I. Basic Research Strategy
The area leader and the principal investigators of planned research projects all have a good command of both molecules (A01) and light (A02) within their own systems, and their responsibilities are classified (A01 and A02) merely for the sake of convenience. Moreover, all of them have the ability to create bio-imaging technologies themselves and to demonstrate the proof of concept. We have a policy of not clearly distinguishing between development and practice. Planned research projects in this research area will serve as minimum-size cores that bring together various skills and knowledge, and within and among these cores, a variety of needs and seeds will interact bi-directionally. We expect that this will give rise to novel bio-imaging technologies. That said, rather than leaving everything to the bottom-up approach, the leaders of planned research projects will work toward resolving several important themes that they have laid out.
We will build this research area entirely as a large core that mobilizes different skills and knowledge in cooperation with co-investigators and members of publicly offered research projects. As for publicly offered research projects, we are considering including researchers with expertise in electrons, sonic waves, etc., who could bring about technological development necessary for the advancement of this research area, although those are not directly linked to the main themes of this research area. This would promote collaboration of light and electrons, and that of light and sound. We are also considering encouraging participation from areas other than life sciences (e.g., software development).
Dissemination of research results will be realized basically by the following two strategies. First, results pertaining to technological development, discovery, etc., will be disseminated worldwide through papers and the website. Second, the dissemination of bio-imaging technologies and knowledge (achievement within the field as well as general findings) will be directed toward researchers in Japan. In facilitating dissemination across Japan, we will take up image processing as a new attempt. As part of that strategy, three attempts will be made: “Program contest,” “Database construction with a correct answer,” and “Image processing program sommelier” to spread image processing software that accommodates near-future 4D measurements and surpasses ImageJ.
II. Specific Research Content
We plan to establish a collaboration system with the following four themes.
This technology, which was awarded the Nobel Prize in Chemistry in 2014, is still in its infancy. Among the challenges is “deep-tissue super-resolution,” that is, an attempt to increase the spatial resolution of optical observation of not only the cell surface on the basal side, but also structures that are considerably far away from the cover glass surface (nuclear structures, Golgi apparatus, or cell surface on the apical side). One of the reasons why this technology poses a challenge is that the heterogeneity in refractive index produces aberration, and PSF (point spread function) becomes distorted as the distance from the cover glass surface increases. Kamiya and Nemoto will work collaboratively to address this issue. In this area, the application of Nemoto’s multi-beam scanning methodology (vector beams and wavefront correction technique) to the “localization method” with illumination of the entire field of view for blinking probes developed by Kamiya will be examined. In contrast, Kamiya will work on the development of fluorescent dyes for molecular orientation imaging, which Nemoto is trying to achieve in this area. Miyawaki will also participate in this collaboration. Miyawaki has developed a reagent that realizes clearing of a fixed sample while preserving its microstructure; collaboration with Kamiya is expected to improve PSF in the entire fixed cell. Miyawaki has also succeeded in the orientation-specific labeling of intracellular organelles using fluorescent proteins, and will be working with Nemoto as well.
In vivo deep imaging
To make deep imaging of living tissues possible, we will collaboratively work on creating long-wavelength (>700 nm) light-emitting proteins for practical application. Soga, who has developed an inorganic fluorescent dye (OTN-NIR), will explore surface modification (biocompatibility, delivery, and targeting of the dye) in response to the needs of those who perform in vivo imaging (Miyawaki and colleagues). Miyawaki will collaborate with Maki (co-investigator) in creating light-emitting proteins that work at longer wavelengths (> 700 nm). It is still a long way off before red fluorescent proteins can be used without permission from the license holder, and a series of development projects such as these are, in a way, an important national mission in the sense that the products of research will be spread to the Japanese industry. As an application, Miyawaki will work with Matsuda to render FRET biosensors excitable at longer wavelengths, and with Imamura and Nemoto to conduct two-photon excitation imaging using the new laser light source. Moreover, the entire area will work toward developing an optical microscope system to detect (excite) these long-wavelength probes, and by using the reagents, etc., that attenuate light scattering in living tissues, which is currently under development by Nemoto and colleagues, Matsuda and Imamura will make an attempt to perform in vivo deep imaging that exceeds conventional limits.
