Osaka University achieves high-sensitivity observation of molecules in biological samples through development of a new Raman microscope

Raman microscopy can measure internal structure and chemical composition without preprocessing, but its application to biological samples has been limited. A research group led by Doctoral Student Kenta Mizushima (currently at Johns Hopkins University) has succeeded in developing a Raman microscope that can freeze biological samples and observe molecules with high sensitivity. The research group includes Professor Katsumasa Fujita, and Specially Appointed Associate Professor Masahito Yamanaka of the Graduate School of Engineering, and Associate Professor Yasuaki Kumamoto of the Institute for Open and Transdisciplinary Research Initiatives at Osaka University, with collaborators including Associate Professor Nicholas Smith of the Osaka University Immunology Frontier Research Center, Specially Appointed Professor Hideo Tanaka of Kyoto Prefectural University of Medicine, and Group Director Mikiko Sodeoka of the RIKEN Center for Sustainable Resource Science. Fujita said, "I expect that this technology will prove useful in a wide range of fields from basic research to practical cell industry applications, and I will start a company next year to put it into practical use." The study was published in Science Advances.

Raman spectroscopy allows for non-destructive observation of the internal structure and chemical composition in various states, such as solids, liquids, and gases, without any preprocessing by applying a laser and measuring the Raman scattered light from the sample. Although widely used in materials and related fields, its application to biological samples such as cells has been limited because the Raman scattered light is so weak. Reasons for the limited scope of application include material damage by the use of high-intensity laser irradiation and change of chemical composition of the sample caused by sample fixation during long-time measurement.

The research group has developed a method for long-term Raman observation by rapidly freezing biological samples such as cells and fixing them at liquid nitrogen temperature. Specifically, they developed a unique sample chamber to quickly freeze the sample with liquid propane at −185℃. The sample is then fixed under temperature control by continuously cooling it with surrounding liquid nitrogen channels or warming it with a heater. The cryofixed sample is then irradiated simultaneously with 100-milliwatt lasers at several hundred points (several milliwatts per point) to observe a wide area for an extended period. This method enabled Raman observation with a high signal-to-noise ratio, high resolution, and a field of view several hundred times larger than conventional methods. Mizushima says, "We had a hard time developing the freezing method. With cryogens for cryo-electron microscopy, Raman scattering comes out from the cryogen, so we could not obtain proper imaging data. The most difficult part was that we had to repeat experiments before finally identifying liquid propane."

In the experiment, they used the newly developed microscope for long-term Raman observation of cryofixed cells and confirmed that the Raman signal was about eight times higher than that obtained by the conventional method. Furthermore, they performed Raman observation of the redox state of cytochrome c in cryofixed ischemic rat heart tissue. For the first time, they obtained the microscopic images showing differences in the redox state of cytochrome c in ischemic and normal heart tissue samples, which could not be observed in unfixed live samples. They also succeeded in increasing the number of recognizable molecular species in cell observation to nine, which is about twice as many as that with the conventional method.

Fujita said, "We had the idea of freezing samples for some time, but the experiment was difficult. We initially tried to bring the samples frozen elsewhere to the microscope for observation, but their state changed. So, in 2018, I came up with the idea of incorporating the cryostat into a microscope, and we could make it happen because the project was accepted for CREST next year. In this research, we reported the results from 10 to 20 hours of observation, but we can increase the range of observation and the number of identifiable molecular species if we observe the sample for a longer period."

Various biological samples are frozen in a wide range of fields, including drug discovery, reproductive medicine, cell therapy, regenerative medicine, animal husbandry, and basic research, and the new method can be applied to quality control and evaluation in these fields. In fact, Osaka University and Iwatani Corporation have jointly revealed that temperature fluctuations during sample transfer can reduce cell viability. Moreover, the new method has opened up the possibility of high-sensitivity Raman observation of drugs and other molecules present at low concentrations in biological samples and has also made it possible to observe the chemical state of molecules in biological samples at the moment of being frozen. The method is expected to be useful for a wide range of research.

Journal Information
Publication: Science Advances
Title: Raman microscopy of cryofixed biological specimens for high-resolution and high-sensitivity chemical imaging
DOI: 10.1126/sciadv.adn0110