected to confer health effects based on data obtained in medical and/or nutritional studies. Japan became the first country to adopt this system in 1991. "Health claim" for FOSHU utilizing lactobacilli and bifidobacteria is limited to regulation of intestinal function at present, but there are data suggesting the potential for future enhancement of health claims to include reduction of cancer risk via immunoreactivity, prevention of atopic dermatitis,prevention of respiratory infection, regulation of blood glucose level, blood pressure, and cholesterol, prevention and improvement of gastric ulcers, and inhibition of the causative bacteria of diseases such as periodontal disease. "The boom in FOSHU is not merely a transient fad, but is founded on solid evidence presented by the elucidation of the functions of microorganisms such as lactobacilli and bifidobacteria along with the whole analysis of intestinal microbiota," says Dr. Benno. While praising the boom highly on the one hand, he also warns that it may be going too far. "Too many people confuse food for specified health uses with drugs." The keys are individual efforts to improve one's lifestyle, including diet, thereby altering the overall balance of intestinal microbiota, and to practice health promotion and disease prevention. Dr. Benno proves this maxim by practicing it himself. "At one time, to be honest, although I gave people similar advice, I was not such a big fan of vegetables and yogurt. However, I modified my lifestyle three years ago. I take a one-hour walk in the morning wearing seven kilograms of weights on my arms and legs. My diet now centers on vegetables and I eat five hundred grams of yogurt every day. I succeeded in reducing my body fat from 28% to 22%, and I overcame the hay fever and gout I suffered from for years." |
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| Linking intestinal microbiota research to health | |||
"I have been engaged in intestinal microbiota research for thirty years, and I have witnessed the field undergo drastic change during the last three years. I do not feel that I am overstating the case by saying that we are the ones who have made that change happen." The Molecular Microbial Functions Division has accepted many students from the food industry since incorporating molecular biological techniques."I often remind young researchers that one cannot learn anything about bacteria by looking solely at DNA. One can truly understand the function of DNA only by studying living bacteria. As a microbiologist, I choose not to use the phrase 'impossible to culture,' since any bacteria can be cultured eventually (Figures 4)," said Dr. Benno, who concluded with the following. "We could not have reached this point without a more-than-fifty-year history of intensive culturing efforts. We are trying to conduct the research that was not possible with past culturing methods with the help of molecular biological techniques. Intestinal microbiota research in the 20th century was research for research's sake, but now we have finally reached the point where it can be applied to preventive medicine. I would like to find a practical means to bridge the efforts of all the past researchers to the future of human health." 
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Visualizing nanostructures on solid surfaces at the atomic level
RIKEN Frontier Research System |
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| Irradiation of light causes electrons to "jump" out from the surface | |||
"I often have trouble getting people to understand what is interesting in my work," began Dr. Ushioda with a grin. "My work actually has major implications for the currently 'hot' field of nanotechnology and various other fields, and more importantly, is fascinating in and of itself."
The Laboratory for Surface-Photodynamics studies the interactions between light and solid surfaces at the atomic level; there are actually all kinds of "interactions" taking place on solid surfaces (Figure 1). "Irradiation of light to substances causes electrons to jump out from the surface. This phenomenon, called the "photoelectric effect," is one of our research subjects."Dr. Ushioda explained the photoelectric effect using the following metaphor. "Let's use a bucket to represent a substance to which light is shined. The rim of the bucket is the surface of the substance. Electrons fill the bucket to just below the rim, but they cannot jump out of the bucket. If light is shined to the electrons, they receive the light energy, are excited to higher energy levels, and jump out from the rim of the bucket, or the surface of the substance, as a result. That is a simple representation of the photoelectric effect. This effect is used by the photomultipliers at Kamiokande, which were made famous when their creator, Dr. Masatoshi Koshiba, won the Nobel Prize in Physics." A photomultiplier is a light detection device that multiplies electrons jumping out from metal struck by light and converts the light into electric current. Kamiokande was designed to detect the light induced by electrons colliding with neutrinos from a supernova explosion as they react with water molecules. However, because of the low frequency of reactions and miniscule quantity of light emitted, researchers built the world's largest photomultiplier (50 cm in diameter) and achieved a great discovery. "This Japanese photomultiplier is undoubtedly the world's best, without which Dr. Koshiba's discovery would not have been possible." |
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| Physical and chemical phenomena on surfaces | |||
