Fast motions of biological molecules, such as lipids and proteins, have been observed with gold nanoparticles that work
as contrast agent for optical microscopy. Because of the strong scattering signal of gold nanoparticles, motions of the
biomolecules can be investigated with high temporal resolution and spatial precision. To further analyze complex
interactions of multiple biological molecules, development of multicolor imaging system with metallic nanoparticles is
important. Here, we developed a multicolor imaging system using silver and silver-gold alloy nanoparticles together with
gold nanoparticles. We used multiple lasers that matched with plasmon resonance of each metal nanoparticles, and
constructed a total internal reflection multicolor dark-field microscope with spectrophotometer. Fast and complex motions
of multiple phospholipids in supported membranes, and multiple kinesins were observed at 100 μs time resolution.
Gold nanoparticles (AuNPs) have been widely used as optical probes to observe motions of biomolecules such as lipids and proteins in biological systems. They efficiently scatter light without photobleaching, and provide high contrast in optical images. Motions of single biomolecules, labeled with AuNPs, have been investigated by tracking center positions of AuNPs in optical image. Nanoscale stepping motions of motor proteins and fast diffusional motions of lipids in membranes have been observed. To understand the working mechanism of tiny and complex biological molecules in detail, further improvement of the localization precision, and imaging capability for multiple biomolecules will be important. We developed an annular illumination total internal reflection dark-field microscope with axicon lens to achieve localization precision at angstrom level and temporal resolution at microsecond order. Localization precisions at 0.3 nm was achieved with 40 nm AuNPs at temporal resolution of 100 µs. We used this system for the observation of bio-molecular motors, such as kinesins and dyneins. We also developed a multicolor single particle tracking system using silver and silver-gold alloy nanoparticles (AgNPs and AgAuNPs) together with AuNPs. We constructed a total internal reflection multicolor dark-field microscope with multiple lasers that match the plasmon resonance wavelength of AgNPs, AgAuNPs, and AuNPs, respectively. A spectrophotometer was used in imaging optics, to project scattering images at each wavelength on the different portions of two-dimensional high-speed CMOS camera. Motions of multiple phospholipids in supported lipid membranes, and multiple kinesins were simultaneously observed at 100 µs time resolution.
Raman microscopy is a powerful tool to observe molecular distribution in live cells. Here, we propose a technique to detect small molecule drugs using alkyne-tag surface-enhanced Raman scattering (SERS) imaging. To obtain SERS effect, we use gold nanoparticles as SERS probes so that we can enhance Raman scattering of small molecule drugs at low concentration with several orders of magnitude. We use alkyne tag to selectively detect the drug molecules by using the Raman peak of alkyne in the spectral silent region. Home-built slit-scanning Raman microscopy enables us to perform rapid SERS imaging. We successfully detect SERS signal from an alkyne-tagged inhibitor of a lysosomal enzyme with gold nanoparticle modified glass substrate.
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
functions.
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-1). Because the alkyne signal is located in the silent region of the cell (1800-2700 cm-1), 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.
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