10 research outputs found
Dynamic SERS Imaging of Cellular Transport Pathways with Endocytosed Gold Nanoparticles
Dynamic SERS imaging inside a living cell is demonstrated with the use of a gold nanoparticle, which travels through the intracellular space to probe local molecular information over time. Simultaneous tracking of particle motion and SERS spectroscopy allows us to detect intracellular molecules at 65 nm spatial resolution and 50 ms temporal resolution, providing molecular maps of organelle transport and lisosomal accumulation. Multiplex spectral and trajectory imaging will enable imaging of specific dynamic biological functions such as membrane protein diffusion, nuclear entry, and rearrangement of cellular cytoskeleton
Dynamic SERS Imaging of Cellular Transport Pathways with Endocytosed Gold Nanoparticles
Dynamic SERS imaging inside a living cell is demonstrated with the use of a gold nanoparticle, which travels through the intracellular space to probe local molecular information over time. Simultaneous tracking of particle motion and SERS spectroscopy allows us to detect intracellular molecules at 65 nm spatial resolution and 50 ms temporal resolution, providing molecular maps of organelle transport and lisosomal accumulation. Multiplex spectral and trajectory imaging will enable imaging of specific dynamic biological functions such as membrane protein diffusion, nuclear entry, and rearrangement of cellular cytoskeleton
Visualizing Cell State Transition Using Raman Spectroscopy
<div><p>System level understanding of the cell requires detailed description of the cell state, which is often characterized by the expression levels of proteins. However, understanding the cell state requires comprehensive information of the cell, which is usually obtained from a large number of cells and their disruption. In this study, we used Raman spectroscopy, which can report changes in the cell state without introducing any label, as a non-invasive method with single cell capability. Significant differences in Raman spectra were observed at the levels of both the cytosol and nucleus in different cell-lines from mouse, indicating that Raman spectra reflect differences in the cell state. Difference in cell state was observed before and after the induction of differentiation in neuroblastoma and adipocytes, showing that Raman spectra can detect subtle changes in the cell state. Cell state transitions during embryonic stem cell (ESC) differentiation were visualized when Raman spectroscopy was coupled with principal component analysis (PCA), which showed gradual transition in the cell states during differentiation. Detailed analysis showed that the diversity between cells are large in undifferentiated ESC and in mesenchymal stem cells compared with terminally differentiated cells, implying that the cell state in stem cells stochastically fluctuates during the self-renewal process. The present study strongly indicates that Raman spectral morphology, in combination with PCA, can be used to establish cells' fingerprints, which can be useful for distinguishing and identifying different cellular states.</p></div
Raman images of cell-lines with differentiation capability.
<p>Raman images of Neuro2a (A) and 3T3L1 (D) cells before (left panel) and after (right panel) the induction of differentiation (inset; bright-field image). (B, E) Averaged Raman spectra of N2a (B) and 3T3L1 (E) cells before (blue) and after (red) induction of differentiation. Spectra are average of 15–27 cells. Spectra from the fibroblast cell-line NIH3T3 are also plotted (black). Peaks characteristic to cytochrome C are indicated with asterisks. (C, F) Score plots of Neuro2a (C) and 3T3L1 (F) cells before (blue) and after (red) the induction of differentiation calculated by PCA. For PCA analysis, raw spectra without averaging was used. Data from the fibroblast cell-line NIH3T3 are also plotted (black). Each marker shows averaged score values of the spectra obtained from single nuclei. Error bar shows SD of the score values from the same nuclei.</p
Difference in Raman spectra between cell-lines.
<p>(A) Averaged Raman spectra of NIH3T3 (blue), EPH4 (purple) and Hepa1-6 (orange) cells in the fingerprint region (700–1800 cm<sup>−1</sup>). Raman spectra are average of 10–34 cells for each cell-line. The lower envelope, which was estimated by a 4<sup>th</sup>-order polynomial fitting, was subtracted from all spectra in order to make the spectral differences clearer for comparison <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084478#pone.0084478-Lieber1" target="_blank">[32]</a>. (B) Score plots calculated by PCA for three cell-lines. For PCA analysis, raw spectra without averaging was used. Each symbol represents a single cell. NIH3T3 (blue), EPH4 (purple) and Hepa1-6 (orange).</p
Transition of ESC state during differentiation.
<p>(A–E) Score plots of Raman spectra at various stages of differentiation spanning a period of 2 weeks. Each marker shows averaged score values of the spectra obtained from single nuclei. Error bar shows SD of the score values from the same nuclei. (F) Histograms of the population distribution of the SD relative to PC1 and PC2 before (blue), 1 week (red) and 2 weeks (green) after LIF removal. Solid lines show the results of the fitting achieved with two Gaussian functions.</p
Raman images of ESCs before and after induction of differentiation.
