Columns that reveal the world
- Getting up close and personal with the researchers -

Molecules and the atoms that make up molecules cannot be seen with the naked eye, let alone with the world's most precise microscope. Physical chemistry is the study of chemical knowledge, such as the structure and changes of invisible molecules, using physical methods such as quantum mechanics and thermodynamics to interpret these knowledge. This may seem strange to those who have been taught that chemistry and physics are separate subjects at school, but in the world of science, there are no boundaries between chemistry, physics, mathematics, and biology. Our research is driven by a desire to unravel natural phenomena by mobilizing all knowledge.

In my laboratory, we use light called lasers to investigate changes in molecular structure, chemical reactions, energy flows, and so on in extremely short time domains (time resolution). Here, the first important thing is "light." Light with wavelengths visible to the human eye (visible light) is recognized as "colors" of each wavelength, ranging from violet with a short wavelength of 380 nanometers* to red with a long wavelength of 780 nanometers. In addition, there are ultraviolet and infrared rays outside the visible range, and "spectroscopy" is the process of capturing these differences in light as colors (light spectrum).
*Nanometer: 1 billionth of a meter

In research, a laser is aimed at a target molecule, and the structure and chemical reactions of the molecule are examined from the spectrum of the light absorbed by the molecule. When a molecule is hit by laser light, it absorbs the energy of the light and changes from its lowest energy, stable ground state to a higher energy excited state. In this excited state, the molecule either undergoes a chemical reaction and changes into a different molecule, or releases the energy it has received and attempts to return to its stable ground state. What we want to see are the various changes that occur at each moment.

An easy example is molecules that release energy in the form of light emission. However, there are many molecules that do not emit light, so we focused on the fact that the energy of light is converted into heat. We developed an original experimental method by expanding on an analytical technique called "photothermal spectroscopy," which examines the slight heat emitted by molecules in an excited state.

Photothermal spectroscopy technology has the potential to be applied in unexpected fields. In April 2020, through joint research between our laboratory and the Tokyo Metropolitan Police Department's Crime Science Laboratory, we developed a new method for finding traces of document tampering.

In crime investigations, being able to visualize intentionally blacked-out areas or characters added later on evidential documents such as receipts can be useful in uncovering illegal activities. Conventional forensic investigations have used infrared light to detect tampering based on the way ink glows, but this is not effective for ink materials that do not emit light. Initially, we focused on photothermal spectroscopy technology, but since it was difficult to directly detect the slight heat emitted by ink when exposed to light, we instead used a method called "photoacoustic spectroscopic imaging," which detects the "sound" produced when heat is generated.

Have you ever watched fireworks up close and felt a shock wave reverberating through your body the moment the fireworks went up and lit up? Photoacoustic spectroscopy works in the same way as fireworks, capturing the sound emitted by molecules exposed to light. In reality, the sound does not come directly from the molecules, but rather from the heat instantly released by excited molecules (shock waves).

We aimed a laser at ink written on paper and detected the sound produced with a microphone. We were able to read the differences in ink from the differences in the sounds captured, and successfully detected tampering of the text. Although we have not yet fully understood the mechanism, we have found that the pitch and frequency of the sound produced differs depending on the type of ink. As the sound also changes over time, it is possible to distinguish if something has been added later, even if it is the same ink.

Developing a new method for analyzing "document tampering" in collaboration with the National Research Institute of Police Science

Among the interactions between light and molecules, one area that has drawn our greatest interest is "nucleobases." All living things, including humans, have DNA, a biopolymer made up of four types of nucleobases: A (adenine), G (guanine), C (cytosine), and T (thymine). Have you ever heard that important genetic information is written in DNA? In fact, amino acids are made by combining the bases arranged in DNA. Amino acids are then linked together to synthesize proteins, which function in the body as materials for building the body and as enzymes that cause chemical reactions, maintaining life.

Nucleic acid bases are molecules that do not emit light, but we know that they absorb ultraviolet light and enter a high-energy excited state. When a base molecule enters an excited state, it instantly releases its energy and transfers it to its surroundings, returning to its original, stable ground state. When exposed to ultraviolet light, the base molecule remains in an excited state for only a very brief moment, a femtosecond (one quadrillionth of a second). Even when it enters an excited state, it does not undergo any chemical reactions and quickly returns to its original, stable state, making it well suited to protecting important genetic information.

