We propose the combination of alkyne-tag and surface-enhanced Raman scattering (SERS) spectroscopy to perform
highly-sensitive and selective drug imaging in live cells. Gold nanoparticles are introduced in lysosomes through
endocytosis as SERS agents, and the alkyne-tagged drugs are subsequently administered in cells. Raman microscopic
observation reveals the arrival of drug in lysosome through enhanced Raman signal of alkyne. Since the peak of alkyne
appears in Raman-silent region of biomolecules, selective detection of drugs is possible without background signal of
endogenous molecules. From endocytosed gold nanoparticles in living HeLa cells, we observed distinct Raman signal
from alkyne-tagged inhibitor of lysosomal enzyme.
Raman microscopy is useful for molecular imaging and analysis of biological specimens. Here, we used alkyne containing a carbon-carbon triple bond as a Raman tag for observing small molecules in live cells. Alkyne tags can maintain original properties of target molecules with providing high chemical specificity owing to its distinct peak in a Raman-silent window of biomolecules. For demonstrations, alkyne-tagged thymidine and coenzyme Q analogue in live cells were visualized with high-spatial resolution. We extended the application of alkyne-tag imaging to visualize cell organelles and specific lipid components in artificial monolayer membranes.
Surface enhanced Raman scattering (SERS) has been used to detect biological molecules at a low concentration. We
developed a rapid Raman imaging system, which can image dynamic activity of SERS agents, such as gold nanoparticles,
in a living cell and the temporal behaviors of SERS spectra. Combination of slit scanning and an EM-CCD camera for
measuring SERS spectra enables us to obtain a SERS image in a few seconds. The system can also be used to track a
single particle moving in a cell with a laser focus and measure SERS spectra with a temporal resolution of 50 msec. By
using the developed microscope systems, we monitored the change of SERS spectra associated cell transportation
Role of small molecules such as drugs or metabolites in cells is commonly studied by fluorescence microscopy in which
a fluorescent label is attached to the molecule. However, fluorescent labels are typically large that often interfere with the
normal cellular function of the molecule. To avoid the use of bulky fluorescent labels, we introduce a technique that uses
a simple small chemical tag called alkyne consisting of two carbons connected by a triple bond. The alkyne-tagged
molecule is imaged using Raman microscopy that detects the strong Raman signal from the CC triple bond stretching
vibration (~2120 cm<sup>-1</sup>). Because the alkyne signal is located in the silent region of the cell (1800-2700 cm<sup>-1</sup>), it does not
interfere with any intrinsic cellular Raman signals. Here, we demonstrate this technique by showing Raman images of an
alkyne-tagged component of DNA in a living cell using a slit-scanning confocal Raman microscope. This fast imaging
technique is based on a line-shaped focus illumination and simultaneous detection of the Raman spectra from multiple
points of the sample. Using this microscope, we obtained time-course Raman images of the incorporation of EdU in the
DNA of HeLa cells in just several tens of 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.