Near-Infrared Bioimaging

Amid the remarkable recent progress in bioresearch, bioimaging—a technology for visualizing only what one wants to see within a living organism—is developing rapidly. The primary targets of this research are proteins, DNA, and other substances that play crucial roles inside cells and living organisms. It is a technology for observing various target molecules to understand harmful parts of the human body, such as cancerous tissues, and the mechanisms by which they develop. Among these techniques, bioimaging using fluorescence is considered particularly important in modern research. In this process, fluorescent substances—small molecules or proteins that emit fluorescence—play a vital role as bioimaging tools. These include reporter fluorescent molecules and fluorescent proteins that label only the desired areas, as well as fluorescent molecular probes that have the function of enabling the visualization of specific targets. When performing bioimaging with fluorescent molecules or probes, light from deep within the body is absorbed by substances in the living tissue. Therefore, light in the wavelength range from 650 nm to 900 nm, known as the near-infrared window (NIR Window I for bioimaging), has good biological transparency. Fluorescent molecules that emit light in this region are needed for research, such as animal experiments.

In our analytical chemistry laboratory, we have been working on the development of fluorescent molecules that are highly bright and emit strongly in the near-infrared window region, and we have developed a bright fluorescent molecule called KFL (Keio Fluorophore). There are currently 14 types of this molecule, developed as materials that emit high-intensity fluorescence in various colors from approximately 500 nm to 740 nm in the near-infrared window region. KFL, as shown in the figure, has a structure in which borondipyrromethene is extended with a heterocycle. Among the more than 100,000 different types of fluorescent dye molecules available today, it belongs to the class of the brightest. (To express the optical performance of this KFL fluorescent molecule in physical constants, its molar extinction coefficient is 200,000–300,000, and its fluorescence quantum yield is 0.5–1.0, making it an exceptionally bright fluorescent molecule). When used as a reporter molecule, it can brightly stain the inside of cells and tissues. Furthermore, by modifying parts of the molecule, it becomes possible to visualize the distribution of calcium, other ions, pH, and more.

The biggest drawback of bioimaging using such fluorescence is that it requires irradiating light to observe the fluorescence, which inevitably leads to a phenomenon called background fluorescence, where the surrounding area becomes slightly bright. Therefore, in recent years, our laboratory has been working on artificial bioluminescence, which does not have this problem of background light, as an alternative to fluorescence. Organisms such as fireflies and luminous jellyfish have proteins and substrates in their bodies that emit light through their own chemical reactions (oxidation reactions), and these are the source of bioluminescence. In the field of bioluminescence research, the work of Nobel laureate Dr. Osamu Shimomura is renowned. The essence of chemistry, and its role in the 21st century, is arguably to create things that were previously impossible: materials that surpass those found in living organisms (natural products), environmentally friendly products, and useful items from safe materials that are abundant in the environment. Research into artificial bioluminescence is one such endeavor, and its importance lies in creating molecules and materials that surpass those of living organisms. Our analytical chemistry laboratory has been conducting organic synthesis of artificial proteins and novel substrates (small molecules) in collaboration with the University of Tokyo, the University of Tsukuba, Kagoshima University, and others. Among these efforts, we created a new small molecule using coelenterazine as a substrate. When this was combined with ALuc, an artificial enzyme advantageous for artificial bioluminescence, it was found to produce brighter bioluminescence than conventional natural products. Furthermore, we are striving to establish an artificial bioluminescence system using these components that allows for the observation of deep biological sites, such as cancerous areas, with near-infrared light. Students in the laboratory are designing and synthesizing novel substrate molecules with various structures for these purposes, and research toward new artificial bioluminescence is steadily progressing. Among these developments, a near-infrared bioluminescence system using a derivative of coelenterazine (a synthetic small-molecule substrate), the light source of luminous jellyfish, and an artificial enzyme has also been created. It is expected to be used for actual in vivo imaging in the future.