“Stress” is an ambiguous word. This area aims to explore a new avenue for stress by using various probes that allow for visualization of stress-related phenomena. Cultured cells on cover glass (derived from multicellular organisms), which are viable under a considerable amount of stress, represent a perfect theme for this area that straddles both in vitro and in vivo worlds. As an example, we consider oxidative stress. Miyawaki has produced a variety of oxidative stress probes by utilizing fluorescent proteins. On the other hand, the antioxidative effects of cells have also to be visualized. Kamiya will work on the development of probes that can quantify GSH (reduced glutathione), an indicator of antioxidative effects in the cytoplasm. Miyawaki will develop probes to quantify bilirubin (unconjugated), an indicator of antioxidative effects in the membrane, using UnaG derived from Japanese freshwater eel (Anguilla japonica). Matsuda and colleagues have developed FRET biosensors for measuring stress kinase activity, and using these probes, Imamura will lead a collaborative study to deepen the understanding of stress phenomena in cancer.
Zooming in and out
The three themes listed above are mainly set for 4D imaging, and their targeted spatial scales vary. The spatial scales of interest to the principal investigators of planned research projects are roughly organized in the following order: intracellular microstructure (Kamiya) – cell (Kamiya, Matsuda) – tissue (Nemoto, Imamura, Matsuda) – organ (Nemoto, Imamura, Soga, Matsuda) – individual organism (Soga). While processing imaging data obtained at different spatial scales, we will also attempt to incorporate data in a small spatial scale into data in a large spatial scale in a given context. The area as a whole will work collaboratively to build databases that provide meaningful zooming in and out. This collaborative effort will be led by Yokota and Miyawaki.
III. Specific Methods to Promote Organic Collaboration
Within this research area, researchers who control biological molecules and those who control electromagnetic waves will interact with each other in such a way that technologies related to “resonance” will improve dramatically, which can then be applied and extended to the area of biological sciences. Each researcher will tackle basic biological and technical problems, and by combining the achievements of researchers who share common and complementary problems within the area, we hope to achieve new technological development and application.
The results of studies in the proposed research area will be transmitted to the world with the aim of revolutionizing certain areas of biological sciences. Furthermore, our goal is to enhance the understanding of life sciences researchers regarding the benefits of developed technologies. Researchers in Group A01 will pursue the development of probes, whereas researchers in Group A02 will engage in the development of optical devices (software) optimized for such probes, thereby facilitating the provision of expertise and information. On the other hand, publicly offered research groups will be expected to produce breakthrough innovations in the areas of life sciences through the use of developed technologies, and to provide feedback regarding imaging techniques.
In order to promote organic collaboration at various levels, the following activities will be carried out on a regular basis as a research area: 1) research meetings and internal evaluation meetings, 2) young investigator workshops, 3) co-hosted symposiums and workshops at a variety of academic conferences, 4) organization of international symposiums, 5) practical workshops on imaging, 6) technical support system on the Web, etc.
Specific goals and objectives of the research area and planned research projects
The goal of this research area, as well as the planned research projects, is to revolutionize various fields of biological sciences by facilitating interactions between researchers who design molecules and those who control light, in investigating dramatic interactions between light and molecules (resonance), developing innovative imaging technologies, and promoting collaborative interdisciplinary research. To that end, we will strengthen the system of collaboration among researchers to achieve a new paradigm shift in biological sciences, by focusing on four themes: “super-resolution imaging,” “in vivo deep imaging,” “stress imaging,” and “zooming in and out.”