<p>Bright-field (left panel) and Raman images (right panel) of undifferentiated (A) and differentiated (B) ESCs. (C) Averaged Raman spectra of undifferentiated ESCs (blue), differentiated ESCs (red), and MSCs (green) in the fingerprint region (700–1800 cm<sup>−1</sup>). For PCA analysis, raw spectra without averaging was used. Spectra shown are average of 18–44 cells.</p
Alkyne-Tag Raman Imaging for Visualization of Mobile Small Molecules in Live Cells
Alkyne has a unique Raman band that does not overlap
with Raman
scattering from any endogenous molecule in live cells. Here, we show
that alkyne-tag Raman imaging (ATRI) is a promising approach for visualizing
nonimmobilized small molecules in live cells. An examination of structure–Raman
shift/intensity relationships revealed that alkynes conjugated to
an aromatic ring and/or to a second alkyne (conjugated diynes) have
strong Raman signals in the cellular silent region and can be excellent
tags. Using these design guidelines, we synthesized and imaged a series
of alkyne-tagged coenzyme Q (CoQ) analogues in live cells. Cellular
concentrations of diyne-tagged CoQ analogues could be semiquantitatively
estimated. Finally, simultaneous imaging of two small molecules, 5-ethynyl-2′-deoxyuridine
(EdU) and a CoQ analogue, with distinct Raman tags was demonstrated
Alkyne-Tag SERS Screening and Identification of Small-Molecule-Binding Sites in Protein
Identification of
small-molecule-binding sites in protein is important
for drug discovery and analysis of protein function. Modified amino-acid
residue(s) can be identified by proteolytic cleavage followed by liquid
chromatography–mass spectrometry (LC–MS), but this is
often hindered by the complexity of the peptide mixtures. We have
developed alkyne-tag Raman screening (ATRaS) for identifying binding
sites. In ATRaS, small molecules are tagged with alkyne and form covalent
bond with proteins. After proteolysis and HPLC, fractions containing
the labeled peptides with alkyne tags are detected by means of surface-enhanced
Raman scattering (SERS) using silver nanoparticles and sent to MS/MS
to identify the binding site. The use of SERS realizes high sensitivity
(detection limit: ∼100 femtomole) and reproducibility in the
peptide screening. By using an automated ATRaS system, we successfully
identified the inhibitor-binding site in cysteine protease cathepsin
B, a potential drug target and prognostic marker for tumor metastasis.
We further showed that the ATRaS system works for complex mixtures
of trypsin-digested cell lysate. The ATRaS technology, which provides
high molecular selectivity to LC–MS analysis, has potential
to contribute in various research fields, such as drug discovery,
proteomics, metabolomics and chemical biology
Au-Protected Ag Core/Satellite Nanoassemblies for Excellent Extra-/Intracellular Surface-Enhanced Raman Scattering Activity
Silver
nanoparticles (AgNPs) and their assembled nanostructures such as core/satellite
nanoassemblies are quite attractive in plasmonic-based applications.
However, one biggest drawback of the AgNPs is the poor chemical stability
which also greatly limits their applications. We report fine Au coating
on synthesized quasi-spherical silver nanoparticles (AgNSs) with few
atomic layers to several nanometers by stoichiometric method. The
fine Au coating layer was confirmed by energy-dispersive X-ray spectroscopy
elemental mapping and aberration-corrected high-angle annular dark-field
scanning transmission electron microscopy. The optimized minimal thickness
of Au coating layer on different sized AgNSs (22 nm [email protected] nm Au,
44 nm [email protected] nm Au, 75 nm [email protected] nm Au, and 103 nm [email protected] nm Au) was
determined by extreme chemical stability tests using H<sub>2</sub>O<sub>2</sub>, NaSH, and H<sub>2</sub>S gas. The thin Au coating
layer on AgNSs did not affect their plasmonic-based applications.
The core/satellite assemblies based on Ag@Au NPs showed the comparable
SERS intensity and uniformity three times higher than that of noncoated
Ag core/satellites. The Ag@Au core/satellites also showed high stability
in intracellular SERS imaging for at least two days, while the SERS
of the noncoated Ag core/satellites decayed significantly. These spherical
Ag@Au NPs can be widely used and have great advantages in plasmon-based
applications, intracellular SERS probes, and other biological and
analytical studies