Yusuke Murakami

Coherent Raman imaging (CRI) enables label-free chemical imaging based on intrinsic molecular vibrations, but its applicability is often limited by low sensitivity, hindering the detection of low-abundance biomolecules. Although electronic resonance enhances sensitivity, most resonance CRI implementations rely on narrowband excitation and/or detection, which limits spectral coverage and complicates the differentiation of target molecules from complex backgrounds. Here, we address these challenges by developing broadband electronic resonance coherent anti-Stokes/Stokes Raman scattering (BER-CARS/CSRS) microscopy. We show that BER-CARS/CSRS enables highly sensitive, label-free imaging of endogenous chromophores with broad spectral coverage of the entire fingerprint region. Specifically, we captured time-lapse images, visualizing low-abundance cytochromes alongside abundant biomolecules (lipids, proteins, and nucleic acids) in living human embryonic kidney (HEK) 293 cells. Furthermore, we applied the method to mouse tissues, highlighting characteristic localizations of cytochromes in the brain cortex, the cerebral ventricle wall, and the liver. Our results demonstrate that BER-CARS/CSRS provides highly sensitive, label-free chemical imaging from organelle dynamics to tissue mapping, enabling quantitative phenotyping and slide-scale histopathology.

Lipids constitute approximately half of the dry weight of the brain and play a crucial role in the rapid conduction of action potentials and neurotransmission. However, the maturation of the lipid composition underlying complex brain functions matures during development remains unclear. We developed a sensitive coherent Raman spectroscopic imaging system in which the laser beam is always aligned with the optical axis of the microscope. This system enables seamless stitching-free imaging with a scalable field-of-view and broadband wavelength coverage. Using this unique combination system, we analyzed temporal and spatial changes in lipid composition from the fetal stage to adulthood across mouse brain regions. Our findings revealed that regions with a high total lipid ratio gradually expanded throughout the brain during size growth. While the total lipid ratio continuously increased, changes in lipid composition, particularly ceramide accumulation, were observed in the fiber regions during periods of increasing axonal myelination. High-resolution coherent Raman imaging of the fetal brain revealed that radial glial cells (the most undifferentiated cells) exhibited a higher total lipid ratio than the more differentiated cells. These results can be detected using this system and provide fundamental insights into lipid maturation across different brain regions and developmental stages.

Neuronal primary cilia play important roles in brain development, sensory perception, and neurogenesis. Rootletin, a fibrous protein composed of coiled-coil motifs, is a key structural component of ciliary rootlets and is essential for understanding ciliary functions. However, the precise mechanisms by which Rootletin influences ciliary dynamics and impacts neuronal function remain largely unknown, primarily due to the challenges associated with visualizing these structures. In this study, we present a label-free, rapid, and highly sensitive method for visualizing Rootletin molecules in brain tissue. This platform integrates a second harmonic generation (SHG) microscope with background reduction by a one-step chemical pretreatment. Additionally, we employed coherent anti-Stokes Raman scattering imaging to simultaneously determine the cellular regions and intracellular locations of SHG signals. Applying this multimodal multiphoton imaging approach to the mouse hippocampus revealed that neuronal ciliary rootlets exhibited highly organized and specific intracellular distributions. Moreover, measurements of cultured neurons revealed that ciliary rootlets were detected even in immature neurons. These findings highlight the utility of our label-free imaging platform in developmental and neuroscience research, providing a powerful tool for characterizing ciliary dynamics and neuronal function.