To obtain “deep-tissue super-resolution images,” the following approaches will be taken in a collaborative fashion: application of Nemoto’s multi-beam scanning methodology (vector beams and wavefront correction technique) to the “localization method” with illumination of the entire field of view for blinking probes developed by Kamiya; development of fluorescent dyes by Kamiya, which are suitable for molecular orientation imaging that Nemoto is trying to achieve; development and application of novel clearing reagents by Miyawaki, and application of orientation-specific labeling of intracellular organelles using fluorescent proteins, etc. The results of these studies will spur technological innovations in various areas of research, including cell polarity, for example, in cell merging where spatial resolution can be improved for the optical observation of not only the cell surface on the basal side but also structures that are considerably far away from the cover glass surface (e.g., nuclear structures, Golgi apparatus, or cell surface on the apical side).
In vivo deep imaging
In addition to the development of an inorganic fluorescent dye (OTN-NIR) by Soga, the creation of long-wavelength (>700 nm) light-emitting proteins by Miyawaki, and the creation of long-wavelength FRET biosensors by Miyawaki and Matsuda, Imamura and Nemoto will work on the development of novel two-photon excitation imaging using a new laser light source in order to achieve in vivo deep imaging that exceeds current limits (>1 mm in cancer, >1.5 mm in the brain) in various biological tissues, including cancer tissue and brain tissue. This will enable us to perform imaging of cancer cell invasion and/or metastasis processes, which has only been possible in some cancer models, and imaging of hippocampal cells in deep brain, for example.
Kamiya will develop probes that can quantify GSH (reduced glutathione), an indicator of antioxidative effects in the cytoplasm. Miyawaki will develop oxidative stress probes based on fluorescent proteins and probes that can quantify bilirubin (unconjugated), an indicator of antioxidative effects in the membrane. Matsuda and colleagues will develop FRET biosensors for measuring stress kinase activity. Moreover, Matsuda and Imamura will create cancer models for imaging by these probes, and Imamura and Nemoto will develop optical systems optimized for such imaging. These efforts will help us pioneer a new aspect of stress research by enabling in vivo imaging of various types of stress, which have not yet been analyzed in vivo.
Zooming in and out
This project mainly targets 4D imaging. Yokota and Miyawaki will take the lead in processing image data obtained at different spatial scales while concurrently working on the construction of revolutionary databases that incorporate data in a small spatial scale into data in a large spatial scale. Through collaborations by researchers in this area as a whole, these databases will be applied to each imaging technology.
As described above, the application of this advanced technology to biological sciences will be performed swiftly while promoting novel technological development, thereby leading to the elucidation of pathophysiology as well as the search for physiological functions, with the aim of achieving breakthroughs in life sciences.
Specific plans and approaches
The main purpose of this research area is to facilitate interactions between researchers who control biological molecules and those who control electromagnetic waves within the research area to spur dramatic improvements in technologies associated with “resonance,” thereby allowing for their application and development in biological sciences.
Each researcher will tackle basic biological and technical problems, and by combining the achievements of researchers who share common and complementary problems within the research area, new technological applications and development will be achieved. The results of studies in the proposed research area will be transmitted to the world with the aim of revolutionizing certain areas of biological sciences. Moreover, efforts will be made to enhance the understanding of many life sciences researchers regarding the benefits of developed technologies.
Provision of expertise and information will be facilitated as researchers in Group A01 develop probes, whereas researchers in Group A02 optimize software and optical devices. On the other hand, researchers engaged in publicly offered research will be expected to utilize the developed technologies to generate breakthroughs in life sciences, and will be requested to provide valuable feedback. The research area as a whole will regularly hold the following meetings in unison, forging organic collaboration between planned research and publicly offered research.
(1) Research meetings and internal evaluation meetings
(2) Young investigator workshops
(3) Co-hosted symposiums and workshops at a variety of academic conferences
(4) Organization of international symposiums
(5) Practical workshops on imaging
(6) Technical support system on the Web, etc.