Capturing the Natural States of Biomolecules in Crowded Environments: Label-free in situ Quantitative Analysis Using Raman Microscopy

Unlike representative structural techniques (X-ray crystallography, cryo-EM, NMR, and fluorescence), Raman microscopy enables the label-free, in situ detection of biomolecules, even in disordered or heterogeneous states. It also allows the observation of all molecules within the field of view, depending on their concentrations. This review highlights our Raman research that leverages these advantages, demonstrating the ability to quantitatively measure the concentration of molecules in complicated biological systems in a label-free and in situ manner. A key technique introduced in this review is the use of the Raman band of water as an internal intensity standard. Water outside droplets (condensates) in measurements of liquid-liquid phase separation or outside living cells in cell measurements has a constant density regardless of the sample. Thus, the O-H stretching bands of these waters can be used as intensity standards, allowing for the quantitative interpretation of Raman intensities and enabling the determination of biomolecular concentrations in droplets or cells under a microscope. In addition, by analyzing the shape of the O-H stretching band of intracellular water, the temperature inside a single cell can be determined in situ without labeling. The method established in this study, which comprises three approaches utilizing Raman imaging, label-free, in situ observation, and quantitative visualization of components, is expected to have diverse applications, including the elucidation of disease onset mechanisms.