So what would happen if we slightly changed the structure of this base molecule? The chemical formula for thymine, one of the bases, is "C5H6N2O2," but we replaced one of the two oxygen atoms (O) with sulfur (S). In the periodic table, the element sulfur is located below the oxygen element and has similar properties, so it was thought that replacing O with S would not change the properties of thymine very much.

However, this prediction was incorrect. When thymine with O substituted with S (called thiothymine) was irradiated with ultraviolet laser light, the time it remained in an excited state increased to microseconds (one millionth of a second), one billion times longer than before. Furthermore, it was found that while normal nucleic acids absorb UVB (ultraviolet B rays), which has a short wavelength of ultraviolet light, thymine with O substituted with S absorbs UVA (ultraviolet A rays), which has a long wavelength.

Research by a British research group that, like us, is focusing on thionucleobases, has shown the possibility of cancer treatment. Even if a nucleic acid base in which O has been replaced with S is introduced into a cancer cell, the cancer cell will accept it as is and incorporate it into its own DNA. If UVA is irradiated in this state, it will be possible to attack only the cancer cells without damaging normal cells that do not absorb UVA.

Molecular model of thiothymine

There are still many mysteries to be solved regarding the excited state of nucleic acid bases. As explained above, excited nucleic acid bases immediately release energy and lose their excited state, but where does the energy released from the nucleic acid base go? In fact, part of the energy is transferred to stable oxygen molecules.

Just as humans breathe, cells need oxygen. When oxygen molecules in cells receive energy from nucleic acid bases and reach a high-energy state, they become a type of highly reactive oxygen called singlet oxygen. While ultraviolet rays are necessary for the synthesis of vitamin D, the generation of singlet oxygen by ultraviolet rays can also damage cells, causing wrinkles and other problems. This phenomenon is called photoaging.

A device that measures the emission of singlet oxygen (which can detect the production of singlet oxygen)

It is believed that this mechanism may also be involved in photosensitivity and photoallergy, which are common side effects of nonsteroidal anti-inflammatory drugs used in plasters. It is possible that an allergy to light develops when the molecules used in the drug undergo a photochemical reaction due to ultraviolet light. At this point, it is unknown what the allergen is or what the mechanism is. However, if we can understand the mechanism behind the symptoms at a molecular level, investigating the reaction to light may lead to the development of drugs that cause fewer side effects.

I currently serve as Trustee of the Japanese Society of Photomedicine and Photobiology, more than half of whose members are dermatologists. I hope to shed light on biological phenomena related to light through the exchange of ideas between the different fields of medicine and physical chemistry, as well as discover knowledge that will be useful in the medical field.

Our joint research with the National Research Institute of Crime Science and our pharmaceutical research into nucleic acid bases and drug molecules are research that has a clear application. However, the main part of our research is in the field of very basic science. Even our various applied research projects are born out of the knowledge gained from basic research.

One of the representative research results from our laboratory is the "two-photon absorption spectrum of nucleic acid bases." Normally, molecules absorb one photon (light can be considered as a collection of energy particles), so nucleic acid bases only absorb ultraviolet light. However, by irradiating a laser, it is possible to absorb two photons (two-photon absorption) of long wavelengths (low energy) that are not normally absorbed. Two-photon absorption is particularly difficult in nucleic acid bases, and there was no technology to measure this phenomenon.

In April 2020, we applied the photoacoustic method used in the document falsification investigation by the National Research Institute of Criminal Investigation, which captures heat emitted by light absorption as sound, and succeeded in precisely measuring the two-photon absorption spectrum of nucleic acid bases for the first time in the world. The device we developed uses two types of lasers. The acoustic waves generated when the excitation laser is irradiated onto the molecule are captured as fluctuations in the light of the other laser, and the acoustic waves are measured with high sensitivity and high speed. Our laboratory is the only one in the world that has this device, so we must continue to generate more knowledge from here. The molecular properties and principles discovered here will never fade and can become the basis of new technologies.

World's first successful measurement of "two-photon absorption spectrum of nucleic acid bases"

Our research stance will remain unchanged: to interpret natural phenomena using a wide range of scientific knowledge. Moreover, this is a never-ending field of research; for every one thing that is learned, two questions arise. Scientists tell you what they have learned, but this is only a very small part of natural phenomena; most things are still unknown. Together with the students, I would like to continue my research by adhering to the principles of "continuing to enjoy doing things that interest you" and "not believing things that you have not seen with your own eyes, even if they are said to be common sense." (Published in March 2021)