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There are other dynamic physical and chemical phenomena aside from the photoelectric effect taking place on the surfaces of solids, many of which have applications in various industrial technologies. One of these phenomena is crystal growth. Cooling a liquid from the bottom causes it to gradually solidify from the bottom. Crystals grow as the atoms moving freely within the liquid are incorporated onto the surface of the solid. "The semiconductor industry is eager to know how atoms behave at the solid-liquid interface." Silicon (Si) is an element found abundantly in the earth's crust, and is also the most commonly-used material for semiconductors. However, to be used in semiconductor devices, Si must be in the form of a monocrystal, in which atoms are formed into a regular three-dimensional array. One of the difficulties facing the semiconductor industry today is the creation of perfect monocrystals. "If we can understand exactly what is happening on the surfaces of solids during crystal growth, we may be able to control monocrystalline growth," says Dr. Ushioda. Research into the properties of solid surfaces has applications in our daily lives as well. "Automobile mufflers have built-in catalytic converters that also use the chemical phenomena occurring on surfaces to convert toxic substances in exhaust gases such as nitrogen oxides (NOx) to nontoxic substances." |
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| Observation of individual atoms with STM | |||
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"The surfaces and interfaces of substances are quite fascinating. However, because studying events occurring on surfaces at the atomic level is extremely difficult, the field remains underdeveloped," states Dr. Ushioda. Constant interactions are taking place between various molecules and atoms present in the air, such as oxygen and carbon dioxide, on the surfaces of substances. For example, minute alcohol molecules present in the air exhaled by a researcher as leftovers from the night before might influence the experimental results. Thus, conditions and outcomes can vary with every experiment, but science requires reproducible results. In order to eliminate unwanted effects in the study of surfaces, experiments must be performed using uncontaminated samples in an ultrahigh vacuum apparatus (Figure 2). This task became relatively easy in the 1970s. The arrival of the STM (Scanning Tunneling Microscope) greatly contributed to the advancement of research into surface physics. The STM was invented at IBM's Zurich Research Laboratory in 1981 by Gerd Binnig and Heinrich Rohrer, who were awarded the Nobel Prize in Physics for the achievement in 1986. The basics of STM are as follows. A sharp metal probe is placed approximately 1 nm (10-9 m) from the surface of a sample, and a voltage is applied to cause a small electric current called a "tunnel current" to flow between the probe and the surface. Scanning the surface with the probe to obtain a constant tunnel current generates an STM image of the topography of the surface, i.e., individual atoms (Figure 3). "STM has a resolution of 0.01 nm in the vertical direction, which is more than adequate to show individual atoms, since the diameter of hydrogen, the smallest atom, is 0.1 nm."
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| Characterization of nanostructures using STM light emission spectroscopy | |||
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"STM allows us to see individual atoms, but not to identify the atoms," explained Dr. Ushioda, pointing out one of the limitations of STM. "My goal at present is to see the nanometer-scale minute structures created on solid surfaces and the properties of atoms and molecules adsorbed on surfaces."
Dr. Ushioda focused on STM light emission spectroscopy (Figure 4). Reports began to filter out of IBM's Zurich Research Laboratory around 1988 indicating that STM has an associated weak emission of light. Electrons surrounding an atomic nucleus located under the STM probe receive energy from the tunnel current , are excited to higher energy levels, and emit this energy as light when they return to their original energy levels. "Spectroscopic observation of STM light emission should be able to identify the atoms under the probe, since the electrons of each element emit a unique spectrum of light." Dr. Ushioda remained quite confident even in the face of the extremely weak light emitted by the STM. "Japan has the world's best photomultipliers. More importantly, I am good at collecting weak light to see things that cannot be seen ordinarily." True to his word, Dr. Ushioda's group demonstrated that STM light emission spectroscopy can identify oxygen (O) adsorbed on a copper (Cu) substrate in 2001. This was the world's first experiment proving that STM light emission can be used to identify individual molecules and atoms adsorbed on solid surfaces. Recently, Dr. Ushioda's group discovered that hydrogen (H) atoms adsorbed on the surface of a nickel (Ni) substrate [Ni(110)] can also be identified using STM light emission spectroscopy (Figure 5). Adsorbed species can be visualized by ordinary STM so long as the electronic states are at the proper energy level. Ordinary STM cannot visualize H atoms adsorbed on a Ni(110) substrate since they do not have such an electronic state. Thus, Dr. Ushioda's discovery underscores the significant superiority of STM light emission spectroscopy to ordinary STM in terms of ability to identify adsorbed species. "We know that we can make nanostructures, but we can't find uses for them if we can't determine their properties. Our work helps to enable the evaluation of individual nanostructures, which was previously impossible."
