Raman spectral imaging has become a more and more popular technique in biological studies because it can extract
chemical information from living cells in a label-free manner. One of the most challenging issues in the label-free
Raman imaging of biological samples is to increase the molecular specificity in the spectra for better chemical contrast.
Usually, the Raman spectrum from a cell is dominated by a few strong Raman bands such as the amide I band around
1650 cm<sup>-1</sup>, CH<sub>2</sub> bend around 1445 cm<sup>-1</sup> or the amide III band around 1300 cm<sup>-1</sup> and it is not easy to get chemical contrast from other Raman bands that overlap with them. In this study, we aim to manipulate the chemical contrast in a living cell by exploiting the polarisation effects in Raman spectroscopy. By simply putting an analyser before the spectrometer, we can take the Raman image at the parallel and perpendicular polarisation against the incident light at the sample. The Raman spectra at the two orthogonal polarisations represent the Raman signals with different molecular orientation and symmetry of vibrations. Our experimental results demonstrate that at certain Raman shifts the two orthogonal polarisations indeed present different chemical contrasts. This indicates that polarized Raman imaging can help us visualise the different chemical contrasts that overlap at the same Raman shift and therefore increase the amount of chemical information we can get from cells.
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.
Raman spectroscopy has been utilized to investigate properties of biomolecules due to its capability of detecting
molecular vibrations that represent molecular species, structures and environmental conditions. In this research, we used
Raman scattering to image dynamics of molecules in living cells. By combining the 532 nm excitation and a slitscanning
detection technique, we observed dynamics of molecular distributions of cytochrome, protein beta sheets, and
lipids in an unstained HeLa cell during mitosis with a frame rate of 5 minutes.
We observed spatial and temporal behaviors of surface enhanced Raman scattering (SERS) signals with gold
nanoparticles in living cells. Gold nanoparticles with the diameter of 50 nm were introduced into macrophage cells
through endocytosis. We performed observation of SERS signals from a macrophage with 785 nm excitation. Strong
SERS signal from the particles in the cell was observed, and spectrum from each particle shows difference in Raman
peaks and intensity. We also observed time-lapse SERS and dark-field image with a frame rate of 3 min. We confirmed
that strong SERS signal from the particle in the cell and the spectral changes with positions of the particles in the cell.
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.
In recent years, various types of molecular imaging technologies have been developed, but many of them require probes
and may have some influence on the distribution of the target molecules. In contrast, Raman microscopic analysis is
effective for molecular identification of materials, and molecular imaging methods employing Raman scattering light can
be applied to living organisms without use of any exogenous probes. Unfortunately, Raman microscopic imaging is
rarely used in the biomedical field due to the weakness of Raman signals. When the conventional Raman microscopes
are used, the acquisition of an image of a cell usually takes several hours. Recently, a slit-scanning confocal Raman
microscope has been developed. It can acquire images of living cells and tissues with faster scanning speed. In this study,
we used the slit-scanning confocal Raman microscope (RAMAN-11) to image the distribution of a drug in living cells.
We could acquire images of the distribution of an anticancer reagent in living cells within several minutes. Since the
wavelength of Raman scattering light is determined by the frequency of molecular vibration, the in situ mapping of the
intracellular drugs without use of a probe is possible, suggesting that laser Raman imaging is a useful method for a
variety of pharmacokinetic studies.
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<sup>-1</sup> with the temporal resolution of 3