We demonstrate dynamic imaging of molecular distribution in unstained living cells using Raman scattering. By combining slit-scanning detection and optimizing the excitation wavelength, we imaged the dynamic molecular distributions of cytochrome c, protein beta sheets, and lipids in unstained HeLa cells with a temporal resolution of 3 minutes. We found that 532-nm excitation can be used to generate strong Raman scattering signals and to suppress autofluorescence that typically obscures Raman signals. With this technique, we reveal time-resolved distributions of cytochrome c and other biomolecules in living cells in the process of cytokinesis without the need for fluorescent labels or markers.

We perform time-resolved observation of living cells with gold nanoparticles using surface-enhanced Raman scattering (SERS). The position and SERS spectra of 50-nm gold nanoparticles are simultaneously observed by slit-scanning Raman microscopy with high spatial and temporal resolution. From the SERS observation, we confirm the attachment of the particles on the cell surface and the entry into the cell with the subsequent generation of SERS signals from nearby molecules. We also confirm that the strong dependence of SERS spectra on the position of the particle during the transportation of the particle through the cell. The obtained SERS spectra and its temporal fluctuation indicate that the molecular signals observable by this technique are given only from within a limited volume in close proximity to the nanoparticles. This confirms the high spatial selectivity and resolution of SERS imaging for observation of biomolecules involved in cellular events in situ.

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We developed a Raman microscope using a slit-scanning technique for observation of biological samples. A sample was illuminated by a line-shaped laser light, and Raman spectra were measured at different points in the line simultaneously by a spectrometer equipped with a 2D detector. The parallel detection of the Raman spectra boosts the image acquisition rate, which enable us to observe a living biological sample with high temporal and spatial resolution. We also applied a noise reduction technique using singular value decomposition. We recorded motion of intracellular components of living HeLa cells as sequential Raman images in a spectral region between 600 -3000 cm -1 with the temporal resolution of 3 minutes.

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