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| Investigating the physical properties of quantum wells | |||
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STM light emission spectroscopy can also be used to investigate the physical properties of the nanostructures of semiconductors; quantum wells, which are semiconductors with small energy gaps sandwiched by semiconductors with greater energy gaps. These structures are called "wells" since electrons tend to fall into areas of semiconductors with small energy gaps. Energy gaps represent the energy required for electrons in valence bands to jump to the conduction band (electronic transition). "Quantum physics defines electrons as waves. Narrower quantum wells cause the wavelengths of electrons to shorten. Since shorter wavelengths have higher energy, reducing the size of quantum wells causes electrons to have increased energy levels. This is called the 'quantum confinement effect,'" explained Dr. Ushioda. "We have successfully observed the quantum confinement effect of individual quantum wells using STM light emission spectroscopy with the unprecedentedly high resolution of approximately 1 nm." Dr. Ushioda's group observed cross-sections of crystals composed of alternating layers of aluminum gallium arsenide (AlGaAs) and gallium arsenide (GaAs). The layers of GaAs are sandwiched by the AlGaAs, which has greater energy gaps, forming quantum wells. The widths of the quantum wells can be freely adjusted by changing the thickness of the GaAs layer. The experiment clearly demonstrated that electronic transition in narrower quantum wells involves higher energy and electronic transition in wider quantum wells involves lower energy, as predicted by quantum mechanics (Figure 6). There are other quantum structures in addition to the layered structure of the quantum well, including quantum dots, quantum wires, and quantum boxes. "Nanostructures making use of the quantum confinement effect are already used in semiconductor devices, but there had been no methods available by which to evaluate individual quantum structures. STM light emission spectroscopy provides us with a powerful tool."
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| Technological revolution of liquid crystals | |||
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The Laboratory for Surface-Photodynamics is also studying liquid crystals. "Today, liquid crystals are an important part of our lives, but the fact is, liquid crystal displays are still fabricated using very primitive methods, and there is much room for improvement," stated Dr. Ushioda. The current method of creating liquid crystals involves attaching a polyamide membrane to a glass substrate and rubbing it with a cloth to cause the polyamide molecules to align in the direction of rubbing. Liquid crystal molecules are then poured onto this aligned membrane. However, the rubbing process is difficult to control, and is associated with problems of static electricity and dust generation. Research Scientist Kiyoaki Usami of the Laboratory for Surface-Photodynamics is developing a new method in which liquid crystal molecules are placed on aligned membranes created using light, without rubbing. This method is expected to be a technological innovation, as it eliminates the problems associated with the rubbing process. |
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| Protein folding is fascinating | |||
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"I typically don't make definite research plans," said Dr. Ushioda. "One can only plan things which lie within the scope of one's own imagination. Finding something interesting among external information that happens to come along could lead to developments surpassing those of your own imagination."
Dr. Ushioda names protein folding as his current topic of greatest curiosity. "Protein folding is the process of a single amino acid chain forming a three-dimensional protein. I liken it to a straight piece of yarn spontaneously transforming into a sweater. How this occurs cannot be easily explained by physics." Dr. Ushioda predicts that observations of weak light called "Raman scattering," which is generated by interactions between light and substance, may enable us to witness the folding process at the atomic level. "Physicists have primarily been concerned with solid matter, but now we are reaching a stage where physicists will have to deal with soft matter, such as liquids and amorphous substances, which are difficult to cope with using conventional theories. This is another reason I find protein folding to be particularly compelling." Dr. Ushioda also serves as Research Director of the "Creation of Innovative Technologies through Integration of Information, Biotechnology, and the Environment with Nanotechnology" a project of the Japan Science and Technology Agency (JST), providing further proof that surface physics research sits firmly at the core of technology creation in the 21st century. "Sometimes, I just don't know what to say when someone asks me what my research is good for," laughed Dr. Ushioda. "We physicists are interested in looking at these kinds of things just because they are interesting. At the same time, we need technologists who accept tasks based on needs. We need both groups to mesh together to obtain good results." The Laboratory for Surface-Photodynamics recently began to use STM to study the physical properties of carbon nanotubes at the atomic level. "I want to see the differences between the properties of atoms at the ends of carbon nanotubes and in the places where carbon nanotubes bend sharply. Isn't it fascinating?" And another seed of innovative technology is sown...
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