The nonlinear variant of Raman spectroscopy, coherent anti-Stokes Raman scattering (CARS) microscopy, combines powerful Raman signal enhancement with several other advantages such as label-free detection and has been used to image various cellular processes including host-pathogen interactions and lipid metabolism. Cellular biomolecules contain unique molecular vibrations that can be visualized by coherent anti-Stokes Raman scattering (CARS) microscopy without the need for labels. Here we review the application of CARS microscopy for label-free imaging of cells and tissues using the natural vibrational contrast that arises from biomolecules like lipids as well as for imaging of exogenously added probes or drugs. High-resolution CARS microscopy combined with multimodal imaging has allowed for dynamic monitoring of cellular processes such as lipid metabolism and storage, the movement of organelles, adipogenesis and host-pathogen interactions and can also be used to track molecules within cells and tissues. The CARS imaging modality provides a unique tool for biological chemists to elucidate the state of a cellular environment without perturbing it and to perceive the functional effects of added molecules.

Increasing use of silver nanoparticles (Ag-NPs) in various products is resulting in a greater likelihood of human exposure to these materials. Nevertheless, little is still known about the influence of carbohydrates on the toxicity and cellular uptake of nanoparticles. Ag-NPs functionalized with three different monosaccharides and ethylene glycol were synthesized and characterised. Oxidative stress and toxicity was evaluated by protein carbonylation and MTT assay, respectively. Cellular uptake was evaluated by confocal microscopy and ICP-MS. Ag-NPs coated with galactose and mannose were considerably less toxic to neuronal-like cells and hepatocytes compared to particles functionalized by glucose, ethylene glycol or citrate. Toxicity correlated to oxidative stress but not to cellular uptake. Carbohydrate coating on silver nanoparticles modulates both oxidative stress and cellular uptake, but mainly the first has an impact on toxicity. These findings provide new perspectives on modulating the bioactivity of Ag-NPs by using carbohydrates.

Multiplexed SERS imaging of receptor proteins on the surface of mammalian cells has been carried out using functionalized silver nanoparticles. Deconvolution of four differently functionalized nanoparticles is readily achieved, and using this approach, receptor co-localization can be probed and protein–protein interactions can be elucidated at the surface of cells.

HMA2 is a Zn²⁺-ATPase from Arabidopsis thaliana. It contributes to the maintenance of metal homeostasis in cells by driving Zn²⁺ efflux. Distinct from P_1B-type ATPases, plant Zn²⁺-ATPases have long C-terminal sequences rich in Cys and His. Removal of the 244 amino acid C terminus of HMA2 leads to a 43% reduction in enzyme turnover without significant effect on the Zn²⁺ K_½ for enzyme activation. Characterization of the isolated HMA2 C terminus showed that this fragment binds three Zn²⁺ with high affinity (K_d = 16 ± 3nm). Circular dichroism spectral analysis indicated the presence of 8% α-helix, 45% β-sheet, and 48% random coil in the C-terminal peptide with noticeable structural changes upon metal binding (8% α-helix, 39% β-sheet, and 52% random coil). Zn K-edge XAS of Zn-C-MBD in the presence of one equivalent of Zn²⁺ shows that the average zinc complex formed is composed of three His and one Cys residues. Upon the addition of two extra Zn²⁺ ions per C-MBD, these appear coordinated primarily by His residues thus, suggesting that the three Zn²⁺ binding domains might not be identical. Modification of His residues with diethyl pyrocarbonate completely inhibited Zn²⁺ binding to the C terminus, pointing out the importance of His residues in Zn²⁺ coordination. In contrast, alkylation of Cys with iodoacetic acid did not prevent Zn²⁺ binding to the HMA2 C terminus. Zn K-edge XAS of the Cys-alkylated protein was consistent with (N/O)₄ coordination of the zinc site, with three of those ligands fitting for His residues. In summary, plant Zn²⁺-ATPases contain novel metal binding domains in their cytoplasmic C terminus. Structurally distinct from the well characterized N-terminal metal binding domains present in most P_1B-type ATPases, they also appear to regulate enzyme turnover rate.