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

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

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

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